Detection and Analysis of Genetic Alterations in Normal Skin and Skin Tumours 91-7283-379-3

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Detection and Analysis of Genetic Alterations in Normal Skin and Skin Tumours

Åsa Sivertsson Med.Lic.

Royal Institute of Technology Department of Biotechnology Stockholm 2002

 Åsa Sivertsson Department of Biotechnology Royal Institute of Technology Stockholm Center for Physics, Astronomy and Biotechnology SE-106 91 Stockholm Sweden Printed at Universitetsservice US AB Box 700 14 100 44 Stockholm Sweden

ISBN 91-7283-379-3

Åsa Sivertsson (2002): Detection and analysis of genetic alterations in normal skin and skin tumours Department of Biotechnology, Royal Institute of Technology Stockholm, Sweden ISBN 91-7283-379-3 ABSTRACT The investigation of genetic alterations in cancer-related genes is useful for research, prognostic and therapeutic purposes. However, the genetic heterogeneity that often occurs during tumour progression can make correct analysis challenging. The objective of this work has been to develop, evaluate and apply techniques that are sufficiently sensitive and specific to detect and analyse genetic alterations in skin tumours as well as in normal skin. Initially, a method based on laser-assisted microdissection in combination with conventional dideoxy sequencing was developed and evaluated for the analysis of the p53 tumour suppressor gene in small tissue samples. This method was shown to facilitate the analysis of single somatic cells from histologic tissue sections. In two subsequent studies the method was used to analyse single cells to investigate the effects of ultraviolet (UV) light on normal skin. Single p53 immunoreactive and nonimmunoreactive cells from different layers of sunexposed skin, as well as skin protected from exposure, were analysed for mutations in the p53 gene. The results revealed the structure of a clandestine p53 clone and provided new insight into the possible events involved in normal differentiation by suggesting a role for allele dropout. The mutational effect of physiological doses of ultraviolet light A (UVA) on normal skin was then investigated by analysing the p53 gene status in single immunoreactive cells at different time-points. Strong indications were found that UVA (even at low doses) is indeed a mutagen and that its role should not be disregarded in skin carcinogenesis. After slight modifications, the p53 mutation analysis strategy was then used to complement an x-chromosome inactivation assay for investigation of basal cell cancer (BCC) clonality. The conclusion was that although the majority of BCC’s are of monoclonal origin, an occasional tumour with apparently polyclonal origin exists. Finally, a pyrosequencing-based mutation detection method was developed and evaluated for detection of hot-spot mutations in the N-ras gene of malignant melanoma. More than 80 melanoma metastasis samples were analysed by the standard approach of single strand conformation polymorphism analysis (SSCP)/DNA sequencing and by this pyrosequencing strategy. Pyrosequencing was found to be a good alternative to SSCP/DNA sequencing and showed equivalent reproducibility and sensitivity in addition to being a simple and rapid technique.

Keywords: single cell, DNA sequencing, p53, mutation, UV, BCC, pyrosequencing, malignant melanoma, N-ras  Åsa Sivertsson, 2002

There is much pleasure to be gained from useless knowledge Bertrand Russell

ISBN 91-7283-379-3 This thesis is based on the following publications, which in the text will be referred to by their Roman numerals:

LIST OF PUBLICATIONS I.

Persson Å, Ling G, Williams C, Bäckvall H, Ponten J, Ponten F, Lundeberg J. (2000) Analysis of p53 mutations in single cells obtained from histological tissue sections. Anal Biochem 287(1): 25-31.

II.

Persson Å/Ling G, Berne B, Uhlén M, Lundeberg J, Ponten F. (2001) Persistent p53 mutations in single cells from normal human skin. Am J Pathol 159:1247-1253.

III.

Persson Å, Wiegleb Edström D, Bäckvall H, Lundeberg J, Pontén F, Ros AM, Williams C. (2002) The Mutagenic Effect of Ultraviolet A1 in Human Skin-demonstrated by sequencing the p53 gene in single keratinocytes. Photodermatology, Photoimmunology and Photomedicine. In press.

IV.

Asplund A, Sivertsson Å, Lundeberg J, Pontén F (2002) Analysis of xchromosome inactivation patterns in human basal cell carcinoma reveals diverse clonal organization: evidence of multicellular origin. Manuscript.

V.

Sivertsson Å, Platz A, Hansson J, Lundeberg J. (2002) Pyrosequencing as an alternative to SSCP for detection of N-ras mutations in human melanoma metastases. Clin Chem. In press.

INTRODUCTION ___________________________________________________ 1 Mutation detection_______________________________________________________ 1 Scanning methods _______________________________________________________ 2 DNA sequencing ______________________________________________________________ 2 Sanger sequencing _____________________________________________________________ 2 SSCP _______________________________________________________________________ 4 Other conformation-based methods ________________________________________________ 6 Cleavage –based techniques _____________________________________________________ 8

Specific methods ________________________________________________________11 Hybridisation-based techniques__________________________________________________ 11 Methods based on allele-specific amplification ______________________________________ 12 Oligonucleotide ligation assays __________________________________________________ 12 Techniques based on polymerase extension _________________________________________ 13 Pyrosequencing ______________________________________________________________ 16

CARCINOGENESIS________________________________________________ 19 Cancer of the skin _______________________________________________________21 Ultraviolet radiation __________________________________________________________ 21 Normal skin _________________________________________________________________ 24 The tumour suppressor p53 _____________________________________________________ 26 The p53 patch _______________________________________________________________ 27 Non-melanoma skin cancer _____________________________________________________ 29 Malignant melanoma __________________________________________________________ 30

PRESENT INVESTIGATION________________________________________ 32 Genetic analysis of skin and skin tumour samples. _____________________________32 Sample handling and preparation for analysis_______________________________________ 32 Sample preparation _________________________________________________________ 32 Microdissection____________________________________________________________ 33 Laser microdissection _______________________________________________________ 33

p53 gene analysis ________________________________________________________35 Development of method for single cell genetic analysis (I). _____________________________ 35

Applications ____________________________________________________________38 Effects of UV radiation on the p53 gene in normal human epidermis (II, III) _______________ 38 Sun-exposure and persistent p53 mutations (II) _____________________________________ 38 p53 mutations induced by UVA1 (III) _____________________________________________ 41 The clonality of BCC (IV) ______________________________________________________ 43 Detection of N-ras hot-spot mutations in melanoma using pyrosequencing (V) ______________ 45

Abbreviations______________________________________________________ 47 Acknowledgements _________________________________________________ 48 References ________________________________________________________ 50

Å Sivertsson

INTRODUCTION The human body consists of approximately 2x101 2 cells, each containing the hereditary information of the genome in the form of 3x10 9 basepairs of deoxyribonucleic acid (DNA). This DNA is arranged into 46 chromosomes and the composition of the DNA bases within these macromolecules determines the genotype and contributes to the phenotype of an individual. Consequently, alterations in chromosomes may lead to genetically related diseases. Cells are constantly exposed to agents and events that can be harmful to the DNA. Depurination, deamination, oxidation and methylation of bases are endogenous events that can change the base composition of DNA, while exogenous agents such as UV light, ionising radiation and genotoxic substances may damage DNA by creating pyrimidine dimers, doublestrand breaks and adducts. Several different gatekeeper and DNA repair systems exist to maintain DNA integrity, but occasionally these backup systems fail and mutations arise. Mutations can be divided into two classes; gross alterations and subtle alterations. Gross alterations involve changes in chromosome number, partial deletions of chromosomes, chromosomal translocations and gene duplications, while subtle alterations comprise of single base substitutions, insertions and deletions. Mutations are common events in cancer and the ability to monitor them is therefore of great interest for diagnostic and prognostic purposes as well as in understanding gene function.

Mutation detection Gross chromosomal aberrations can usually be detected by cytogenetical methods or by DNA fragment analysis, while methods that are more sensitive and usually more expensive must be used for detection of subtle alterations. A wide range of methods already exists for detection of single (or a few) basepair changes. Specificity, sensitivity, technical complexity and cost vary between the different methods and the multitude of different techniques present indicates that the perfect technique for detection of subtle alterations has not yet been described. The mutation detection strategies can be divided into two categories depending on the alteration they identify. Scanning methods detect uncharacterised sequence variations, while specific methods

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Detection and analysis of genetic alterations in normal skin and skin tumours

identify previously characterised sequence variations such as polymorphisms and mutation hotspots. Some of the more widely used methods as well as some promising upcoming techniques will be described briefly below.

Scanning methods In order to perform a comprehensive mutation analysis of a stretch of DNA the use of scanning methods or DNA sequencing is required. The principle of some scanning methods described below is shown in Figure 1. DNA sequencing is considered the golden standard for identification of mutations while any mutation found by a scanning method is also usually confirmed by DNA sequencing. DNA sequencing In 1977, two different DNA sequencing techniques based on the generation of singlestrand DNA ladders were described. Maxam-Gilbert sequencing takes advantage of chemicals that cleave DNA at specific bases to create fragments of different lengths subsequently separated by electrophoresis (Maxam, 1977). The toxicity of the chemicals used for cleavage has precluded widespread use of this method, but it provides an approach for sequencing templates unavailable for enzymatic sequencing such as adduct modified DNA (Wilkins, 1985; Mao, 1995; Mao, 1992). The Sanger sequencing technique, which is more widely used, relies on enzymatic chain termination to generate a sequencing ladder for size separation (Sanger, 1977). Sanger sequencing Although Sanger sequencing was introduced 25 years ago, to date it is still the only method able to scrutinise each position in an unknown sample of up to 800 basepairs in length. This feature has lead to the continuous development of the technique via automation, nucleotide labelling and enzyme engineering, despite it being a laborious and expensive method. The method is based on primer extension in the presence of a dideoxynucleotide, which causes chain termination. Four parallel reactions are performed in which a primer is hybridised to the single-stranded DNA template and extended by a DNA polymerase in the presence of a low concentration of a chain terminating dideoxynucleotide (ddNTP) (one in each reaction) and all four deoxynucleotides (dNTPs). The synthesis is terminated whenever a ddNTP is incorporated instead of

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the corresponding dNTP resulting in a ladder of fragments terminated at stepwise intervals. The fragments are separated according to size by electrophoresis and the sequence of the target DNA is determined by reading the band pattern. Conventionally, radioactively labelled nucleotides or primers have been used to facilitate detection of the band patterns, but these labels have now generally been replaced by fluorescent-based labelling strategies. Fluorescent dyes can be introduced by using primers with a 5´ end-label (dye-primers) (Smith, 1986; Ansorge, 1986) or alternatively by incorporation of labelled ddNTPs (dye-terminators) (Prober, 1987) or labelled dNTPs (internal labelling) (Ansorge, 1992) during extension. The dye-primer and the dye-terminator set-ups are compatible with the two different formats available for slab-gel electrophoresis i. e. the four lane-one dye and the one lane-four dye strategies. The four lane-one dye format generates easily interpreted data, since a single dye is used and the mobility is equal in all lanes. The throughput, however, is slow but an automated set-up is available in the automated laser fluorescence sequencer (ALF) from Amersham Pharmacia. In the one lane-four dye format, four different fluorophores are used and all four sequencing ladders are separated simultaneously in a single lane using the set-up described by Smith (Smith, 1986). This approach has become more common due to its higher throughput and is used in the automated sequencers commercially available from manufacturers such as Applied Biosystems. The requirement for processing raw data using base calling algorithms may however slightly hamper the sensitivity of mutation detection and quantification. The ever-increasing need for higher throughput and cost reduction in DNA sequencing has lead to the gradual replacement of the slab gels by capillary gel electrophoresis (CGE) (Smith, 1991). CGE requires smaller sample volumes allowing for a reduction in the amount of template DNA and reagents, thereby resulting in a reduction in overall costs. In addition, faster sequencing runs are facilitated, since the capillaries can more effectively dissipate heat allowing the use of higher voltages during electrophoresis. Also in terms of sample analysis capillaries have an advantage over slab gels in that tracking of lanes no longer needs to be considered. In addition to the development of fluorescent labelling and automation, a significant advance in the technology has been the modification and engineering of the enzymes used in the DNA sequencing reaction. The first enzyme used was the Klenow

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Detection and analysis of genetic alterations in normal skin and skin tumours

fragment, which due to its sequence dependent discrimination between ddNTPs generated variations in the raw data and thus uneven peaks (Klenow, 1970). Higher quality and more easily interpreted data was generated using the T7 DNA polymerase, which showed even incorporation of all four ddNTPs (Tabor, 1987; Tabor, 1989; Kristensen, 1988). This enzyme still offers the most uniform sequence data but has the disadvantage of being temperature sensitive and easily degraded. However, after the introduction of the thermostable polymerase derived from Thermus aquaticus (Taq DNA polymerase) (Chien, 1976) the use of a linear PCR amplification mode for the dideoxy reactions has become very popular. In cycle sequencing, one primer is used to generate the Sanger fragments in an amplification reaction, which gives the advantages of lower template requirement, generation of single stranded DNA without time-consuming denaturation of double-strands and ease of automation (Innis, 1988; Murray, 1989). Manipulations of the Taq DNA polymerase solved initial problems with discrimination of the enzyme for dNTPs over ddNTPs and a uniform peak pattern comparable to that of T7 DNA polymerase can now be generated (Reeve, 1995; Tabor, 1995). SSCP Scanning methods usually provide a simple and low-cost assay to simultaneously compare larger regions of DNA between normal and unknown samples to rapidly eliminate non-mutant samples. Since its introduction in 1989, single strand conformation polymorphism (SSCP) analysis has become one of the most popular scanning strategies, which is probably due to its technical simplicity in combination with its low cost and relatively high sensitivity (Orita, 1989). The method is based on the sequence dependent mobility of single stranded DNA during electrophoresis. Amplified DNA fragments are first denatured using agents such as formamide, sodium hydroxide, urea or methylmercuric hydroxide (Humphries, 1997; Xie, 1997). Despite the superior performance of methylmercuric hydroxide in some applications its toxicity precludes its widespread use, with the result that formamide is the most commonly used reagent today (Weghorst, 1993; Hongyo, 1993). The single stranded DNA is then subjected to electrophoresis through a non-denaturing gel with the mobility of the DNA fragment dependent on its adopted conformation, which is influenced by its sequence and size. Thus, a single base alteration can be detected by SSCP if a conformer displaying different mobility through the gel is generated.

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However, due to the absence of theoretical models to predict either the threedimensional structure of single-stranded DNA under a given set of conditions or the resulting mobility of any given conformer, the optimal conditions for discriminating between any two fragments differing by a single base must be empirically determined. In addition to the sequence context and size of the DNA fragment, several parameters such as the gel matrix composition, temperature during electrophoresis and concentration of DNA have been found to affect the sensitivity of SSCP analysis. The pH, ionic strength and composition of the electrophoresis buffer can also affect the mobility of DNA during SSCP. However, it has been postulated that virtually all mutations can be detected by using a combination of the three following conditions; electrophoresis at room temperature with or without 5%-10% glycerol and at 4°C without glycerol (Hayashi, 1993; Hayashi, 1991; Sheffield, 1993). The composition of the gel matrix is important and optimal sensitivity have been achieved using high concentrations of acrylamide with low crosslinking or alternatively by using the commercially available Mutation Detection Enhancement (MED) gel (Ravnik-Glavac, 1994; Glavac, 1993). The difference in migration between fragments longer than 300 basepairs can be further enhanced by addition of 10-15 % glycerol or sucrose, or 5 % of a denaturing agent such as urea. Addition of glycerol decreases the pH and thereby weakens the electrostatic repulsion between the negative charges in the nucleic acid backbone, which in turn may allow for greater stabilisation of the tertiary structure (Kukita, 1997). The same effect can also be achieved by lowering the temperature during electrophoresis or by using buffers with a low pH or higher salt concentration (Kukita, 1997). Addition of primers to the SSCP template has also been shown to stabilise the single stranded conformation resulting in better separation of the DNA fragments (Almeida, 1998). In relation to the DNA fragment length, Sheffield and coworkers has reported an effective size limit of 150-200 basepairs in order to achieve a maximum sensitivity of 97 % (Sheffield, 1993), while other studies have shown sensitivity greater than 95 % for fragments ranging from 200 to 900 basepairs (Fan, 1993; Ravnik-Glavac, 1994; Ravnik-Glavac, 1994; Highsmith, 1999). These varying results may in part depend on the sequence composition of the samples. The detection sensitivity is greater in GC rich templates which is due to the higher proportion of hydrogen bonds formed between G and C residues resulting in a more intricate, and thus more easily influenced folding of the strands (Highsmith, 1999).

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Detection and analysis of genetic alterations in normal skin and skin tumours

The detection of fragments after electrophoresis can be achieved by autoradiography, silver staining or through the use of fluorescent dyes (Hoshino, 1992; Hiort, 1994; Iacopetta, 1998; Law, 1996). Autoradiography is time-consuming and involves the use of radioactively labelled primers and has therefore gradually been replaced by other methods. Analysis on automated sequencers using fluorescent-labelled primers is another commonly used alternative which offers high-throughput in addition to high sensitivity. The use of fluorescent dyes further allows for the loading of low concentrations of DNA which prevents disturbing reannealing (Makino, 1992; Iwahana, 1996; Ellison, 1996; Ellison, 1993; Iwahana, 1994). Depending on the sequence analysed and the conditions used it has been reported that between 60 % to 100 % of mutations present can be detected by SSCP (Martincic, 1996; Vidal-Puig, 1994; Ellis, 2000; Moore, 2000; Ellison, 1993; Ravnik-Glavac, 1994). Usually a sensitivity of 90% or less is reported using a single condition for electrophoresis while a sensitivity of greater than 95 % is reached when several different conditions are applied. Recently, high-throughput methods combining SSCP and heteroduplex analysis (HA) have been described (Kozlowski, 2001; Kourkine, 2002). By thermally denaturing and rapidly cooling a mixture of wild type and mutant double-stranded DNA, single stranded conformers as well as homo -and heteroduplexes will be formed. The sample is then subjected to simultaneous SSCP and HA using capillary electrophoresis or capillary array electrophoresis and fluorescent detection. This tandem approach results in 100 % sensitivity for mutation detection, compared to a sensitivity of 9093% for SSCP and 75-81 % for HA. Other conformation-based methods Denaturing gradient gel electrophoresis (DGGE), HA and denaturing high performance liquid chromatography (DHPLC) belong to a group of scanning methods which are based on mobility shifts created during electrophoresis between DNA fragments of different sequences but of equal lengths. Denaturing gradient gel electrophoresis (Myers, 1985)[(Fischer, 1980; Fisher, 1983) uses a linearly increasing gradient of a denaturing agent to exploit differences in the melting properties of DNA duplexes with different sequences. During migration in the gel the duplexes will progressively dissociate at discrete domains that have lower melting temperatures (dependent on the sequence composition) which will cause a marked

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retardation of the fragments. A single base change may result in a different migration pattern, thus allowing mutations to be detected. If homo- and heteroduplexes of wildtype and mutant sequences are formed via denaturation and reannealing of the amplified fragments and a GC-clamp is introduced (Myers, 1985; Sheffield, 1989; Myers, 1985) (to prevent complete denaturation of high melting domains), the sensitivity can be further increased. Under these conditions, detection of almost 100 % of single nucleotide changes in fragments ≤ 500 basepairs has been shown (Myers, 1985). Several variants of DGGE have been developed such as genomic DGGE (Borresen, 1988), temperature gradient gel electrophoresis (TGGE) (Wartell, 1990) and constant denaturant gel electrophoresis (CDGE) (Hovig, 1991). Denaturing gradient gel electrophoresis is a sensitive method and has an advantage over other similar methods in that it is possible to optimise the method through computer simulation. A limitation of DGGE is that establishing the method is labour intensive and it can only analyse fragments of less than 600 basepairs. Heteroduplex analysis (Nagamine, 1989; Keen, 1991) is based on the difference in mobility through a native gel due to conformational differences between homoduplex and heteroduplex DNA. The sensitivity is high for detection of insertions and deletions but lower for detection of single base substitutions (Bhattacharyya, 1989; Bhattacharyya, 1989). By using a special gene matrix, the sensitivity for single base changes can be improved reaching an estimated mutation detection rate of 80% (Perry, 1992). Conformation-sensitive gel electrophoresis (CSGE) is a variant of this method where conditions are created to increase the local denaturation around a mismatch to enhance the retardation (Ganguly, 1993; Ganguly, 2002). Denaturing high-performance liquid chromatography is based on the same principle, but the separation is performed in a special HPLC column and thus gel pouring is avoided (Liu, 1998). Under partly denaturing conditions, the method detects single-base substitutions as well as small deletions and insertions in fragments of 100 to 1,500 basepairs with high efficiency and sensitivity (Wagner, 1999; Jones, 1999). Detection of mutations in fragments smaller than 100 basepairs is also possible but completely denaturing conditions must be used (Oefner, 2000). The method has recently been adapted so that it can be performed in capillaries, which gives the same degree of separation but increases the throughput and decreases the sample and solution volumes (Xiao, 2001; Huber, 2001). Dideoxy fingerprinting (ddF) combines a modified DNA sequencing reaction with non-denaturing gel electrophoresis (Sarkar, 1992). In ddF, PCR products are

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Detection and analysis of genetic alterations in normal skin and skin tumours

subjected to a sequencing reaction containing only one dideoxy terminator and the analysis is based on migration differences or the loss or gain of fragments. Although a detection sensitivity of up to 100 % for detection of p53 mutations has been demonstrated, the establishment of the method is difficult and time-consuming (Sarkar, 1992; Martincic, 1996; Blaszyk, 1995). Cleavage –based techniques Another group of scanning methods relies on chemical or enzymatic cleavage of mismatches. Chemical cleavage of mismatch (CCM) was developed by Cotton in 1988 as a modification of the Maxam-Gilbert DNA-sequencing method (Cotton, 1988). Amplified DNA fragments are first denatured and reannealed (forming homoand heteroduplexes), followed by chemical modification of mismatched cytosines and thymines by hydroxylamine and osmium tetroxide, respectively. The modified bases can then be cleaved by hot piperidine treatment, which facilitates the localisation of mismatches through detection of the cleavage products. The throughput of CCM has been increased through the use of single tube reactions and by adapting the method to a solid phase assay format (Hansen, 1996; Ramus, 1996). Fluorescence assisted mutation assay (FAMA) is a modification of CCM that uses fluorescently labelled primers in the PCR to achieve a higher sensitivity in visualisation of the cleaved product (Verpy, 1994). Chemical cleavage of mismatch is the most sensitive and specific cleavage method (Cotton, 1989) and these modifications has allowed CCM to be performed in a semi-automated fashion. However, the biohazardous chemicals involved make the method less attractive for routine use, although improvements have been made by for example substituting osmium tetroxide for potassium permanganate (Lambrinakos, 1999). Methods based on enzymatic cleavage have an advantage over CCM in that they avoid the use of hazardous chemicals. However, they display varying sensitivities and most enzymes do not cleave all types of mismatches. The types of enzymes commonly used in various cleavage assays include endonucleases, ribonucleases and exonucleases. Several different methods take advantage of enzymatic cleavage by endonucleases. Cleavage Fragment Length Polymorphism (CFLP) (Brow, 1996) relies on the enzyme Cleavase which induces endonucleolytic cleavage at the base of single stranded stem-loop structures formed after cooling of the DNA without

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reannealing. These stem-loop structures are dependent on the primary sequence and thus changes in the sequence may result in alterations of the cleavage profile, which subsequently can be detected by capillary or denaturing gel electrophoresis. In contrast to CFLP, other endonuclease assays rely on cleavage at or near the site of mismatches in heteroduplexes. The bacteriophage proteins T4 endonuclease VII (T4E7) and T7E1, sometimes referred to as resolvases, can localise and cleave mismatches in large DNA duplexes with a sensitivity approaching 100 % (Mashal, 1995; Youil, 1995; Youil, 1996). Ribonuclease A cleavage is another screening method for large fragments (1Kb), which was first described by Myers using DNA:RNA hybrids (Myers, 1985). Mismatches formed after hybridisation of labelled RNA probes with amplified DNA are cleaved by RnaseA and visualised by electrophoresis. The sensitivity of the original method is approximately 60 % but a sensitivity of 88-90 % can be achieved using a modified method described by Goldrick. In the Non-Isotopic RNase Cleavage Assay (NIRCATM) (Goldrick, 1996; Goldrick, 2001), RNA:RNA heteroduplexes formed by in vitro transcription of PCR products are cleaved by RnaseTI and Rnase1 which cleave a wider range of mismatches than RnaseA. The cleaved products are then separated by non-denaturing agarose gel electrophoresis and visualised by ethidium bromide. Exonuclease protection assays take advantage of mismatch-binding proteins for mutation detection. The E. coli mismatch recognition protein MutS detects and binds preferentially to single base mismatches and the resulting protein /DNA complex can be visualised by a gel mobility shift assay (Lishanski, 1994). However, MutS is unable to detect C:C mismatches and the low stability of MutS/DNA complexes may also result in a loss of signal (Jiricny, 1988). MutS can also be used in the MutEx exonuclease protection assay to localise mutations (Ellis, 1994) and in an in vitro constructed MutHLS system to detect mismatches in heteroduplexes after PCR amplification (Smith, 1996). Another E. coli protein Mut Y, has also been used by itself or in combination with thymine glycosylase for mismatch detection (Hsu, 1994; Lu, 1992). The enzyme is highly sensitive but recognises only G:A mismatches and therefore detection of G:G or C:C mismatches is not possible even when glycosylase is used. In Base Excision Sequence Scanning (BESS) (Hawkins, 1997; Hawkins, 1999) a limiting amount of dUTP is present during a single PCR amplification. The amplified product is then cleaved at sites of dUTP incorporation and at dGTP sites

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Detection and analysis of genetic alterations in normal skin and skin tumours

using two different excision reactions involving uracil-N-glycosylase/E. coli endonuclease IV and dGTP modifications respectively. The resulting sets of fragments correspond to the positions of deoxyguanine and deoxythymidine in the sequence and can be analysed using standard sequencing gels or on an automated sequencer. All mutation types can be detected and in most cases, exact identification of the mutation and determination of its position is possible.

Scanning methods Normal

Mutant

SSCP

Gelshift

DGGE

Gelshift

HA

Gelshift

CCM

Cleavage

CFLP

Cleavage

RNase

Cleavage

R S MutS

Gelshift Exo

Exo

MutEx

S

Cleavage protection Exo

Exo

S H L

MutHLS

Cleavage

MutY

Cleavage

Y

BESS

U

U

G

Cleavage

Figure 1. Principles of scanning methods used for detection of mutations at non-defined positions.

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Specific methods In contrast to scanning technologies, specific methods identify alterations at predefined sites. Two types of alterations are of particular interest; mutations and single nucleotide polymorphisms. Mutations can be distributed across the genome, but at some sites a clustering tendency is observed. These so-called “hotspot mutations” have been identified in for example cancer-related genes such as ras and p53. Single nucleotide polymorphisms (SNP) have been estimated to occur once in every thousand bases in the human genome. They represent nucleotide variations at certain positions in coding and non-coding regions of the genome. SNPs in coding regions may account for differences in drug response between individuals and also may be a contributing factor in disease susceptibility (Evans, 1999), (Davignon, 1988; Bertina, 1994). The possible effect of SNPs in non-coding regions is not yet determined but they are extremely useful as markers in population and genetics studies (Jorde, 2000; Hacia, 1999). Some methods that have been developed to analyse both mutations and SNPs and are tailor-made for detection of variations at specific sites are described below and shown in Figure 2. Hybridisation-based techniques The possibility of detecting a single-base mismatch using hybridisation with allele specific oligonucleotides (ASO) was demonstrated as early as 1979 (Wallace, 1979). Since the invention of PCR, the principle of ASO has become widely used in various assays. The method relies on the differences in hybridisation efficiency to the target DNA between a fully complementary ASO probe and a probe containing a single mismatch. The early ASO assays were performed in a dot-blot or reverse dot-blot format immobilising either the amplified target DNA or the oligonucleotide probe set respectively on a membrane (Saiki, 1988; Saiki, 1989). The hybridisation product was detected using autoradiography or enzyme conjugates. The assay was later adapted to a high-density microarray format, but neither format allows for perfect allele discrimination (Wang, 1998; Cho, 1999). Increased stringency can be achieved using DASH (dynamic allele-specific hybridisation) where the hybridisation is monitored over a temperature gradient (Ririe, 1997),(Prince, 2001) or by using higher affinity LNA (locked nucleic acid) probes instead of DNA (Orum, 1999). In some recently developed real-time PCR-based ASO assays, fluorescence is emitted as a result of a change in the physical distance between a fluorophore and a quencher molecule. In

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Detection and analysis of genetic alterations in normal skin and skin tumours

the Molecular Beacon assay (Giesendorf, 1998; Vet, 1999; Tyagi, 1996; Smit, 2001) fluorescence is emitted when the stem-loop structure of the probe is opened upon perfect hybridisation to the DNA target sequence during the primer-annealing phase. In the TaqManTM assay release of the quencher after exonuclease degradation of perfectly annealed probes by the polymerase allows the fluorophore to emit a fluorescent signal (Livak, 1995; Livak, 1999). Both assays are compatible with 96well or 384-well microtiter plate formats and facilitate the use of multiple fluorophores. However, the need for fluorescent and quencher moieties on the probes make these assays rather expensive. The concept of ASO was the first step towards today’s oligonucleotide arrays, which are used in various applications ranging from mutation detection to gene expression studies. The theoretical principle of sequencing by hybridisation (SBH) was independently described by two groups in the late 1980’s and is based on arrays of all possible combinations of short immobilised oligonucleotides (Drmanac, 1989) (Lysov Iu, 1988). Labelled target DNA is then hybridised to the array and the hybridisation pattern is used to in silico reconstruct the target sequence. This method proved less suitable for de novo sequencing but has been successfully used for mutation detection in the p53 gene (Drmanac, 1998). Methods based on allele-specific amplification Several allele-specific PCR-based methods have also been used for sequencing of defined alterations. Allele specific PCR primers with the 3´ terminus annealing at the variant position are used in the PCR reaction, which results in amplification with only the perfectly matched primer. Assays based on this principle are for example ASA (allele specific amplification) (Okayama, 1989), ASPCR (allele specific PCR) (Wu, 1989) and PASA (PCR amplification of specific alleles) (Sommer, 1992). Oligonucleotide ligation assays To increase the specificity of ASO hybridisation a number of assays based on ASO ligation have been developed. In the oligonucleotide ligation assay (OLA) (Landegren, 1988) a ligation probe and probes specific to wild type and mutant alleles are hybridised adjacent to each other on the target sequence. Since ligases can effectively discriminate against mismatches, only perfectly matched probes will be

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ligated. Depending on the label used, the detection of the ligated products can be carried out in an ELISA format or by electrophoretic separation in a DNA sequencer instrument (Nickerson, 1990; Samiotaki, 1994), (Grossman, 1994). The OLA assays have also been used in different microarray formats (Broude, 2001; Gerry, 1999) but the need for several differently modified probes increases the cost significantly. In ligase chain reaction (LCR) two pairs of probes are used together with a thermostable ligase in a cyclic ligation reaction resulting in amplification of the target sequence in cases where the probes are perfectly matched (Barany, 1991). Padlock probes (Nilsson, 1994) (Nilsson, 1997) are linear oligonucleotides that have complementary target sequences at both ends separated by a random DNA sequence. Upon hybridisation to a target, the probe ends are ligated to form a circularised probe if there is complete homology to the target. Signal amplification can then be achieved by using the circularised product of successful padlock probe ligation as a template for rolling circle amplification (RCA) which creates a long single stranded DNA composed of tandem-repeats complementary to the padlock probe (Fire, 1995; Baner, 1998; Lizardi, 1998). A single tube assay combining ligation and rolling circle amplification has also been described which increases the throughput significantly (Qi, 2001). Techniques based on polymerase extension Minisequencing, also denoted PEX for single nucleotide primer extension (Syvänen, 1990), is based on discrimination of variants by single nucleotide extension at the site of a mutation. A primer is hybridised to the target sequence immediately adjacent to the variable position and a DNA polymerase is then used to extend the 3´ end of the primer with a labelled dideoxynucleotide complementary to the nucleotide at the variable site. The method has been adapted to various formats and detection strategies, resulting in ELISA, electrophoresis and microarray based formats (Nikiforov, 1994; Pastinen, 1996; Tully, 1996; Pastinen, 1997; Lindroos, 2001). The minisequencing concept is also used in the commercially available SNaPshotTM assay (Applied Biosystems) and in the improved variant MAPA (multiplex automated primer extension) described by Makridakis and Reichardt (Makridakis, 2001). MALDI-TOF–MS (matrix-assisted laser desorption ionisation - time-of-flight - mass spectrometry) emerged as a method for DNA sequencing in the late 1980’s. An innovation in the field of ionisation of macromolecules made it possible to perform

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Detection and analysis of genetic alterations in normal skin and skin tumours

analysis of DNA in a system which was earlier limited to peptide analysis (Karas, 1988). DNA samples are desorbed, ionised and subjected to an electric field where the molecules are accelerated proportional to their mass/charge ratio. Detection is based on the time required for the molecules to reach the detector. MALDI-TOF-MS is a rapid sequence analysis technique that permits simultaneous analysis of many DNA strands in a heterogenous mixture. However, the limited read-length of 100 nucleotides resulting from sequencing of dideoxy generated DNA ladders make it less attractive for de novo sequencing (Wu, 1994; Taranenko, 1998; Monforte, 1997). The method has been successfully used to sequence exon 5 to 8 of the p53 gene (Fu, 1998), but the true potential probably lies in typing single point mutations and SNPs (Griffin, 2000; Griffin, 2000; Li, 1999; Tang, 1999; Griffin, 1999). Several different approaches for MALDI-TOF-MS have been described. In the PINPOINT assay (Haff, 1997; Haff, 1997; Ross, 1998) the target is subjected to primer extension by a single base in the presence of all four ddNTPs and the mass added onto the primer then defines the variable base in the subsequent MALDI-TOF-MS analysis. In MassEXTEND (Little, 1997; Little, 1997), (Braun, 1997; Braun, 1997) and VSET (very short extension)(Sun, 2000) which are similar assays, dNTP mixes containing a single ddNTP or ddNTP mixes containing a single dNTP respectively, are used in the primer extension reaction. Apyrase-Mediated Allele-Specific Extension (AMASE) is another single-step extension approach recently described (Ahmadian, 2001). This assay relies on extension of paired allele-specific primers in the presence of the nucleotide degrading enzyme apyrase. Only a perfectly matched primer will give rise to extension, since the slower reaction kinetics of a mismatched primer-template configuration will allow for the apyrase to degrade the nucleotides before extension can occur. Thus the acknowledged difficulties that polymerases have in discriminating between certain mismatches can be circumvented. The assay has recently been adapted to a microarray format and successfully used for SNP typing (O'Meara, 2002).

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Specific methods

Mutant

Normal

No duplex

ASO

Beacons

A T

A C

Fluorescence

ASA

No amplification

OLA

No ligation

LCR

No ligation / amplification

Padlocks

No ligation / amplification

PEX

No extension

AMASE

No extension

Figure 2. Principles of methods used for detection of mutations/variations at defined positions.

15

Detection and analysis of genetic alterations in normal skin and skin tumours

Pyrosequencing The principle of sequencing-by-synthesis was described in 1985 (Melamede, 1985) and led to the development of two approaches for DNA sequencing; direct detection of labelled nucleotides (Canard, 1994; Metzker, 1994) and indirect detection of released pyrophosphate (Hyman, 1988; Nyren, 1987), respectively. In the direct approach, the low efficiency of incorporation of labelled nucleotides results in unsynchronised extension and limits the read length to a few bases (Metzker, 1994). The indirect approach relies on the use of enzymes to convert inorganic phosphate, released upon sequential addition and incorporation of nucleotides, into light. This approach was further developed and in 1993 a real-time sequencing method was described (Nyren, 1993). Two important modifications to this real-time sequencing strategy were the substitution of dATPαS for dATP and the introduction of the nucleotide degrading enzyme apyrase which resulted in an improved method denoted Pyrosequencing (Ronaghi, 1996; Ronaghi, 1998; Ronaghi, 2001) (Figure 3). The use of dATPαS solved the problem of non-specific signals generated by luciferase after each addition of dATP, while addition of apyrase dispensed with the need for extensive washing to remove the excess of nucleotides after each addition. Pyrosequencing is based on polymerase-assisted primer extension using sequential addition and incorporation of nucleotides in the presence of adenosine phosphosulfate (APS), D-luciferin, ATP sulfurylase, luciferase and apyrase. The pyrophosphate (PPi) released upon incorporation of a nucleotide is used as substrate for ATP sulfurylase generating ATP. This ATP is then used in a reaction catalysed by luciferase resulting in the release of detectable light. After each addition, the excess nucleotides are degraded by apyrase before the next nucleotide is added. If the added nucleotide does not form a basepair with the DNA template, it is not incorporated by the polymerase and thus no light will be generated. The nucleotide is then rapidly degraded by apyrase. Pyrosequencing may also be performed directly on double-stranded DNA using enzymatic removal of amplification primers and nucleotides (from the PCR) as described by Nordström et al (Nordstrom, 2000). To obtain high-quality pyrosequencing data, efforts are required to optimise the relative amounts of the different enzymes involved. The most critical reactions in pyrosequencing are those that compete for nucleotides i. e. DNA polymerisation and nucleotide removal. The DNA polymerase should be in excess to ensure that polymerisation takes place immediately upon nucleotide addition. Furthermore, the

16

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nucleotide concentration must be above the KM of the DNA polymerase to obtain rapid polymerisation, but it should not be so high that it affects the fidelity of the polymerase (Eckert, 1990; Cline, 1996). Finally, the degradation of nucleotides by apyrase must be slower than the incorporation by DNA polymerase. An interesting feature of pyrosequencing is the possibility to predetermine the dispensation order of the nucleotides. For analysis of an unknown sequence, cyclic dispensation of AGTC might be the best choice, while programmed dispensation can be more suitable for sequencing of known mutations or SNPs. A programmed nucleotide delivery approach can be used to keep synchronised extension of different alleles in (or out of) phase and thus improve the discrimination between different alleles. A fully automated microtiter plate-based instrument, simultaneously analysing 96 samples is commercially available (Pyrosequencing AB) allowing sequencing of SNPs in less than 10 minutes and analysis of short stretches of DNA in less than an hour. Due to product accumulation and decreased enzymatic activity over time, the pyrosequencing read-length is limited and despite reports of read-lengths above 100 bases, the effective limit is around 40 bases (Garcia, 2000; Gharizadeh, 2002). Pyrosequencing has thus so far mostly been used for SNP typing (Ahmadian, 2000; Gustafsson, 2001; Gustafsson, 2001; Fakhrai-Rad, 2002; Wasson, 2002; Andreasson, 2002) but it has been used in the typing of bacteria (Monstein, 2001) and viruses (Gharizadeh, 2001; O'Meara, 2001), mutation screening (Garcia, 2000) and tag library sequencing (Agaton, 2002; Nordstrom, 2001).

17

Detection and analysis of genetic alterations in normal skin and skin tumours

Polymerase 5'

TAGAAGGACCGTTTAAGT

3' 5'

ATCTT

Primer dTTP PPi Sulfurylase Apyrase

ATP Luciferase dTMP + 2Pi

Light

Real-time monitoring

Nucleotides added:

DNA sequence:

A

T

C

TT

Figure 3. Schematic diagram of pyrosequencing.

18

Å Sivertsson

CARCINOGENESIS The disease of cancer is described in the papyri from Egypt (written around 1600 BC) which are the earliest medical records known and probably date from sources as early as 2500 BC. The ancient Egyptians used not only surgery but also pharmacological and magical treatments to fight the disease and were able to distinguish between benign and malignant tumours. However, up until the 17th century, cancer was considered an illness caused by an excess of black bile and curable only in its earliest stages. Although enormous progress has been made in understanding cancer in the last century, the disease remains elusive. Cancer is now defined as a cellular disease, characterised by a transformed cell population that displays net cell growth and an anti-social behaviour, which has originated from one single cell. Many of the events involved in this multi-step process, both genetic and epigenetic, have been defined but the complexity of the cell machinery makes it difficult to determine the definitive outcome of any single event. In general terms, carcinogenesis can be divided into three stages: initiation, promotion and progression. Initiation of a cell is the result of events leading to permanent genetic alterations that will be transferred to the daughter cells, such as mutations in one or more proto-oncogenes or tumour-suppressor genes. During promotion, the initiated cell or cells go through selective clonal expansion due to a growth advantage over normal cells, e.g. by evading apoptosis. The end result of promotion is a benign tumour and the events that turn this tumour malignant are denoted progression. The genetic events during these different stages have been described in detail for the development of metastasising colon cancer via mild and severe dysplasia and adenoma, leading to the postulation that between three and six mutations are needed to complete the carcinogenic process (Vogelstein, 1993). In the first successful attempt to transform normal human cells to cancer cells using only defined genetic events, it was shown that a minimum of four pathways involving telomerase, the two tumour-suppressors p53 and pRb and the oncogene ras had to be interrupted (Weitzman, 1999). From a cell physiological perspective, Hanahan and Weinberg have identified and described six traits that are probably common to all cancers (Hanahan, 2000). These traits are defined as; self-sufficiency in growth signals, insensitivity to anti-growth signals, evasion of apoptosis, a limitless replicative potential, sustained angiogenesis and the capacity of tissue invasion and metastasis. Self-sufficiency in growth signals can be achieved through alterations in

19

Detection and analysis of genetic alterations in normal skin and skin tumours

growth signalling, for example by receptor over-expression, autocrine stimulation or activation of the ras oncogene or members of its pathway. Disruption of the pRb pathway, for example by mutation of pRb or TGF-β or inactivation of the tumour suppressor p53, renders cells insensitive to anti-growth signals and thus stimulates proliferation. In addition, inactivation of p53 is one way of evading apoptosis, which can also be accomplished by production of survival factors such as IGF-2. An unlimited replicative potential is achieved by maintaining telomere length by upregulation of the enzyme telomerase or by interchromosomal exchange of sequences. Furthermore, loss of p53 or activation of ras can give rise to the capability of inducing and sustaining angiogenesis through downregulation of the angiogenesis inhibitor thrombospondin and release of the negative transcriptional control on the proangiogenic factor vascular endothelial growth factor (VEGF) respectively. The final essential characteristic of a cancer cell is the ability to invade and metastasise, which is acquired by alterations in expression of cell-cell adhesion molecules such as E-cadherin and integrins and by upregulation of proteases. According to this information and from experimental evidence, it is apparent that multiple genetic hits are necessary for tumour development. However, due to the meticulous maintenance of genomic integrity by monitoring and repair enzymes, mutations are such rare events that the probability of acquiring the required number of genetic alterations during a lifetime would be diminutive unless there are some changes in genomic stability. Genome instability can be achieved by altering the function of proteins involved in the genomic caretaker systems, thus creating cells with higher mutability and the so-called “mutator phenotype”. The most prominent and most studied member of these caretaker systems is the tumour suppressor p53 (discussed below) that plays an important role by inducing either growth arrest or apoptosis in response to DNA damage. Thus alterations in p53 or members of its pathway are not only involved in reaching several of the above mentioned biological endpoints necessary for cancer development but are also a prerequisite in creating genomic instability.

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Cancer of the skin

Skin cancer, which is the generic term for basal cell cancer (BCC), squamous cell cancer (SCC) and malignant melanoma, is the most common cancer in the western world. In the U.S.A. the incidence of non-melanoma skin cancer (NMSC) i. e. BCC and SCC is estimated to be about 700 cases per 100,000 individuals per year with a ratio of BCC to SCC of roughly 4:1, while the incidence of melanoma is 50 cases per 100,000 per year (Miller, 1994). This reflects an increase in incidence of 3-8% per year since the 1960´s (Green, 1992; Glass, 1989). In Sweden, the numbers are lower with an incidence of NMSC estimated to be about 100 cases per 100,000 individuals and of melanoma estimated to be approximately 12 cases per 100,000 per year (Nylén, 2002). The increase in incidence over the last ten years is similar to that in the U.S.A. and is at about 3-4 % per year. A connection between skin cancer and weather-beaten skin has been discussed since the end of the 19th century and the relation to chronic sun-exposure was promptly suggested (Hyde, 1906). Today, ultraviolet radiation (UVR) is well established as a carcinogen and knowledge of the underlying molecular mechanisms is constantly and rapidly increasing. Ultraviolet radiation Ultraviolet radiation in the solar electromagnetic spectrum extends from the UVA band (315-400 nm) through the UVB band (280-315 nm) down to the high-energy UVC band (190-280 nm) and vacuum UV (below 190 nm). The most energetic and harmful UV radiation (wavelengths below 240 nm) is absorbed by oxygen in the outer reaches of the atmosphere, while the stratospheric ozone formed in the process absorbs part of the UVB radiation. UVA, which comprise the major part of the total spectral energy of solar UV, is not absorbed at all (Diffey, 1999). Ultraviolet radiation is highly photochemically active and reacts, depending on the wavelength, directly or indirectly with different biomolecules such as DNA and proteins. UVB is directly absorbed by DNA and exerts a genotoxic effect mainly by dimerizing pyrimidines giving rise to cyclobutan pyrimidine dimers (CPD) and 6-4 pyrimidine-pyrimidone photoproducts (6-4 PP) (Tornaletti, 1996) (Figure 4). If these products are not removed during DNA repair, these lesions will induce the UV signature mutations CT and CC-TT (Hauser, 1986; Brash, 1987; Drobetsky, 1987; Armstrong, 1992). Recent experimental studies have also shown that UVB can cause deletions and that

21

Detection and analysis of genetic alterations in normal skin and skin tumours

UV (A/B) can cause chromosomal aberrations in mammalian cells (Horiguchi, 2001; Emri, 2000). UVA is usually subdivided into UVA1 (340-400 nm) and UVA2 (315340 nm), since they differ in their effect from a biological point of view (Fitzpatrick, 1986). The shorter wavelength range of UVA2 has a similar effect as UVB, while UVA1 is not absorbed by DNA but is oxidative in nature and damages DNA by generating reactive oxygen species (ROS) (Young, 1998; Cadet, 1997). Endogenous photosensitisers such as porphyrines and NADH become excited by absorbing UVA radiation and can then react directly with DNA (type I reaction) or give rise to ROS by reacting with oxygen (type II reaction) (De Gruijl, 2000; Kawanishi, 2001). The DNA damage caused by both type I and type II reactions appears most frequently to generate 8-oxo-7,8-dihydroxyguanine (8-oxo-dG), which is a miscoding lesion inducing G-T transversions (Cheng, 1992) (Figure 4). However, type II reactions to a lesser extent may also induce the formation of the 5-OH-dC adduct leading to C-T mutations (Wang, 1998; Jenkins, 2001) (Figure 4). It is generally accepted that DNA damage caused by UV radiation is responsible for the induction of skin cancer. Both UVB and UVA radiation have been proven to be fully carcinogenic in animal studies with UVB radiation giving rise to actinic keratosis, SCC and BCC (De Gruijl, 1983), while UVA radiation mainly induces papillomas and SCC (Kelfkens, 1991). Induction of malignant melanoma has not yet been correlated to any particular UVR wavelength but studies on opossum as well as retrospective studies both on users of tanning salons and users of sunscreen implicates UVA (Ley, 1997; Westerdahl, 1995; Wolf, 1998; Swerdlow, 1988; Westerdahl, 1994). UVA radiation is also the cause of photoageing of the skin and affects all layers down to the dermis. UVB radiation, on the other hand, is mainly absorbed by the epidermis with only 2-10 % reaching the basal layer compared to 20 % for UVA (Tyrrell, 1996).

22

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O

A

H C

N

C

C

N

S

O

C

H

CH3

P

O N

C

C

C

N

C

C

C

N H

C

H

UV radiation

H

O

OH

C C

CH3

P

S

N

S

O

C

H

H N

C

N

S

O

H C

CH3

TT-dimer

O O

H

CH3

C

N

C

C

N

S

C

H

Adjacent thymines

O

CH3

P

H

O

S

C

N

C

C

N

C

H

B

OH

CH3

6-4 photoproduct

S

S

P

P

5-OH-dC

8-OXO-dG

Figure 4. Structure of UV-induced photoproducts (A) and the DNA lesions commonly formed after UVA irradiation (B).

23

Detection and analysis of genetic alterations in normal skin and skin tumours

Normal skin The skin including the underlying subcutis amounts to 15% of our bodyweight and is thereby our largest organ and also the most important barrier to protect us from the environment. Its major functions are to prevent dehydration, physical protection, protection against chemical agents and micro-organisms, heat regulation and sensory functions. The skin is a constantly renewing tissue that consists of an epidermis overlying a dermal layer in conjunction with subcutaneous tissues mostly made up of fat (Figure 5). The epidermis is subdivided into four layers, the stratum basale (basal layer), the stratum spinosum, the stratum granulosum and the stratum corneum. Keratinocytes are the principal cell type of the epidermis and undergo cell division, differentiation and maturation while gradually migrating to the skin surface to be shed resulting in a turnover time of 15-30 days for epidermis. These cells are proposed to make up the fine mosaic of epidermal proliferative units (EPU) which consists of a column of transit-amplifying and differentiated keratinocytes overlaying approximately 10 basal cells, in which one epidermal stem cell is responsible for maintenance (Clausen, 1990; Potten, 1981; Potten, 1988). An investigation of the EPU size by looking at the x-chromosome inactivation pattern in skin revealed a mosaic pattern with clones of 20-350 basal cells, suggesting an EPU size of less than 35 cells in diameter (Asplund, 2001). Epidermal stem cells are slow-cycling cells that reside in the basal layer of epidermis as well as in the bulge area of the hair follicle (Figure 5). The latter have been shown to be bipotent and are thus able to give rise not only to the hair follicle but also to the epidermis and may therefore be the ultimate stem cells of the epidermis/hair follicle system (Taylor, 2000; Oshima, 2001). The progeny of basal layer stem cells is transit amplifying cells that undergo a finite number of divisions to generate the terminally differentiating cells that move upwards from the basal layer. In the case of hair follicular stem cells, the progeny undergoes two independent pathways of migration and differentiation. Cells can move upwards to be part of the maintenance of the epidermis or migrate downwards during the growth phase of the hair follicle giving rise to the lower part of a new hair follicle (Taylor, 2000). The basal layer also contains melanocytes producing melanin for protection against UV radiation. Melanin is produced in melanocyte-specific organelles (melanosomes), which are phagocytised by the keratinocytes and accompanies them to the skin surface. Melanin exerts its protective effect by absorbing, reflecting and scattering UVR and possibly also acts as a free radical

24

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quencher. The production of melanin is induced by UV-light and by melanocyte stimulating hormone excreted from the pituitary gland and from keratinocytes. The melanocyte stemcells in mouse have recently been described as being localised in the lower permanent portion of the hair follicle (Nishimura, 2002).

A Cornified layer Granular layer

Epidermis

Spinous layer Dermis

Basal layer

Cornified layer

B Granular l. Epidermis

Spinous l. Basal l.

Dermis

Subcutis Hair follicle Sebaceous gland

Sweat gland Fat

Figure 5. Histological section of normal epidermis (A) and a schematic picture of the different layers of the skin (B).

25

Detection and analysis of genetic alterations in normal skin and skin tumours

The tumour suppressor p53 The tumour suppressor gene p53 is an 11 exon gene spanning over 20,000 basepairs on the long arm of chromosome 17. P53 has been proposed as the guardian of the genome and is the most frequently mutated gene in all human cancers (Lane, 1992). P53 mutations fall into two classes, those that affect the DNA-binding domain (Class I) and those that change the conformation of the protein (Class II) (Ory, 1994). The latter are associated with a more severe phenotype in vitro and exert a dominant negative effect over the wild-type protein (Ory, 1994). The gene encodes a 393 amino acid nuclear phosphoprotein that functions as a stress and DNA damage-inducible sequence specific transcription factor that selectively activates genes involved in differentiation, cell cycle control, DNA repair, senescence, angiogenesis and apoptosis (el-Deiry, 1992; Hupp, 1992; Hall, 1996; Levine, 1997; Ford, 1997). The p53 protein is constitutively expressed in small amounts and is under normal conditions present in a latent form with a half-life of 5-20 minutes due to rapid degradation. In response to DNA damaging agents e.g. gamma radiation, UV radiation, alkylating or oxidising agents (Huang, 1996; Nelson, 1994), (Siegel, 1995) or cellular stress in the form of hypoxia, hyperthermia, nucleotide depletion, serum starvation, massive DNA demethylation or activated oncogenes (Graeber, 1994; Linke, 1996; Matsumoto, 1997; Canman, 1997; Jackson-Grusby, 2001) the protein is rapidly activated. Upon activation, p53 is altered to allow for homotetramerisation which makes the protein functionally active as a transcription factor. Depending on the type and duration of damage or stress, genes that cause either growth arrest, apoptosis, altered DNA repair or altered differentiation are selectively activated. In response to low doses of DNA damage or milder forms of stress, genes involved in growth arrest and DNA repair including the cyclin-dependent kinase inhibitor p21Waf1

(G1 arrest), 14-3-3 Sigma (G2 arrest), Gadd45 (repair) and PCNA (repair) are

activated to allow for repair of the damage (Kastan, 1991; McKay, 1999). Severe DNA damage or extreme stress instead triggers genes involved in the apoptotic pathway such as bax, PIDD, NOXA, PUMA and p53AIP (Miyashita, 1995; Polyak, 1997). Regulation of activity occurs through phosphorylation and through reduction of cysteines in the DNA binding domain. Acetylation was earlier thought to be deeply involved in activation, but recent data does not support this view (Espinosa, 2001; Nakamura, 2000). Instead it is speculated that acetylation might mark a p53-activated promoter for subsequent inactivation by recruiting deacetylases (Prives, 2001).

26

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Phosphorylation can occur on more than 18 phosphoacceptor sites and although some are phosphorylated under normal growth conditions (Meek, 1990; Gatti, 2000) most of them are modified in response to damage or stress (Canman, 1998; Fuchs, 1998; Chernov, 1998; Hirao, 2000; Siliciano, 1997; Higashimoto, 2000; Chehab, 1999). Multiple kinases, some of which are able to act on the same sites, are involved in the phosphorylation and the type of stress factor involved determines which kinases are active. In response to UV radiation, ATR, Chk1 and JNK are preferentially activated (Unsal-Kacmaz, 2002; Liu, 2000; Buschmann, 2001). Phosphorylation is not only involved in transcriptional activation but also in p53 stability. Serine 15 and serine 20, which are phosphorylated by the kinases ATM/ATR and Chk2 respectively, are involved in the association of p53 with one of its master regulators, mdm-2 (Shieh, 1997). P53 transcriptionally upregulates the proto-oncoprotein mdm-2 that in turn negatively regulates p53 by targeting it for ubiquitin-mediated proteasomal degradation, which thus results in the low amount and short half-life of the p53 protein under normal conditions. The damage-induced phosphorylation of multiple sites, including those involved in mdm-2 binding, releases p53 from mdm-2 and stabilises the protein (Fuchs, 1998; Shieh, 1997). Disruption of the association with mdm-2 is further responsible for the stabilisation of the p53 protein commonly associated with p53 mutation. The p53 patch Typical damage to normal skin that is chronically exposed to UVR includes erythema, edema, hyperplasia, sunburn cells and photoageing. The acute response to UVR is a transient accumulation of the tumour suppressor protein p53 in the skin, which can be detected by immunostaining. The distribution of the protein is wavelength dependent with UVA inducing dispersed p53 immunostaining limited to the basal layer, while UVB induces dispersed immunostaining throughout the entire epidermis (Campbell, 1993; Ren, 1996) (Figure 6). This p53 accumulation, which is due to the prolonged half-life of the wild-type conformation of the protein, can be detected after 4 hours with levels returning to normal within 120 hours (Pontén, 1995). In addition to the dispersed pattern of p53 immunoreactive cells seen after UVR exposure, a compact pattern with strong immunoreactivity localised to all cell nuclei in a sharply demarcated area is also frequently found in skin (Figure 6) (Urano, 1995; Ren, 1996; Jonason, 1996). These so-called “p53 patches” comprise of 10-3000 cells and can

27

Detection and analysis of genetic alterations in normal skin and skin tumours cover as much as 4 % (there are 3-40 patches /cm2 depending on exposure) of the epidermis with their size and frequency increasing in a dose-dependent manner with sun-exposure. Although, increasing age has been correlated with an increase in size of p53 patches an age-related increase in frequency is somewhat disputed (Ren, 1996; Jonason, 1996; Tabata, 1999). The patches consist of clones of morphologically normal cells arising from the epidermal-dermal junction or from hair follicles with their incidence and location suggesting a possible role in skin carcinogenesis. In addition, 30 -70 % of these clones harbour a p53 mutation (Jonason, 1996; Pontén, 1997; Tabata, 1999). However, although they are frequently found adjacent to BCC, CIS (carcinoma in situ) and SCC they have so-far not been found to share the same mutation (Pontén, 1997; Ren, 1996). Despite this fact, the early onset of p53 patches in normal mouse skin following chronic UV irradiation has been suggested as an indicator of tumour risk (Berg, 1996; Rebel, 2001). In another recent animal study, it was demonstrated that the clonal expansion of patches was continually driven by UV radiation and that the clone size represented a murine EPU or a multiple thereof (Zhang, 2001). Thus stem cell compartments seem to act as physical barriers to clonal expansion, but colonisation can occur under the influence of constant UV irradiation. Perhaps an explanation for this is that the p53 mutation allows the patches to escape the UV induced apoptosis which affects cells in neighbouring compartments (Ziegler, 1994). A

B

Figure 6. Immunoreactive keratinocytes showing the compact pattern typical of a p53 patch (A) and the dispersed pattern common in response to UV damage (B).

28

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Non-melanoma skin cancer Basal cell cancer and SCC are keratinocyte-derived tumours that partly share aetiology and have therefore commonly been grouped together as non-melanoma skin cancer. However, some important differences exist. Cutaneous SCC is believed to arise from interfollicular basal stem cells and is what can be called a “classical” cancer that develops through precursor lesions (dysplasia also denoted actinic or solar keratosis) with the potential to progress into cancer in situ and further into metastasising disease. It can develop in different parts of the skin but its behaviour may differ depending on its location. The tumour never spontaneously regresses once developed, but in accordance with the characteristics of precursors, dysplasias frequently do regress (Marks, 1986). Basal cell cancer on the other hand is a slowly growing, locally invasive tumour that lacks known precursors and almost never progresses to metastasising disease. This inability to metastasise has been explained by the unconditional dependence of the tumour on the specific loose connective stroma recruited by the surrounding fibroblasts (van Scott, 1961). Its cellular origin is thought to be either interfollicular basal cells retaining basal morphology or keratinocytes in hair follicles or sebaceous glands (Kruger, 1999; Miller, 1993). Basal cell cancer develops only in hair-bearing skin and mostly in sun-exposed areas, with 85 % appearing in the head and neck region. The major risk factor for developing BCC and SCC is exposure to sunlight and especially UVB radiation, but while SCC is associated with cumulative exposure, BCC development has been linked to early-age sunburn and intermittent, intense exposure (Armstrong, 2001; Diffey, 1979; Kricker, 1995; Franceschi, 1996; Rosso, 1996; Zanetti, 1996). The correlation with UVR is not as strong for BCC as SCC suggesting that other factors might be involved (Kricker, 1995; Kricker, 1995). Other agents associated with BCC and SCC include exposure to ionising radiation, arsenicals, polyaromatic hydrocarbons and psoralenplus-UVA therapy (Gailani, 1997; Stern, 1996). The genetic background of BCC and SCC is similar, with mutation of the p53 gene being an early event during tumour development. Mutations are present in approximately 50% of BCCs and up to 90 % of SCCs with the majority being missense mutations carrying a UV-signature (Soehnge, 1997; Brash, 1991; Brash, 1996). In typical cases of SCC, one p53 allele carries a point mutation and the other is deleted, while point mutations commonly affect both alleles in BCC. Except for p53, the oncogene ras and other tumour suppressors including p14

ARF

, p16

INK4a

and the ptch gene are found mutated in SCC. The ptch

29

Detection and analysis of genetic alterations in normal skin and skin tumours

gene is located on chromosome 9q and is involved in the repression of genes that drive cell growth and differentiation. The ptch protein forms part of a receptor complex together with the product of the Smoothened gene. Upon binding of the sonic hedgehog to this complex, ptch conformation is changed and Smoothened is activated, resulting in upregulation of its target genes. Mutations that inactivate ptch therefore lead to constitutive activation of Smoothened, which in turn leads to overexpression of GLI1 and uncontrolled cell proliferation. Germline mutations in the ptch gene are responsible for the autosomal dominant disorder denoted Gorlin syndrome, which is characterised by developmental abnormalities and multiple early onset BCCs (Gorlin, 1995). The ptch gene is also the most commonly altered gene in sporadic BCC and is lost and /or mutated in at least two thirds of the tumours (Gailani, 1996). However, the frequency of UV signature mutations in this gene is much lower than in p53, which indicates that factors other than UV are important for BCC development. In tumours with intact ptch, other genes involved in ptch signalling, such as Smoothened are mutated, indicating that inactivation of this pathway is a necessary step for tumour formation. Malignant melanoma Malignant melanoma arises from melanocytes and is the most fatal of skin cancers. Tumours mostly occur on the face, ears, neck, shoulders and back and are rare on the buttocks and the soles of the feet (Green, 1993). There is a clear correlation between melanoma and sunlight, especially intermittent sun-exposure that often results in sunburn. The early stages of melanoma development are thought to begin with a lentigo (freckle) that turns into a common aquired melanocytic naevus (mole) with a dermal component which then develops further into a naevus with an aberrant cell pattern. A lentigo results from the accumulation of normal melanocytes at the dermalepidermal junction, while a mole consists of normally appearing melanocytes that extends suprabasally and dermally. The number of moles is proportional to sunexposure during early childhood and a high number is associated with an elevated frequency of dysplatic naevi. However, moles routinely regress after age 15 in males and age 25 in females (Nicholls, 1973). The melanoma begins when the naevus develops a subpopulation of cells with nuclear atypia, denoted a dysplastic naevus, which is then followed by a radial growth phase, a vertical growth phase and metastasis. The rate of progression from naevi to melanoma is rather low, although

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the risk of transformation of a dysplatic naevus is almost 100 times as high as for a common naevi (Kraemer, 1983). Melanomas, especially of the superficial spreading type, are one of few human tumours that may spontaneously regress. Total regression occurs in about 5 % of the tumours, while partial regression is seen in 10-50 % of the tumours depending on their thickness (Blessing, 1992). On the genetic level, it seems that mutations in or loss of the CDKN2A (INK4a) locus and mutations in the oncogenes H-ras and N-ras play the most important roles in the development and progression of malignant melanoma (Herbst, 1997; Healy, 1996; Ortonne, 2002). The INK4a locus encodes both the CDK4/6 inhibitor p16INK4a and the mdm-2 binding protein p14ARF. Thus loss of this locus disrupts both the pRb and the p53 pathways. This is common in melanoma cell lines but in primary tumours the rate of homozygous deletion is only 10 % with a mutation rate of 0-25 % (Pollock, 1995; Ruas, 1998). The ras family of proto-oncogenes contains three functional members (N-ras, H-ras and K-ras) encoding GTP-binding G-proteins, which in their active state stimulate extracellular growth and differentiation (Crespo, 2000). The signalling stops when the intrinsic GTPase activity turns the protein into its inactive GDP-bound state. Mutations that lock the protein in the active state will result in continuous activation of downstream targets and thus continuous proliferation. Such activating mutations preferentially occur in codons 12, 13, 61 and 63 (Barbacid, 1987) of ras and exert their effect by reducing the intrinsic GTPase activity of the protein in addition to making it insensitive to GTPase-activating proteins (GAPs) (Der, 1986; Polakis, 1993; Sweet, 1984). N-ras is the ras gene most commonly mutated in cutaneous melanoma with a reported incidence rate of between 5% and 70 % (Barbacid, 1987; van 't Veer, 1989; Albino, 1989; Jiveskog, 1998; Ball, 1994; Demunter, 2001). This variability in the frequency of ras mutations can be explained by the fact that the mutation rate varies with sun-exposure as well as with the location of the tumour (Jiveskog, 1998; van 't Veer, 1989). Recently another gene in the ras pathway, B-raf, was reported to harbour missense mutations in 66 % of malignant melanomas (Davies, 2002). B-raf is one of three raf genes that are just downstream of ras and code for cytoplasmic serine/threonine kinases regulated by binding ras.

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Detection and analysis of genetic alterations in normal skin and skin tumours

PRESENT INVESTIGATION This thesis is based on five papers describing the development, evaluation and application of techniques for detection and analysis of genetic alterations in normal skin and in various skin tumours. Initially a method based on conventional dideoxy sequencing was developed and evaluated for the analysis of the p53 gene in small tissue samples down to the single cell level. The method was then used to investigate the effects of ultraviolet light on normal skin providing new insight into the possible events involved in normal differentiation and skin carcinogenesis. After slight modifications of the technique it was then used to complement an x-chromosome inactivation assay for investigation of BCC clonality. Finally a pyrosequencing-based method was developed and evaluated for hot-spot mutation detection in the N-ras gene of malignant melanoma.

Genetic analysis of skin and skin tumour samples. The investigation of genetic alterations in cancer-related genes is useful for research, prognostic and therapeutic purposes and in the future may become a routine procedure in cancer therapy and diagnostics (Harvey, 1995; Lowe, 1994). Sensitive and reliable detection methods are of major importance for obtaining relevant results, but a critical issue is the quality of the starting material, which largely affects the outcome of the analysis. Sample handling and preparation for analysis To obtain good quality genetic material a number of factors such as sample selection, preservation, preparation and handling are important. The tissue must be correctly treated to be useful for pathological and immunohistochemical analysis as well as to prevent DNA damage or degradation. Sample preparation Skin tumours such as BCC and melanoma can be surgically removed whereupon the tissue is fixed and paraffin embedded or snap-frozen. Formalin fixation followed by paraffin embedding is the traditional method and has resulted in invaluable archival stores of different tissue samples. This method preserves cell morphology excellently 32

Å Sivertsson

and makes pathological analysis possible decades after excision, but however does not protect the genetic material in the tissue. Formalin fixation has long been known to damage DNA by making protein-DNA cross-links (Chalkley, 1975) as well as decreasing the PCR amplification efficiency (Ben-Ezra, 1991). In recent years evidence has also arisen of artefactual mutations appearing during the amplification of DNA originating from formalin fixed material (Williams, 1999). Alcohol-based fixatives improve the integrity and recovery of nucleic acids, but preservation of cell morphology is somewhat impaired (Alaibac, 1997; Giannella, 1997). When frozen unfixed tissues are used the preservation of morphology is satisfactory for most applications (although it is inferior to that achieved with formalin) with a high recovery rate of nucleic acid. Therefore, in this work only freshly frozen material from excised skin tumours and skin punch biopsies have been used. Microdissection The ability to select specific areas, cell populations or even individual cells from an excised material is of great interest. During the investigation of tumour material, measures must be taken to limit the number of normal cells present in the sample to prevent the wild type DNA in these cells from masking possible genetic alterations in the DNA of tumour cells. This can be achieved by cryosectioning of the biopsies followed by microdissection of the material from the sections. The tissue sections are histologically stained and microdissection is performed under a microscope using a fine surgical scalpel. Using this technique it is possible to specifically dissect tumour nests by removing the surrounding normal tissue and to remove larger strokes of normal cells infiltrating tumour tissue. However, the dissection of more complex tissues is beyond the capability of this technique. Laser microdissection In recent years, a new technique based on laser-assisted microdissection microscopy has addressed the issues of exact and defined sampling and has made possible the retrieval of cell populations and even the retrieval of single cells from heterogenous material. There are two main principles for this type of microdissection, laser microbeam microdissection/laser pressure catapulting (LMM/LPC) developed by Schutze (Schutze, 1994; Schutze, 1998) and laser capture microdissection (LCM) developed by Liotta and co-workers (Emmert-Buck, 1996). In LMM, a 337-nm pulsed

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Detection and analysis of genetic alterations in normal skin and skin tumours

nitrogen laser is used to ablate undesired biological material surrounding the cell or cells of interest. The laser beam allows for fine focused microdissection (focus point < 2µm) without damage to surrounding cells. Tissue sections mounted on membranes or ordinary glass slides can be used and the isolated cell (or cells) can be retrieved using a needle, glass capillary or through laser catapulting into the cap of a microfuge tube (LPC). LCM uses a heat-generating infrared laser to fuse a thin temperaturesensitive transparent film with selected parts of the tissue on an underlying histological section. The film is mounted on a rigid flat cap and upon removal the selected parts of the tissue are detached. These techniques have provided new possibilities for analysis of tissue components and have become standard methods for retrieving defined cell populations, including single cells from heterogeneous samples, e. g. complex tumor and normal tissues (Suarez-Quian, 1999). Microfuge tube cap Laser pressure catapulting

Laser-assisted dissection

PALM laser

Microscope slide

Nailpolish

Tissue section Membrane

Figure 7. A schematic view of LMM/LPC for dissection of a small tissue sample.

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p53 gene analysis

Development of a method for single cell genetic analysis (I). The development of laser-assisted dissection techniques opened up the possibility of performing genetic analysis on a single cell allowing light to be shed on a multitude of biological issues. In a previous study, the heterogeneity of a BCC harbouring two p53 mutations was studied by analysing the p53 sequence of the two affected exons in single cells retrieved from tissue sections of the tumour (Pontén, 1997). However, the amplification of DNA from these single cells was not satisfactory in terms of amplification rate and exon and allele representation. Both alleles of the two exons analysed were amplified in only a fraction of the cells (14%). There may be several reasons for the apparently inefficient amplification of these single cells. A total lack of amplification may reflect damage or loss of the cell during transfer from the microscope slide to the PCR tube, while allelic dropout (ADO) may indicate chemical or physical alterations of the template. These alterations could include DNA degradation, loss of chromosomes during sectioning and microdissection or structural changes in the template rendering it unavailable for amplification. An alternative explanation may also be the preferential amplification of amplicons generated in the first few cycles over the original template. In the first paper of this thesis (I) these issues were addressed in the development and evaluation of a method for analysing exons 4 - 9 of the p53 gene in single cells obtained by laser-assisted microdissection of histological sections. The protocol is based on a previously described multiplex/nested approach where an outer PCR simultaneously amplifying exons 4 -9 of the p53 gene is followed by an inner nested amplification specific for each exon (Berg, 1995). The inner PCR product is then subjected to direct sequencing on automated DNA sequencers such as ALF, ABI 377 or ABI 3700. The PCR protocol was adjusted to increase the probability of primer annealing and extension in the first critical amplification cycles by decreasing the reaction volume and increasing annealing and extension times. To diminish the risk of misincorporation and subsequent artefactual mutations Taq DNA Polymerase was replaced with Pfu DNA polymerase, which has an error rate of 1 in 6.5 x107 base-pairs (Andre, 1997). Evaluation of the optimised protocol using single cells picked from a fresh culture of the HaCaT cell line showed a dramatic increase in amplification efficiency. To determine the allele pick-up frequency, the amplified products of exons 5 and 8, each

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Detection and analysis of genetic alterations in normal skin and skin tumours

containing an inherent p53 mutation affecting different alleles, were sequenced. Sequencing was carried out using dye terminators on the automated ABI 377 DNA sequencer. A mutation signal of 50% for both mutations proved that both alleles were amplified, while a 100 % mutation signal for either of the mutations indicated amplification dependent ADO. It was demonstrated that both alleles and all six exons were amplified in 50 % of the cells, which is an increase in efficiency of more than 300%. The improvement was considered satisfactory and the protocol was applied to single cells retrieved from immunohistochemically stained tissue sections. Again the amplification efficiency decreased as all exons were amplified in only 1 of 16 cells (6 %). This indicated that it was the quality of the DNA and not the amplification procedure that was the critical factor. Measures taken to diminish DNA damage by eliminating the immunohistochemical staining procedure and the proteinase K lysis did however not affect the amplification efficiency. The rate of successful amplification of all exons could however be increased to 70 % when EDTA, which has been shown to protect DNA from degradation (Yagi, 1996) was added during the different steps of sectioning and microdissection. Finally, when EDTA was present during the sample preparation, allele representation was determined to 50 % by sequencing single cells retrieved from a BCC with a known heterozygous mutation. The ADO rates reported in the literature range from 10-60% using fresh cells and are expected to be higher using cells from tissue sections (Garvin, 1998; Findlay, 1998; Rechitsky, 1998). Other parameters affecting ADO includes cell type, length of amplified fragments and degree of multiplexing, with an increase in length and number of amplified fragments resulting in an increased ADO rate. Thus we accepted that a lower ADO rate might be difficult to obtain considering the conditions used. With

this improved method, single cells selected on the basis of

immunohistochemistry, morphology or other features that are in stages of normal development or malignant disease can be isolated and subsequently analysed for p53 gene mutations (Figure 8).

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Single cell microdissection

Mt

Ex 4

Ex 5

Ex 6

Ex 7

Ex 8

Ex 9

Ex 10

Ex 11

Ex 10

Ex 11

Outer multiplex PCR

Inner exon specific PCR Mt

Ex 4

Ex 5

Ex 6

Ex 7

Ex 8

Ex 9

Mt 4 5 6 7 8 9 10 11

DNA sequencing

Figure 8. Schematic illustration of the different steps involved in genetic analysis of single cells from tissue sections.

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Detection and analysis of genetic alterations in normal skin and skin tumours Applications Effects of UV radiation on the p53 gene in normal human epidermis (II, III) Sun exposure has been implicated in all types of human skin cancer, as well as in photoageing of normal skin. Ultraviolet B (UVB) radiation is a causative agent of both BCC and SCC, while UVA radiation is implicated in malignant melanoma. In chronically sun-exposed normal human skin, a multitude of p53 immunoreactive clusters of morphologically normal epidermal keratinocytes can be found (Pontén, 1995; Jonason, 1996; Ren, 1996). These so called p53 clones (or p53 patches) have been shown to originate from putative stem cell compartments and commonly have an underlying p53 mutation, which may facilitate their expansion by allowing their escape from UV-induced apoptosis (Brash, 1996). A single dose of UVA or UVB radiation will further induce transient overexpression of p53 protein in normal skin (Pontén, 2001). In addition, scattered p53 immunoreactive keratinocytes can be found after the acute UV response should have disappeared and also in skin that has been UV-protected. The genetic background of these cells is unclear but due to the development of precise laser-assisted microdissection and protocols for single cell amplification these cells can now be analysed. In the following two papers, the effect of sun exposure and UVA1 radiation on normal epidermis was investigated.

Sun-exposure and persistent p53 mutations (II) Natural sun-exposure leads to a transient increase in the number of p53 immunoreactive cells in normal skin, a response believed to result from overexpression of normal p53 protein and cell-cycle arrest in response to DNA damage. However, it has previously been shown that in skin protected from sunexposure for two months (thus allowing for a complete skin turn-over) scattered p53 immunoreactive keratinocytes can also be found (Berne, 1998). To further investigate the nature of these persistent morphologically normal p53 immunoreactive keratinocytes, we applied the p53 gene analysis strategy on single cells from skin subjected to ordinary daily summer sun and from adjacent skin that had been totally protected from solar radiation by blue denim fabric (sun protection factor 1700). The skin originated from three volunteers of different age and gender and immunohistochemistry revealed a dispersed pattern of scattered p53 immunoreactive cells throughout the epidermis with fewer immunoreactive cells in shielded skin

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compared to sun-exposed skin. A total of 72 scattered single cells from the different layers of epidermis were successfully amplified and sequenced, which revealed the presence of one or more p53 mutations in eight keratinocytes. In cells from sunexposed skin, three out of four mutations were typical UV signature mutations (C-T at a dipyrimidine site). In shielded skin, a typical UV signature was found in one out of six mutations and the remaining were two C-A transversions, two G-A transitions and one C-T transition at non-dipyrimidine sites. The C-T transitions and C-A transversions represent mutation types indicative of oxidative damage and thus possibly related to UVA exposure as discussed in the Introduction and paper III. The major findings of this study were that p53 mutations are frequent in normal skin and that these mutations, which were all missense or truncating mutations, were found in both immunoreactive and non-immunoreactive keratinocytes. The mutations found in the chronically exposed skin may represent events affecting terminally differentiated or transit amplifying cells that are already destined to be shed as a result of the constant epidermal turnover. In this case, possible resulting defects in cell cycle control or in the apoptosis pathway will have no effect and the mutated cells will disappear by the natural course of cell shedding. In the shielded skin, at least two skin turnovers had occurred since the last exposure to UV and therefore the presumably UV-induced mutations appeared to be persistent. This we interpret as being consistent with the mutated cells being the offspring of originally mutated epidermal stem cells. The consequence of these mutations (if the skin had been continuously exposed) might be a selective growth advantage leading to the formation of small p53 clones such as those described below. In one of the biopsies from shielded skin, a small cluster of p53 immunoreactive cells, apprehended as a p53 clone, was found in addition to the scattered cells. The cluster covered an area of 0.05 mm2 involving all layers of the epidermis. From the cluster, 2 5 single cells displaying different p53 immunoreactivity and originating from different epidermal layers were successfully sequenced. Two dominating mutations, affecting all but one of the 18 keratinocytes harbouring mutations, were found among the five different mutations detected. They were typical UV induced mutations affecting the core DNA binding domain of p53 and were present in 12 immunoreactive and 5 non-immunoreactive keratinocytes from all layers of the epidermis (Figure 9). An interesting observation was that cells in the basal layer harboured both mutations to a greater extent compared to suprabasal and superficial

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Detection and analysis of genetic alterations in normal skin and skin tumours

cells. This phenomenon is difficult to fully explain by amplification dependent ADO and might indicate that ADO is a part of the terminal differentiation pathway. The visual appearance of the patch in addition to the sharing of mutations indicated that the p53 cluster had clonal origin as opposed to the scattered cells, which had different mutations. These findings further supported the theory of a mutated stem cell continuously giving rise to transit amplifying cells and terminally differentiated cells. These will spread within an area corresponding to the size of an epidermal proliferative unit (EPU) unless the mutation confers a growth advantage and /or gives the cells the ability to overcome growth restrictions. In this case, the clone was approximately the estimated size of an EPU, which could be interpreted as either that the mutations were not detrimental to growth control or that the clone had regressed after withdrawal of UV radiation (i. e. the potential selective force). In our opinion, since both mutations were missense and affected an important region of the gene the latter interpretation is more plausible. As mentioned earlier, p53 clones are commonly found in chronically sun-exposed skin but the consequence of their presence is so far unknown. Although p53 clones are frequently found adjacent to SCCs and BCCs a genetic link has not been established. However in mice, early emergence of p53 clones has been shown following chronic UV-irradiation and recently their appearance was shown to be an indicator of tumour risk (Berg, 1996; Rebel, 2001). This study provides additional insights into the structure of a clandestine p53 clone and proposes a model for allele dropout during the normal epidermal differentiation process.

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Superficial cells 241

299

281

241

241

241

281

wt

wt

241

wt

wt

Suprabasal cells wt

241+281

241+281

281

241+281 241+281 241+281

3 241+281 wt

281

wt

281+38

Basal cells

281

241+281 +219

p53 immunoreactive keratinocytes

p53 non-immunoreactive keratinocytes

Figure 9. The topography of the p53 clone found in sunshielded normal epidermis. Two missense mutations were present in all three layers of epidermis and found in immunoreactive as well as non-immunoreactive cells.

p53 mutations induced by UVA1 (III) UVA irradiation has long been used in the treatment of skin diseases such as atopic dermatitis and schleroderma, as well as for cosmetic tanning. UVA radiation contains less energy than UVB, does not give rise to the typical UV mutations frequently found in SCCs and BCCs and has been considered to have a minor role in photocarcinogenesis. However, UVA has been shown to induce squamous cell cancer in mice (Matsui, 1991), malignant melanoma in opossum (Ley, 1997) and has also been implicated in the increased risk of malignant melanoma in tanning bed (Autier, 1991; Walter, 1990; Westerdahl, 1994) and sunscreen users (Autier, 1995; Westerdahl, 1995; Wolf, 1998). Similar to UVB radiation, UVA exposure is known to result in a transient increase in p53 in normal human epidermis (Burren, 1998). Recently persistently p53 immunoreactive keratinocytes were also shown after suberythemal doses of UVA1 (Edstrom, 2001; Seite, 2000).

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Detection and analysis of genetic alterations in normal skin and skin tumours

Using our single cell analysis strategy (extended to include exons 10 and 11) we investigated the p53 gene status of the dispersed p53 immunoreactive keratinocytes found in normal epidermis after physiological doses of UVA1. Three volunteers with skin types II-III were during a period of two weeks exposed to six physiological doses (40 J/cm2) of UVA1 on a 40 cm2 area of the buttock. Punch biopsies were taken before and at three different time points after exposure. The first time-point (directly after exposure) was selected in order to detect the transient p53 response, while the second (five days after exposure) was selected in order to detect possible persistent p53 immunoreactivity after disappearance of the transient response. The last timepoint (8 weeks after last exposure) was selected so that at least one skin turnover would have occurred after exposure. Immunostained sections from the different biopsies all revealed a dispersed pattern of p53 immonoreactive cells with the amount of reactive cells significantly increased directly after exposure and five days following exposure in comparison to the skin prior to the treatment. Eight weeks after exposure when skin shedding had occurred, the amount of reactive cells had returned to background levels. The increase in the number of p53 overexpressing cells directly after exposure could be explained by the transient response, while the persistence after five days was interpreted as a sign that damage may have occurred in p53 or parts of its pathway. Single keratinocytes displaying p53 immunoreactivity were microdissected from sections of unexposed skin as well as from sections taken directly after exposure and five days following exposure. Among the exposed cells, three mutations were found in 26,000 basepairs sequenced, while no mutations were detected after sequencing 15,900 basepairs of cells from unexposed skin. All three mutations were G-T transversions, a mutation type that has been related to oxidative damage and implicated as a result of UVA exposure (see Introduction)(Cheng, 1992; De Gruijl, 2000; Kvam, 1997; Kielbassa, 2000; Kawanishi, 2001). Two of the mutations, one missense mutation and one affecting intron sequence were found in cells taken five days after exposure, while another intron sequence mutation was found among the cells taken directly after completed exposure. The finding of mutations in intron sequence were in accordance with our view that these mutations had not yet been selected for and thus represented random events. Affected cells would presumably be shed at the next skin turnover, unless these cells had stem cell properties, in which case the mutations would be transferred to daughter cells.

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Support for the former theory was suggested in sections from 8 weeks after exposure where skin shedding had occurred and a reduction of immunoreactive cells were observed. In addition, the mutation rate was calculated using data from the study, which revealed the obtained value to be more than 10,000 times higher than the estimated value for spontaneous mutations. This further suggested the involvement of UVA radiation in the induction of these mutations. Furthermore, the number of mutations per base-pair was found to be well in accordance with the number found for oxidative damage type of mutations in paper II representing the UVA mutation load from normal sun-exposure. We interpret the findings of this study to show that even low-dose UVA gives rise to a multitude of mutations randomly spread across the genome, some of which will probably affect genes and their function and therefore the role of UVA should not be disregarded in malignant skin disease.

The clonality of BCC (IV) In paper IV, p53 gene analysis was used to complement an X-chromosome inactivation assay to investigate the clonal nature of BCC. BCC usually appears to consist of multiple foci surrounded by a characteristic connective stroma, which its growth is heavily dependent on. This multifocal appearance may indicate a polyclonal nature but both genetic studies and the results of tumour reconstitution from serial sections (Pontén, 1997; Imayama, 1987) has lead to the belief that BCC is of monoclonal origin. However, the tumour’s heavy dependence on stroma suggested a possible parallel to another tumour, phylloid tumour of the breast, which also has both stromal and epithelial components. This tumour has been shown to consist of a compartment of monoclonal stromal cells, which give rise to polyclonal proliferation of epithelial cells (Noguchi, 1993). In paper IV the clonal arrangement in both tumours and their cognate stroma was resolved by studying the x-chromosome inactivation patterns using a microsatellite marker in the HUMARA locus of the androgen receptor gene. The x-chromosome inactivation assay is based on the natural process of lyonization, which leads to random inactivation of one of the xchromosomes in all cells of the female body. In the in vitro assay the active allele is fragmented by enzymatic cleavage, from which the methylated inactive allele is protected. Subsequent amplification of a polymorphic microsatellite yields product only from the intact inactivated allele and the obtained fragment length distinguishes

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Detection and analysis of genetic alterations in normal skin and skin tumours

the maternal from the paternal allele. In this study, manual or laser-assisted microdissection was utilised to obtain pure samples from 13 tumours and their cognate stroma. X-chromosome analysis showed that 12 of these 13 tumours were monoclonal and that all samples from these tumours showed the same inactivation pattern. One tumour however, displayed two distinct x-chromosome inactivation patterns, indicating it was of polyclonal origin. Three samples from this tumour showed non-random inactivation of one allele, while the other allele was inactivated in a fourth sample. This case was further investigated by loss of heterozygosity (LOH) analysis of the ptch and p53 loci and additional sequencing of the p53 gene in microdisssected samples from serial sections (Figure 10). Loss of heterozygosity of the p53 and ptch loci as well as mutation of the p53 gene are frequent events in skin cancer and may thus be used as possible clonality markers. The results of these analyses further supported the conclusions from the x-chromosome inactivation analysis. The three samples sharing inactivation pattern displayed LOH of the same allele and in both loci, while the sample with a deviating x-chromosome pattern was proven unaffected by LOH. Two of the three samples affected by LOH were further shown to harbour a truncating mutation in exon 6 of the p53 gene not detected in the deviating sample. Another intriguing finding in this study was an apparent correlation between xchromosome allelic size difference and which allele was inactivated. This correlation was further investigated by analysing x-chromosome inactivation in eight additional BCC cases. In total, 20 of 21 analysed BCCs showed inactivation of the longer or shorter allele depending on the allelic size difference, while no such bias was seen in an earlier study on normal skin. The implication of this finding is not clear. In conclusion, our findings support the theory that BCC is a tumour of monoclonal origin and indicates that the surrounding connective stroma has a polyclonal nature. However, our data is inconclusive due to the finding of a single polyclonal tumour.

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XL/XS

: inactivation of long or short allele of X-chrom. : long and short allele of p53 : long and short allele of ptch, D9S280 : long and short allele of ptch, D9S180

Dashed lines represent alleles affected by LOH

: p53 mutation XS

XS

XL

XS

Figure 10. A schematic picture of the tumour displaying two distinct patterns of inactivation.

Detection of N-ras hot-spot mutations in melanoma using pyrosequencing (V) Analogous to p53 mutations which are common alterations found early in BCC, mutations in the N-ras gene are frequently found in cutaneous malignant melanoma. In contrast to the p53 gene where mutations are found scattered in the coding region, mutations in N-ras in malignant melanoma have so far been identified at a few prominent positions and most commonly at codons 12, 13 and 61 (Barbacid, 1987; Tormanen, 1992). One of the most common screening assays for such pre-defined positions is the sensitive, simple and low-cost SSCP assay. However, the reliability of this technique is disputed and therefore confirmatory sequencing must be performed. In the final paper of this thesis we investigated the feasability of using pyrosequencing for one-step detection and identification of N-ras mutations in clinical samples of cutaneous melanoma. To ensure that the pyrosequencing strategy was reliable and simple several parameters were initially optimised and evaluated. A PCR approach was developed in which fragments containing the two mutated sites (codon 12 – 13 of exon 1 and codon 61 of exon 2) were co-amplified in an outer multiplex PCR followed by subsequent individual inner PCRs. This approach was used to evaluate two different methods for

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Detection and analysis of genetic alterations in normal skin and skin tumours

obtaining template DNA from tumour samples. Direct proteinase K lysis of the dissected samples is a rapid approach resulting in a crude DNA extract, while a pure extract can be obtained using more expensive and time-consuming commercially available DNA extraction kits. Equal amplification efficiencies and pyrosequencing results were obtained irrespective of which extract was used as a template for the amplification. For the pyrosequencing analysis, nucleotide dispensation orders were created to be able to distinguish between the peak patterns of the six most common mutations at codons 12 and 13, as well as between the four most common mutations at codon 61. The detection limit of the optimised method was then determined by analysis of dilution series of cell line samples containing any of two common codon 61 mutations and was found to be satisfactory. Evaluation on clinical samples were made by analysis of N-ras mutations in DNA extracts from 82 melanoma metastases using the pyrosequencing method and an earlier developed SSCP/Sanger sequencing method. Twenty-two codon 61 mutations, representing four different mutation types, were found among the 71 samples successfully amplified for use in SSCP/Sanger sequencing analysis. These mutations were also detected and correctly identified by pyrosequencing. The high sensitivity of pyrosequencing became evident when the concentrations of some mutations were estimated to be less than 25% according to the relative intensities of the shifted bands in the SSCP analysis. Another four codon 61 mutations were found among the 11 samples not analysed by SSCP and the resulting mutation frequency (32 %) correlates well with previous studies (Jiveskog, 1998; Omholt, 2002; Platz, 1994). We conclude that pyrosequencing is a possible alternative to SSCP /Sanger sequencing for detection of N-ras hot-spot mutations in terms of reproducibility, sensitivity, simplicity and speed. However, a possible limitation of the method is that only a certain number of positions can be analysed simultaneously, in contrast to SSCP where the entire amplified fragment is scanned. Also some effort must be put into creating nucleotide dispensation profiles to obtain maximal sensitivity and specificity. In developing this method we have provided a simple and rapid tool for detection and identification of N-ras hot-spot mutations that may prove useful for large-scale screening of melanoma patients for prognostic and diagnostic purposes.

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Å Sivertsson Abbreviations ADO AMASE ASA ASO BCC BESS CCM CDGE CFLP CGE CIS CSGE DASH ddF ddNTP dNTP DGGE DHPLC DNA EPU FAMA GAP HA LCM LCR LMM/LPC LOH MALDI-TOF–MS NIRCA NMSC OLA PCR PEX PPi SBH SCC SNP SSCP TGGE UVR

allele dropout apyrase-mediated allele-specific extension allele specific amplification allele specific oligonucleotides basal cell cancer base excision sequence scanning chemical cleavage of mismatch constant denaturant gel electrophoresis cleavage fragment length polymorphism capillary gel electrophoresis cancer in situ conformation-sensitive gel electrophoresis dynamic allele-specific hybridisation dideoxy fingerprinting dideoxynucleotidetriphosphate deoxynucleotidetriphosphate denaturing gradient gel electrophoresis denaturing high-performance liquid chromatography deoxyribonucleic acid epidermal proliferative unit fluorescence assisted mutation assay GTPase activating protein heteroduplex analysis laser capture microdissection ligase chain reaction laser microbeam microdissection/laser pressure catapulting loss of heterozygosity matrix-assisted laser desorption ionisation - time-offlight - mass spectrometry non-isotopic RNase cleavage assay non-melanoma skin cancer oligonucleotide ligation assay polymerase chain reaction single nucleotide primer extension pyrophosphate sequencing by hybridisation squamous cell cancer single nucleotide polymorphism single strand conformation polymorphism analysis temperature gradient gel electrophoresis ultraviolet radiation

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Detection and analysis of genetic alterations in normal skin and skin tumours Acknowledgements I would like to thank everyone who has contributed to this thesis and has made these last couple of years so enjoyable. I am especially grateful to: Joakim Lundeberg, my supervisor, for excellent guidance during this scientific journey, for always being so positive, supportive and enthusiastic and for taking so good care of your students. Mathias Uhlén for accepting me as a PhD student and for being a true visionary with a fantastic skill for attracting good people, good money and lots of attention to this department. My collaborators, co-authors and friends in Uppsala: Figge, for inspiring and fruitful discussions on biology, skin cancer and life in general, for being so enthusiastic and, last but not least, for all nice meetings and fantastic X-mas parties. Anna A for interesting discussions on a variety of subjects (some of which involved science…) and for the very funny and very long e-mails with your thoughts on life and science. Thank you so much also for the enormous effort you have put in to get the last paper ready. Helena for being a great and enthusiastic collaborator, for sharing the “passion“ for analysing sequences, life and relationships, and for fun nights at Karlsson´s. Thank you also for letting me use your beautiful UVB damage picture! The late Jan Pontén for being such a great and enthusiasming scientist and for making the solving of the mysteries of EPUs and skin cancer seem like true adventures to me. My collaborators and co-authors in Stockholm: Desiree, for a fun and fruitful collaboration and for being very patient and optimistic during the time our paper was just bouncing around. Anton Platz and Johan Hansson, for a great collaboration and for sharing your knowledge on N-ras and melanoma. Deirdre, for kindly reading and giving valuable comments on this thesis. (Note that no blame should fall on her for errors in this section, which I wrote with no concern whatsoever for spelling and grammar) The “Cancer group”: Jocke, Figge, Afshin, Max, Cecilia L, Cissi, Lotta, Anna G, Helena, Anna, Cecilia for very stimulating and interesting meetings and very nice lunches. My roommates: Maria and Jenny for excellent company and vivid scientific discussions on everything from relationships and shopping to work and philosophy. I would also like to thank Jenny for helping me out with pictures and formats and a whole lot of other things during the writing of this thesis.

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Å Sivertsson Kristofer for bearing with us despite the “girl talk” and for letting us turn up the heating, making our room the only nice and warm place on this floor. Maria, for being not only an excellent roommate, but also a very good and supportive friend AND an extremely nice sister-in-law. Thank you also for your successful effort in the matchmaking business… Anna G and Lotta for filling the corridors with laughter and gossip and for planning events and activities that help to create a great atmosphere. Tove for nice chats, mental support and for sharing the burden of general anxiety which, I think, is a compulsory phase of thesis writing. Niklas Malmqvist for introducing me to “magic wands” and other features of Illustrator. Bahram for great help with sequencing and for not complaining about having to run p53 under special conditions, changing matrices and stuff. Karin and Jenny for help with PCR and sequencing and for adding some culture (not cell culture but REAL culture) to this place. All collegues, former and present, at the Department of Biotechnology for being so nice and helpful and for contributing (everyone in his or her own special way) to the fantastic atmosphere of this place. I feel very privileged to have had the opportunity to get to know you and to work with you, and I would like to mention you all by name but you are so many and it is very late…. My friends, in and out of science, for being such great persons and for all the good times we have shared. Cissi, my best friend, for recruiting me to this department and paving the road for the single cell research, but above all for being such a great friend and for all the good times we have had. My sister with family for always being there for me with good advice, home-made fudge or whatever will do the trick. My parents for endless support and encouragement and for believing in me and wishing me happiness whatever I do. Micke, the love of my life, my husband, for loving me and being all I ever wished for. Finally, I would also like to thank Micke for his generous contribution of genetic material to the most challenging project of our lives so far, which has absolutely nothing to do with any of the projects in this thesis.

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Detection and analysis of genetic alterations in normal skin and skin tumours References Agaton C, Unneberg P, Sievertzon M, Holmberg A, Ehn M, Larsson M, Odeberg J, Uhlen M and Lundeberg J. (2002) Gene expression analysis by signature pyrosequencing. Gene 289:31-39. Ahmadian A, Gharizadeh B, Gustafsson AC, Sterky F, Nyren P, Uhlen M and Lundeberg J. (2000) Single-nucleotide polymorphism analysis by pyrosequencing. Anal Biochem 280:103-110. Ahmadian A, Gharizadeh B, O'Meara D, Odeberg J and Lundeberg J. (2001) Genotyping by apyrasemediated allele-specific extension. Nucleic Acids Res 29:E121. Alaibac M, Filotico R, Giannella C, Paradiso A, Labriola A and Marzullo F. (1997) The effect of fixation type on DNA extracted from paraffin-embedded tissue for PCR studies in dermatopathology. Dermatology 195:105-107. Albino AP, Nanus DM, Mentle IR, Cordon-Cardo C, McNutt NS, Bressler J and Andreeff M. (1989) Analysis of ras oncogenes in malignant melanoma and precursor lesions: correlation of point mutations with differentiation phenotype. Oncogene 4:1363-1374. Almeida TA, Cabrera VM and Miranda JG. (1998) Improved detection and characterization of mutations by primer addition in nonradioisotopic SSCP and direct PCR sequencing. Biotechniques 24:220-221. Andre P, Kim A, Khrapko K and Thilly WG. (1997) Fidelity and mutational spectrum of Pfu DNA polymerase on a human mitochondrial DNA sequence. Genome Res 7:843-852. Andreasson H, Asp A, Alderborn A, Gyllensten U and Allen M. (2002) Mitochondrial sequence analysis for forensic identification using pyrosequencing technology. Biotechniques 32:124-126, 128, 130-123. Ansorge W, Sproat BS, Stegemann J and Schwager C. (1986) A non-radioactive automated method for DNA sequence determination. J Biochem Biophys Methods 13:315-323. Ansorge W, Voss H, Wiemann S, Schwager C, Sproat B, Zimmermann J, Stegemann J, Erfle H, Hewitt N and Rupp T. (1992) High-throughput automated DNA sequencing facility with fluorescent labels at the European Molecular Biology Laboratory. Electrophoresis 13:616-619. Armstrong BK and Kricker A. (2001) The epidemiology of UV induced skin cancer. J Photochem Photobiol B 63:8-18. Armstrong JD and Kunz BA. (1992) Photoreactivation implicates cyclobutane dimers as the major promutagenic UVB lesions in yeast. Mutat Res 268:83-94. Asplund A, Guo Z, Hu X, Wassberg C and Ponten F. (2001) Mosaic pattern of maternal and paternal keratinocyte clones in normal human epidermis revealed by analysis of X-chromosome inactivation. J Invest Dermatol 117:128-131. Autier P, Dore JF, Schifflers E, Cesarini JP, Bollaerts A, Koelmel KF, Gefeller O, Liabeuf A, Lejeune F, Lienard D and et al. (1995) Melanoma and use of sunscreens: an Eortc case-control study in Germany, Belgium and France. The EORTC Melanoma Cooperative Group. Int J Cancer 61:749-755. Autier P, Joarlette M, Lejeune F, Lienard D, Andre J and Achten G. (1991) Cutaneous malignant melanoma and exposure to sunlamps and sunbeds: a descriptive study in Belgium. Melanoma Res 1:69-74. Ball NJ, Yohn JJ, Morelli JG, Norris DA, Golitz LE and Hoeffler JP. (1994) Ras mutations in human melanoma: a marker of malignant progression. J Invest Dermatol 102:285-290. Baner J, Nilsson M, Mendel-Hartvig M and Landegren U. (1998) Signal amplification of padlock probes by rolling circle replication. Nucleic Acids Res 26:5073-5078.

50

Å Sivertsson

Barany F. (1991) Genetic disease detection and DNA amplification using cloned thermostable ligase. Proc Natl Acad Sci U S A 88:189-193. Barbacid M. (1987) ras genes. Annu Rev Biochem 56:779-827. Ben-Ezra J, Johnson DA, Rossi J, Cook N and Wu A. (1991) Effect of fixation on the amplification of nucleic acids from paraffin- embedded material by the polymerase chain reaction. J Histochem Cytochem 39:351-354. Berg C, Hedrum A, Holmberg A, Pontén F, Uhlén M and Lundeberg J. (1995) Direct solid-phase sequence analysis of the human p53 gene by use of multiplex polymerase chain reaction and alphathiotriphosphate nucleotides. Clin Chem 41:1461-1466. Berg RJ, van Kranen HJ, Rebel HG, de Vries A, van Vloten WA, Van Kreijl CF, van der Leun JC and de Gruijl FR. (1996) Early p53 alterations in mouse skin carcinogenesis by UVB radiation: immunohistochemical detection of mutant p53 protein in clusters of preneoplastic epidermal cells. Proc Natl Acad Sci U S A 93:274-278. Berne B, Ponten J and Ponten F. (1998) Decreased p53 expression in chronically sun-exposed human skin after topical photoprotection. Photodermatol Photoimmunol Photomed 14:148-153. Bertina RM, Koeleman BP, Koster T, Rosendaal FR, Dirven RJ, de Ronde H, van der Velden PA and Reitsma PH. (1994) Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 369:64-67. Bhattacharyya A and Lilley DM. (1989) The contrasting structures of mismatched DNA sequences containing looped-out bases (bulges) and multiple mismatches (bubbles). Nucleic Acids Res 17:68216840. Bhattacharyya A and Lilley DM. (1989) Single base mismatches in DNA. Long- and short-range structure probed by analysis of axis trajectory and local chemical reactivity. J Mol Biol 209:583-597. Blaszyk H, Hartmann A, Schroeder JJ, McGovern RM, Sommer SS and Kovach JS. (1995) Rapid and efficient screening for p53 gene mutations by dideoxy fingerprinting. BioTechniques 18:256-260. Blessing K and McLaren KM. (1992) Histological regression in primary cutaneous melanoma: recognition, prevalence and significance. Histopathology 20:315-322. Borresen AL, Hovig E and Brogger A. (1988) Detection of base mutations in genomic DNA using denaturing gradient gel electrophoresis (DGGE) followed by transfer and hybridization with genespecific probes. Mutat Res 202:77-83. Brash DE, Rudolph JA, Simon JA, Lin A, McKenna GJ, Baden HP, Halperin AJ and Ponten J. (1991) A role for sunlight in skin cancer: UV-induced p53 mutations in squamous cell carcinoma. Proc Natl Acad Sci U S A 88:10124-10128. Brash DE, Seetharam S, Kraemer KH, Seidman MM and Bredberg A. (1987) Photoproduct frequency is not the major determinant of UV base substitution hot spots or cold spots in human cells. Proc Natl Acad Sci U S A 84:3782-3786. Brash DE, Ziegler A, Jonason AS, Simon JA, Kunala S and Leffell DJ. (1996) Sunlight and sunburn in human skin cancer: p53, apoptosis, and tumor promotion. J Investig Dermatol Symp Proc 1:136-142. Braun A, Little DP and Koster H. (1997) Detecting CFTR gene mutations by using primer oligo base extension and mass spectrometry. Clin Chem 43:1151-1158. Braun A, Little DP, Reuter D, Muller-Mysok B and Koster H. (1997) Improved analysis of microsatellites using mass spectrometry. Genomics 46:18-23.

51

Detection and analysis of genetic alterations in normal skin and skin tumours Broude NE, Woodward K, Cavallo R, Cantor CR and Englert D. (2001) DNA microarrays with stemloop DNA probes: preparation and applications. Nucleic Acids Res 29:E92. Brow MA, Oldenburg MC, Lyamichev V, Heisler LM, Lyamicheva N, Hall JG, Eagan NJ, Olive DM, Smith LM, Fors L and Dahlberg JE. (1996) Differentiation of bacterial 16S rRNA genes and intergenic regions and Mycobacterium tuberculosis katG genes by structure-specific endonuclease cleavage. J Clin Microbiol 34:3129-3137. Burren R, Scaletta C, Frenk E, Panizzon RG and Applegate LA. (1998) Sunlight and carcinogenesis: expression of p53 and pyrimidine dimers in human skin following UVA I, UVA I + II and solar simulating radiations. Int J Cancer 76:201-206. Buschmann T, Potapova O, Bar-Shira A, Ivanov VN, Fuchs SY, Henderson S, Fried VA, Minamoto T, Alarcon-Vargas D, Pincus MR, Gaarde WA, Holbrook NJ, Shiloh Y and Ronai Z. (2001) Jun NH2terminal kinase phosphorylation of p53 on Thr-81 is important for p53 stabilization and transcriptional activities in response to stress. Mol Cell Biol 21:2743-2754. Cadet J, Berger M, Douki T, Morin B, Raoul S, Ravanat JL and Spinelli S. (1997) Effects of UV and visible radiation on DNA-final base damage. Biol Chem 378:1275-1286. Campbell C, Quinn AG, Angus B, Farr PM and Rees JL. (1993) Wavelength specific patterns of p53 induction in human skin following exposure to UV radiation. Cancer Res 53:2697-2699. Canard B and Sarfati RS. (1994) DNA polymerase fluorescent substrates with reversible 3'-tags. Gene 148:1-6. Canman CE and Kastan MB. (1997) Role of p53 in apoptosis. Adv Pharmacol 41:429-460. Canman CE, Lim DS, Cimprich KA, Taya Y, Tamai K, Sakaguchi K, Appella E, Kastan MB and Siliciano JD. (1998) Activation of the ATM kinase by ionizing radiation and phosphorylation of p53. Science 281:1677-1679. Chalkley R and Hunter C. (1975) Histone-histone propinquity by aldehyde fixation of chromatin. Proc Natl Acad Sci U S A 72:1304-1308. Chehab NH, Malikzay A, Stavridi ES and Halazonetis TD. (1999) Phosphorylation of Ser-20 mediates stabilization of human p53 in response to DNA damage. Proc Natl Acad Sci U S A 96:13777-13782. Cheng KC, Cahill DS, Kasai H, Nishimura S and Loeb LA. (1992) 8-Hydroxyguanine, an abundant form of oxidative DNA damage, causes G----T and A----C substitutions. J Biol Chem 267:166-172. Chernov MV, Ramana CV, Adler VV and Stark GR. (1998) Stabilization and activation of p53 are regulated independently by different phosphorylation events. Proc Natl Acad Sci U S A 95:2284-2289. Chien A, Edgar DB and Trela JM. (1976) Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus. J Bacteriol 127:1550-1557. Cho RJ, Mindrinos M, Richards DR, Sapolsky RJ, Anderson M, Drenkard E, Dewdney J, Reuber TL, Stammers M, Federspiel N, Theologis A, Yang WH, Hubbell E, Au M, Chung EY, Lashkari D, Lemieux B, Dean C, Lipshutz RJ, Ausubel FM, Davis RW and Oefner PJ. (1999) Genome-wide mapping with biallelic markers in Arabidopsis thaliana. Nat Genet 23:203-207. Clausen OP and Potten CS. (1990) Heterogeneity of keratinocytes in the epidermal basal cell layer. J Cutan Pathol 17:129-143. Cline J, Braman JC and Hogrefe HH. (1996) PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases. Nucleic Acids Res 24:3546-3551.

52

Å Sivertsson Cotton RG and Campbell RD. (1989) Chemical reactivity of matched cytosine and thymine bases near mismatched and unmatched bases in a heteroduplex between DNA strands with multiple differences. Nucleic Acids Res 17:4223-4233. Cotton RG, Rodrigues NR and Campbell RD. (1988) Reactivity of cytosine and thymine in singlebase-pair mismatches with hydroxylamine and osmium tetroxide and its application to the study of mutations. Proc Natl Acad Sci U S A 85:4397-4401. Crespo P and Leon J. (2000) Ras proteins in the control of the cell cycle and cell differentiation. Cell Mol Life Sci 57:1613-1636. Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, Teague J, Woffendin H, Garnett MJ, Bottomley W, Davis N, Dicks E, Ewing R, Floyd Y, Gray K, Hall S, Hawes R, Hughes J, Kosmidou V, Menzies A, Mould C, Parker A, Stevens C, Watt S, Hooper S, Wilson R, Jayatilake H, Gusterson BA, Cooper C, Shipley J, Hargrave D, Pritchard-Jones K, Maitland N, Chenevix-Trench G, Riggins GJ, Bigner DD, Palmieri G, Cossu A, Flanagan A, Nicholson A, Ho JW, Leung SY, Yuen ST, Weber BL, Seigler HF, Darrow TL, Paterson H, Marais R, Marshall CJ, Wooster R, Stratton MR and Futreal PA. (2002) Mutations of the BRAF gene in human cancer. Nature 417:949-954. Davignon J, Gregg RE and Sing CF. (1988) Apolipoprotein E polymorphism and atherosclerosis. Arteriosclerosis 8:1-21. De Gruijl F, , Methods in Enzymology, Academic Press 2000, p. 359-366. De Gruijl FR, Van Der Meer JB and Van Der Leun JC. (1983) Dose-time dependency of tumor formation by chronic UV exposure. Photochem Photobiol 37:53-62. Demunter A, Stas M, Degreef H, De Wolf-Peeters C and van den Oord JJ. (2001) Analysis of N- and K-ras mutations in the distinctive tumor progression phases of melanoma. J Invest Dermatol 117:14831489. Der CJ, Finkel T and Cooper GM. (1986) Biological and biochemical properties of human rasH genes mutated at codon 61. Cell 44:167-176. Diffey BJ. (1999) Photodermatology, Arnold, London. Diffey BL, Tate TJ and Davis A. (1979) Solar dosimetry of the face: the relationship of natural ultraviolet radiation exposure to basal cell carcinoma localisation. Phys Med Biol 24:931-939. Drmanac R, Labat I, Brukner I and Crkvenjakov R. (1989) Sequencing of megabase plus DNA by hybridization: theory of the method. Genomics 4:114-128. Drmanac S, Kita D, Labat I, Hauser B, Schmidt C, Burczak JD and Drmanac R. (1998) Accurate sequencing by hybridization for DNA diagnostics and individual genomics. Nat Biotechnol 16:54-58. Drobetsky EA, Grosovsky AJ and Glickman BW. (1987) The specificity of UV-induced mutations at an endogenous locus in mammalian cells. Proc Natl Acad Sci U S A 84:9103-9107. Eckert KA and Kunkel TA. (1990) High fidelity DNA synthesis by the Thermus aquaticus DNA polymerase. Nucleic Acids Res 18:3739-3744. Edstrom DW, Porwit A and Ros AM. (2001) Effects on human skin of repetitive ultraviolet-A1 (UVA1) irradiation and visible light. Photodermatol Photoimmunol Photomed 17:66-70. el-Deiry WS, Kern SE, Pietenpol JA, Kinzler KW and Vogelstein B. (1992) Definition of a consensus binding site for p53. Nat Genet 1:45-49. Ellis LA, Taylor CF and Taylor GR. (2000) A comparison of fluorescent SSCP and denaturing HPLC for high throughput mutation scanning. Hum Mutat 15:556-564.

53

Detection and analysis of genetic alterations in normal skin and skin tumours Ellis LA, Taylor GR, Banks R and Baumberg S. (1994) MutS binding protects heteroduplex DNA from exonuclease digestion in vitro: a simple method for detecting mutations. Nucleic Acids Res 22:27102711. Ellison J, Dean M and Goldman D. (1993) Efficacy of fluorescence-based PCR-SSCP for detection of point mutations. Biotechniques 15:684-691. Ellison JS. (1996) Fluorescence-based mutation detection. Single-strand conformation polymorphism analysis (F-SSCP). Mol Biotechnol 5:17-31. Emmert-Buck MR, Bonner RF, Smith PD, Chuaqui RF, Zhuang Z, Goldstein SR, Weiss RA and Liotta LA. (1996) Laser capture microdissection. Science 274:998-1001. Emri G, Wenczl E, Van Erp P, Jans J, Roza L, Horkay I and Schothorst AA. (2000) Low doses of UVB or UVA induce chromosomal aberrations in cultured human skin cells. J Invest Dermatol 115:435-440. Espinosa JM and Emerson BM. (2001) Transcriptional regulation by p53 through intrinsic DNA/chromatin binding and site-directed cofactor recruitment. Mol Cell 8:57-69. Evans WE and Relling MV. (1999) Pharmacogenomics: translating functional genomics into rational therapeutics. Science 286:487-491. Fakhrai-Rad H, Pourmand N and Ronaghi M. (2002) Pyrosequencing: an accurate detection platform for single nucleotide polymorphisms. Hum Mutat 19:479-485. Fan E, Levin DB, Glickman BW and Logan DM. (1993) Limitations in the use of SSCP analysis. Mutat Res 288:85-92. Findlay I, Matthews P and Quirke P. (1998) Multiple genetic diagnoses from single cells using multiplex PCR: reliability and allele dropout. Prenat Diagn 18:1413-1421. Fire A and Xu SQ. (1995) Rolling replication of short DNA circles. Proc Natl Acad Sci U S A 92:46414645. Fischer SG and Lerman LS. (1980) Separation of random fragments of DNA according to properties of their sequences. Proc Natl Acad Sci U S A 77:4420-4424. Fisher SG and Lerman LS. (1983) DNA fragments differing by single base-pair substitutions are separated in denaturing gradient gels: correspondence with melting theory. Proc Natl Acad Sci USA 80:1579-1583. Fitzpatrick TB. (1986) Ultraviolet-induced pigmentary changes: benefits and hazards. Curr Probl Dermatol 15:25-38. Ford JM and Hanawalt PC. (1997) Expression of wild-type p53 is required for efficient global genomic nucleotide excision repair in UV-irradiated human fibroblasts. J Biol Chem 272:28073-28080. Franceschi S, Levi F, Randimbison L and La Vecchia C. (1996) Site distribution of different types of skin cancer: new aetiological clues. Int J Cancer 67:24-28. Fu DJ, Tang K, Braun A, Reuter D, Darnhofer-Demar B, Little DP, O'Donnell MJ, Cantor CR and Koster H. (1998) Sequencing exons 5 to 8 of the p53 gene by MALDI-TOF mass spectrometry. Nat Biotechnol 16:381-384. Fuchs SY, Adler V, Buschmann T, Wu X and Ronai Z. (1998) Mdm2 association with p53 targets its ubiquitination. Oncogene 17:2543-2547. Fuchs SY, Adler V, Pincus MR and Ronai Z. (1998) MEKK1/JNK signaling stabilizes and activates p53. Proc Natl Acad Sci U S A 95:10541-10546.

54

Å Sivertsson Gailani MR and Bale AE. (1997) Developmental genes and cancer: role of patched in basal cell carcinoma of the skin. J Natl Cancer Inst 89:1103-1109. Gailani MR, Stahle-Backdahl M, Leffell DJ, Glynn M, Zaphiropoulos PG, Pressman C, Unden AB, Dean M, Brash DE, Bale AE and Toftgard R. (1996) The role of the human homologue of Drosophila patched in sporadic basal cell carcinomas. Nat Genet 14:78-81. Ganguly A. (2002) An update on conformation sensitive gel electrophoresis. Hum Mutat 19:334-342. Ganguly A, Rock MJ and Prockop DJ. (1993) Conformation-sensitive gel electrophoresis for rapid detection of single-base differences in double-stranded PCR products and DNA fragments: evidence for solvent-induced bends in DNA heteroduplexes. Proc Natl Acad Sci U S A 90:10325-10329. Garcia CA, Ahmadian A, Gharizadeh B, Lundeberg J, Ronaghi M and Nyren P. (2000) Mutation detection by pyrosequencing: sequencing of exons 5-8 of the p53 tumor suppressor gene. Gene 253:249-257. Garvin AM, Holzgreve W and Hahn S. (1998) Highly accurate analysis of heterozygous loci bysingle cell PCR. Nucleic Acids Res 26:3468-3472. Gatti A, Li HH, Traugh JA and Liu X. (2000) Phosphorylation of human p53 on Thr-55. Biochemistry 39:9837-9842. Gerry NP, Witowski NE, Day J, Hammer RP, Barany G and Barany F. (1999) Universal DNA microarray method for multiplex detection of low abundance point mutations. J Mol Biol 292:251-262. Gharizadeh B, Kalantari M, Garcia CA, Johansson B and Nyren P. (2001) Typing of human papillomavirus by pyrosequencing. Lab Invest 81:673-679. Gharizadeh B, Nordstrom T, Ahmadian A, Ronaghi M and Nyren P. (2002) Long-read pyrosequencing using pure 2'-deoxyadenosine-5'-O'-(1- thiotriphosphate) Sp-isomer. Anal Biochem 301:82-90. Giannella C, Zito FA, Colonna F, Paradiso A, Marzullo F, Alaibac M and Schittulli F. (1997) Comparison of formalin, ethanol, and Histochoice fixation on the PCR amplification from paraffinembedded breast cancer tissue. Eur J Clin Chem Clin Biochem 35:633-635. Giesendorf BA, Vet JA, Tyagi S, Mensink EJ, Trijbels FJ and Blom HJ. (1998) Molecular beacons: a new approach for semiautomated mutation analysis. Clin Chem 44:482-486. Glass AG and Hoover RN. (1989) The emerging epidemic of melanoma and squamous cell skin cancer. Jama 262:2097-2100. Glavac D and Dean M. (1993) Optimization of the single-strand conformation polymorphism (SSCP) technique for detection of point mutations. Hum Mutat 2:404-414. Goldrick MM. (2001) RNase cleavage-based methods for mutation/SNP detection, past and present. Hum Mutat 18:190-204. Goldrick MM, Kimball GR, Liu Q, Martin LA, Sommer SS and Tseng JY. (1996) NIRCA: a rapid robust method for screening for unknown point mutations. BioTechniques 21:106-112. Gorlin RJ. (1995) Nevoid basal cell carcinoma syndrome. Dermatol Clin 13:113-125. Graeber TG, Peterson JF, Tsai M, Monica K, Fornace AJ, Jr. and Giaccia AJ. (1994) Hypoxia induces accumulation of p53 protein, but activation of a G1- phase checkpoint by low-oxygen conditions is independent of p53 status. Mol Cell Biol 14:6264-6277. Green A. (1992) Changing patterns in incidence of non-melanoma skin cancer. Epithelial Cell Biol 1:47-51.

55

Detection and analysis of genetic alterations in normal skin and skin tumours Green A, MacLennan R, Youl P and Martin N. (1993) Site distribution of cutaneous melanoma in Queensland. Int J Cancer 53:232-236. Griffin TJ, Hall JG, Prudent JR and Smith LM. (1999) Direct genetic analysis by matrix-assisted laser desorption/ionization mass spectrometry. Proc Natl Acad Sci U S A 96:6301-6306. Griffin TJ and Smith LM. (2000) Genetic identification by mass spectrometric analysis of singlenucleotide polymorphisms: ternary encoding of genotypes. Anal Chem 72:3298-3302. Griffin TJ and Smith LM. (2000) Single-nucleotide polymorphism analysis by MALDI-TOF mass spectrometry. Trends Biotechnol 18:77-84. Grossman PD, Bloch W, Brinson E, Chang CC, Eggerding FA, Fung S, Iovannisci DM, Woo S, WinnDeen ES and Iovannisci DA. (1994) High-density multiplex detection of nucleic acid sequences: oligonucleotide ligation assay and sequence-coded separation. Nucleic Acids Res 22:4527-4534. Gustafsson AC, Guo Z, Hu X, Ahmadian A, Brodin B, Nilsson A, Ponten J, Ponten F and Lundeberg J. (2001) HPV-related cancer susceptibility and p53 codon 72 polymorphism. Acta Derm Venereol 81:125-129. Gustafsson AC, Kijas JM, Alderborn A, Uhlen M, Andersson L and Lundeberg J. (2001) Screening and scanning of single nucleotide polymorphisms in the pig melanocortin 1 receptor gene (MC1R) by pyrosequencing. Anim Biotechnol 12:145-153. Hacia JG, Fan JB, Ryder O, Jin L, Edgemon K, Ghandour G, Mayer RA, Sun B, Hsie L, Robbins CM, Brody LC, Wang D, Lander ES, Lipshutz R, Fodor SP and Collins FS. (1999) Determination of ancestral alleles for human single-nucleotide polymorphisms using high-density oligonucleotide arrays. Nat Genet 22:164-167. Haff LA and Smirnov IP. (1997) Multiplex genotyping of PCR products with MassTag-labeled primers. Nucleic Acids Res 25:3749-3750. Haff LA and Smirnov IP. (1997) Single-nucleotide polymorphism identification assays using a thermostable DNA polymerase and delayed extraction MALDI-TOF mass spectrometry. Genome Res 7:378-388. Hall PA, Meek D and Lane DP. (1996) p53--integrating the complexity. J Pathol 180:1-5. Hanahan D and Weinberg RA. (2000) The hallmarks of cancer. Cell 100:57-70. Hansen LL, Justesen J and Kruse TA. (1996) Sensitive and fast mutation detection by solid phase chemical cleavage. Hum Mutat 7:256-263. Harvey M, Vogel H, Morris D, Bradley A, Bernstein A and Donehower LA. (1995) A mutant p53 transgene accelerates tumour development in heterozygous but not nullizygous p53-deficient mice. Nat Genet 9:305-311. Hauser J, Seidman MM, Sidur K and Dixon K. (1986) Sequence specificity of point mutations induced during passage of a UV- irradiated shuttle vector plasmid in monkey cells. Mol Cell Biol 6:277-285. Hawkins GA and Hoffman LM. (1997) Base excision sequence scanning. Nat Biotechnol 15:803-804. Hawkins GA and Hoffman LM. (1999) Rapid DNA mutation identification and fingerprinting using base excision sequence scanning. Electrophoresis 20:1171-1176. [pii]. Hayashi K. (1991) PCR-SSCP: a simple and sensitive method for detection of mutations in the genomic DNA. PCR Methods Appl 1:34-38. Hayashi K and Yandell DW. (1993) How sensitive is PCR-SSCP? Hum Mutat 2:338-346.

56

Å Sivertsson Healy E, Sikkink S and Rees JL. (1996) Infrequent mutation of p16INK4 in sporadic melanoma. J Invest Dermatol 107:318-321. Herbst RA, Gutzmer R, Matiaske F, Mommert S, Kapp A, Weiss J, Arden KC and Cavenee WK. (1997) Further evidence for ultraviolet light induction of CDKN2 (p16INK4) mutations in sporadic melanoma in vivo. J Invest Dermatol 108:950. Higashimoto Y, Saito S, Tong XH, Hong A, Sakaguchi K, Appella E and Anderson CW. (2000) Human p53 is phosphorylated on serines 6 and 9 in response to DNA damage-inducing agents. J Biol Chem 275:23199-23203. Highsmith WE, Jr., Nataraj AJ, Jin Q, O'Connor JM, El-Nabi SH, Kusukawa N and Garner MM. (1999) Use of DNA toolbox for the characterization of mutation scanning methods. II: evaluation of single-strand conformation polymorphism analysis. Electrophoresis 20:1195-1203. [pii]. Hiort O, Wodtke A, Struve D, Zollner A and Sinnecker GH. (1994) Detection of point mutations in the androgen receptor gene using non- isotopic single strand conformation polymorphism analysis. German Collaborative Intersex Study Group. Hum Mol Genet 3:1163-1166. Hirao A, Kong YY, Matsuoka S, Wakeham A, Ruland J, Yoshida H, Liu D, Elledge SJ and Mak TW. (2000) DNA damage-induced activation of p53 by the checkpoint kinase Chk2. Science 287:18241827. Hongyo T, Buzard GS, Calvert RJ and Weghorst CM. (1993) 'Cold SSCP': a simple, rapid and nonradioactive method for optimized single-strand conformation polymorphism analyses. Nucleic Acids Res 21:3637-3642. Horiguchi M, Masumura KI, Ikehata H, Ono T, Kanke Y and Nohmi T. (2001) Molecular nature of ultraviolet B light-induced deletions in the murine epidermis. Cancer Res 61:3913-3918. Hoshino S, Kimura A, Fukuda Y, Dohi K and Sasazuki T. (1992) Polymerase chain reaction--singlestrand conformation polymorphism analysis of polymorphism in DPA1 and DPB1 genes: a simple, economical, and rapid method for histocompatibility testing. Hum Immunol 33:98-107. Hovig, Eivind, Smith-Sorensen B, Brogger A and Borrensen AL. (1991) Constant denaturant gel electrophoresis, a modification of denaturing gradient gel electrophoresis, in mutation detection. Mutat Res 263:61. Hsu IC, Yang Q, Kahng MW and Xu JF. (1994) Detection of DNA point mutations with DNA mismatch repair enzymes. Carcinogenesis 15:1657-1662. Huang LC, Clarkin KC and Wahl GM. (1996) Sensitivity and selectivity of the DNA damage sensor responsible for activating p53-dependent G1 arrest. Proc Natl Acad Sci U S A 93:4827-4832. Huber CG, Premstaller A, Xiao W, Oberacher H, Bonn GK and Oefner PJ. (2001) Mutation detection by capillary denaturing high-performance liquid chromatography using monolithic columns. J Biochem Biophys Methods 47:5-19. Humphries SE, Gudnason V, Whittall R and Day IN. (1997) Single-strand conformation polymorphism analysis with high throughput modifications, and its use in mutation detection in familial hypercholesterolemia. Clin Chem 43:427-435. Hupp TR, Meek DW, Midgley CA and Lane DP. (1992) Regulation of the specific DNA binding function of p53. Cell 71:875-886. Hyde J. (1906) On the influence of light in the production of cancer of the skin. Am J Med Sci 131: Hyman ED. (1988) A new method of sequencing DNA. Anal Biochem 174:423-436.

57

Detection and analysis of genetic alterations in normal skin and skin tumours Iacopetta B and Hamelin R. (1998) Rapid and nonisotopic SSCP-based analysis of the BAT-26 mononucleotide repeat for identification of the replication error phenotype in human cancers. Hum Mutat 12:355-360. Imayama S, Yashima Y, Higuchi R and Urabe H. (1987) A new concept of basal cell epitheliomas based on the three-dimensional growth pattern of the superficial multicentric type. Am J Pathol 128:497-504. Innis MA, Myambo KB, Gelfand DH and Brow MA. (1988) DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA. Proc Natl Acad Sci U S A 85:9436-9440. Iwahana H, Fujimura M, Takahashi Y, Iwabuchi T, Yoshimoto K and Itakura M. (1996) Multiple fluorescence-based PCR-SSCP analysis using internal fluorescent labeling of PCR products. Biotechniques 21:510-514, 516-519. Iwahana H, Yoshimoto K, Mizusawa N, Kudo E and Itakura M. (1994) Multiple fluorescence-based PCR-SSCP analysis. Biotechniques 16:296-297, 300-295. Jackson-Grusby L, Beard C, Possemato R, Tudor M, Fambrough D, Csankovszki G, Dausman J, Lee P, Wilson C, Lander E and Jaenisch R. (2001) Loss of genomic methylation causes p53-dependent apoptosis and epigenetic deregulation. Nat Genet 27:31-39. Jenkins GJ, Morgan C, Baxter JN, Parry EM and Parry JM. (2001) The detection of mutations induced in vitro in the human p53 gene by hydrogen peroxide with the restriction site mutation (RSM) assay. Mutat Res 498:135-144. Jiricny J, Su SS, Wood SG and Modrich P. (1988) Mismatch-containing oligonucleotide duplexes bound by the E. coli mutS- encoded protein. Nucleic Acids Res 16:7843-7853. Jiveskog S, Ragnarsson-Olding B, Platz A and Ringborg U. (1998) N-ras mutations are common in melanomas from sun-exposed skin of humans but rare in mucosal membranes or unexposed skin. J Invest Dermatol 111:757-761. Jonason AS, Kunala S, Price GJ, Restifo RJ, Spinelli HM, Persing JA, Leffell DJ, Tarone RE and Brash DE. (1996) Frequent clones of p53-mutated keratinocytes in normal human skin. Proc Natl Acad Sci USA 93:14025-14029. Jones AC, Austin J, Hansen N, Hoogendoorn B, Oefner PJ, Cheadle JP and O'Donovan MC. (1999) Optimal temperature selection for mutation detection by denaturing HPLC and comparison to singlestranded conformation polymorphism and heteroduplex analysis. Clin Chem 45:1133-1140. Jorde LB, Watkins WS, Bamshad MJ, Dixon ME, Ricker CE, Seielstad MT and Batzer MA. (2000) The distribution of human genetic diversity: a comparison of mitochondrial, autosomal, and Ychromosome data. Am J Hum Genet 66:979-988. Karas M and Hillenkamp F. (1988) Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal Chem 60:2299-2301. Kastan MB, Onyekwere O, Sidransky D, Vogelstein B and Craig RW. (1991) Participation of p53 protein in the cellular response to DNA damage. Cancer Res 51:6304-6311. Kawanishi S, Hiraku Y and Oikawa S. (2001) Mechanism of guanine-specific DNA damage by oxidative stress and its role in carcinogenesis and aging. Mutat Res 488:65-76. Keen J, Lester D, Inglehearn C, Curtis A and Bhattacharya S. (1991) Rapid detection of single base mismatches as heteroduplexes on Hydrolink gels. Trends Genet 7:5. Kelfkens G, de Gruijl FR and van der Leun JC. (1991) Tumorigenesis by short-wave ultraviolet A: papillomas versus squamous cell carcinomas. Carcinogenesis 12:1377-1382.

58

Å Sivertsson

Kielbassa C and Epe B. (2000) DNA damage induced by ultraviolet and visible light and its wavelength dependence. Methods Enzymol 319:436-445. Klenow H and Henningsen I. (1970) Selective elimination of the exonuclease activity of the deoxyribonucleic acid polymerase from Escherichia coli B by limited proteolysis. Proc Natl Acad Sci U S A 65:168-175. Kourkine IV, Hestekin CN, Buchholz BA and Barron AE. (2002) High-throughput, high-sensitivity genetic mutation detection by tandem single-strand conformation polymorphism /heteroduplex analysis capillary array electrophoresis. Anal Chem 74:2565-2572. Kozlowski P and Krzyzosiak WJ. (2001) Combined SSCP/duplex analysis by capillary electrophoresis for more efficient mutation detection. Nucleic Acids Res 29:E71. Kraemer KH, Greene MH, Tarone R, Elder DE, Clark WH, Jr. and Guerry Dt. (1983) Dysplastic naevi and cutaneous melanoma risk. Lancet 2:1076-1077. Kricker A, Armstrong BK, English DR and Heenan PJ. (1995) Does intermittent sun exposure cause basal cell carcinoma? a case- control study in Western Australia. Int J Cancer 60:489-494. Kricker A, Armstrong BK, English DR and Heenan PJ. (1995) A dose-response curve for sun exposure and basal cell carcinoma. Int J Cancer 60:482-488. Kristensen T, Voss H, Schwager C, Stegemann J, Sproat B and Ansorge W. (1988) T7 DNA polymerase in automated dideoxy sequencing. Nucleic Acids Res 16:3487-3496. Kruger K, Blume-Peytavi U and Orfanos CE. (1999) Basal cell carcinoma possibly originates from the outer root sheath and/or the bulge region of the vellus hair follicle. Arch Dermatol Res 291:253-259. Kukita Y, Tahira T, Sommer SS and Hayashi K. (1997) SSCP analysis of long DNA fragments in low pH gel. Hum Mutat 10:400-407. Kvam E and Tyrrell RM. (1997) Induction of oxidative DNA base damage in human skin cells by UV and near visible radiation. Carcinogenesis 18:2379-2384. Lambrinakos A, Humphrey KE, Babon JJ, Ellis TP and Cotton RG. (1999) Reactivity of potassium permanganate and tetraethylammonium chloride with mismatched bases and a simple mutation detection protocol. Nucleic Acids Res 27:1866-1874. Landegren U, Kaiser R, Sanders J and Hood L. (1988) A ligase-mediated gene detection technique. Science 241:1077-1080. Lane DP. (1992) Cancer. p53, guardian of the genome. Nature 358:15-16. Law JC, Facher EA and Deka A. (1996) Nonradioactive single-strand conformation polymorphism analysis with application for mutation detection in a mixed population of cells. Anal Biochem 236:373375. Levine AJ. (1997) p53, the cellular gatekeeper for growth and division. Cell 88:323-331. Ley RD. (1997) Ultraviolet radiation A-induced precursors of cutaneous melanoma in Monodelphis domestica. Cancer Res 57:3682-3684. Li J, Butler JM, Tan Y, Lin H, Royer S, Ohler L, Shaler TA, Hunter JM, Pollart DJ, Monforte JA and Becker CH. (1999) Single nucleotide polymorphism determination using primer extension and time-offlight mass spectrometry. Electrophoresis 20:1258-1265. [pii]. Lindroos K, Liljedahl U, Raitio M and Syvanen AC. (2001) Minisequencing on oligonucleotide microarrays: comparison of immobilisation chemistries. Nucleic Acids Res 29:E69-69.

59

Detection and analysis of genetic alterations in normal skin and skin tumours

Linke SP, Clarkin KC, Di Leonardo A, Tsou A and Wahl GM. (1996) A reversible, p53-dependent G0/G1 cell cycle arrest induced by ribonucleotide depletion in the absence of detectable DNA damage. Genes Dev 10:934-947. Lishanski A, Ostrander EA and Rine J. (1994) Mutation detection by mismatch binding protein, MutS, in amplified DNA: application to the cystic fibrosis gene. Proc Natl Acad Sci U S A 91:2674-2678. Little DP, Braun A, Darnhofer-Demar B, Frilling A, Li Y, McIver RT, Jr. and Koster H. (1997) Detection of RET proto-oncogene codon 634 mutations using mass spectrometry. J Mol Med 75:745750. Little DP, Braun A, O'Donnell MJ and Koster H. (1997) Mass spectrometry from miniaturized arrays for full comparative DNA analysis. Nat Med 3:1413-1416. Liu Q, Guntuku S, Cui XS, Matsuoka S, Cortez D, Tamai K, Luo G, Carattini-Rivera S, DeMayo F, Bradley A, Donehower LA and Elledge SJ. (2000) Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint. Genes Dev 14:1448-1459. Liu W, Smith DI, Rechtzigel KJ, Thibodeau SN and James CD. (1998) Denaturing high performance liquid chromatography (DHPLC) used in the detection of germline and somatic mutations. Nucleic Acids Res 26:1396-1400. Livak KJ. (1999) Allelic discrimination using fluorogenic probes and the 5' nuclease assay. Genet Anal 14:143-149. Livak KJ, Flood SJ, Marmaro J, Giusti W and Deetz K. (1995) Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl 4:357-362. Lizardi PM, Huang X, Zhu Z, Bray-Ward P, Thomas DC and Ward DC. (1998) Mutation detection and single-molecule counting using isothermal rolling-circle amplification. Nat Genet 19:225-232. Lowe SW, Bodis S, McClatchey A, Remington L, Ruley HE, Fisher DE, Housman DE and Jacks T. (1994) p53 status and the efficacy of cancer therapy in vivo. Science 266:807-810. Lu AL and Hsu IC. (1992) Detection of single DNA base mutations with mismatch repair enzymes. Genomics 14:249-255. Lysov Iu P, Florent'ev VL, Khorlin AA, Khrapko KR and Shik VV. (1988) [Determination of the nucleotide sequence of DNA using hybridization with oligonucleotides. A new method]. Dokl Akad Nauk SSSR 303:1508-1511. Makino R, Yazyu H, Kishimoto Y, Sekiya T and Hayashi K. (1992) F-SSCP: fluorescence-based polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis. PCR Methods Appl 2:10-13. Makridakis NM and Reichardt JK. (2001) Multiplex automated primer extension analysis: simultaneous genotyping of several polymorphisms. Biotechniques 31:1374-1380. Mao B, Margulis LA, Li B, Ibanez V, Lee H, Harvey RG and Geacintov NE. (1992) Direct synthesis and identification of benzo[a]pyrene diol epoxide- deoxyguanosine binding sites in modified oligodeoxynucleotides. Chem Res Toxicol 5:773-778. Mao B, Xu J, Li B, Margulis LA, Smirnov S, Ya NQ, Courtney SH and Geacintov NE. (1995) Synthesis and characterization of covalent adducts derived from the binding of benzo[a]pyrene diol expoxide to a -GGG- sequence in a deoxyoligonucleotide. Carcinogenesis 16:357-365. Marks R, Foley P, Goodman G, Hage BH and Selwood TS. (1986) Spontaneous remission of solar keratoses: the case for conservative management. Br J Dermatol 115:649-655.

60

Å Sivertsson

Martincic D and Whitlock JA. (1996) Improved detection of p53 point mutations by dideoxyfingerprinting (ddF). Oncogene 13:2039-2044. Martincic D and Whitlock JA. (1996) Improved detection of p53 point mutations by dideoxyfingerprinting (ddF). Oncogene 13:2039-2044. Mashal RD, Koontz J and Sklar J. (1995) Detection of mutations by cleavage of DNA heteroduplexes with bacteriophage resolvases. Nat Genet 9:177-183. Matsui MS and DeLeo VA. (1991) Longwave ultraviolet radiation and promotion of skin cancer. Cancer Cells 3:8-12. Matsumoto H, Takahashi A, Wang X, Ohnishi K and Ohnishi T. (1997) Transfection of p53-knockout mouse fibroblasts with wild-type p53 increases the thermosensitivity and stimulates apoptosis induced by heat stress. Int J Radiat Oncol Biol Phys 39:197-203. Maxam AM and Gilbert W. (1977) A new method for sequencing DNA. Proc Natl Acad Sci U S A 74:560-564. McKay BC, Ljungman M and Rainbow AJ. (1999) Potential roles for p53 in nucleotide excision repair. Carcinogenesis 20:1389-1396. Meek DW, Simon S, Kikkawa U and Eckhart W. (1990) The p53 tumour suppressor protein is phosphorylated at serine 389 by casein kinase II. Embo J 9:3253-3260. Melamede RJ, , US Patent No. 1985. Metzker ML, Raghavachari R, Richards S, Jacutin SE, Civitello A, Burgess K and Gibbs RA. (1994) Termination of DNA synthesis by novel 3'-modified-deoxyribonucleoside 5'-triphosphates. Nucleic Acids Res 22:4259-4267. Miller DL and Weinstock MA. (1994) Nonmelanoma skin cancer in the United States: incidence. J Am Acad Dermatol 30:774-778. Miller SJ, Sun TT and Lavker RM. (1993) Hair follicles, stem cells, and skin cancer. J Invest Dermatol 100:288S-294S. Miyashita T and Reed JC. (1995) Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell 80:293-299. Monforte JA and Becker CH. (1997) High-throughput DNA analysis by time-of-flight mass spectrometry. Nat Med 3:360-362. Monstein H, Nikpour-Badr S and Jonasson J. (2001) Rapid molecular identification and subtyping of Helicobacter pylori by pyrosequencing of the 16S rDNA variable V1 and V3 regions. FEMS Microbiol Lett 199:103-107. Moore L, Godfrey T, Eng C, Smith A, Ho R and Waldman FM. (2000) Validation of fluorescent SSCP analysis for sensitive detection of p53 mutations. Biotechniques 28:986-992. Murray V. (1989) Improved double-stranded DNA sequencing using the linear polymerase chain reaction. Nucleic Acids Res 17:8889. Myers RM, Fischer SG, Lerman LS and Maniatis T. (1985) Nearly all single base substitutions in DNA fragments joined to a GC- clamp can be detected by denaturing gradient gel electrophoresis. Nucleic Acids Res 13:3131-3145.

61

Detection and analysis of genetic alterations in normal skin and skin tumours Myers RM, Fischer SG, Maniatis T and Lerman LS. (1985) Modification of the melting properties of duplex DNA by attachment of a GC-rich DNA sequence as determined by denaturing gradient gel electrophoresis. Nucleic Acids Res 13:3111-3129. Myers RM, Larin Z and Maniatis T. (1985) Detection of single base substitutions by ribonuclease cleavage at mismatches in RNA:DNA duplexes. Science 230:1242-1246. Nagamine CM, Chan K and Lau YF. (1989) A PCR artifact: generation of heteroduplexes. Am J Hum Genet 45:337-339. Nakamura S, Roth JA and Mukhopadhyay T. (2000) Multiple lysine mutations in the C-terminal domain of p53 interfere with MDM2-dependent protein degradation and ubiquitination. Mol Cell Biol 20:9391-9398. Nelson WG and Kastan MB. (1994) DNA strand breaks: the DNA template alterations that trigger p53dependent DNA damage response pathways. Mol Cell Biol 14:1815-1823. Nicholls EM. (1973) Development and elimination of pigmented moles, and the anatomical distribution of primary malignant melanoma. Cancer 32:191-195. Nickerson DA, Kaiser R, Lappin S, Stewart J, Hood L and Landegren U. (1990) Automated DNA diagnostics using an ELISA-based oligonucleotide ligation assay. Proc Natl Acad Sci U S A 87:89238927. Nikiforov TT, Rendle RB, Goelet P, Rogers YH, Kotewicz ML, Anderson S, Trainor GL and Knapp MR. (1994) Genetic Bit Analysis: a solid phase method for typing single nucleotide polymorphisms. Nucleic Acids Res 22:4167-4175. Nilsson M, Krejci K, Koch J, Kwiatkowski M, Gustavsson P and Landegren U. (1997) Padlock probes reveal single-nucleotide differences, parent of origin and in situ distribution of centromeric sequences in human chromosomes 13 and 21. Nat Genet 16:252-255. Nilsson M, Malmgren H, Samiotaki M, Kwiatkowski M, Chowdhary BP and Landegren U. (1994) Padlock probes: circularizing oligonucleotides for localized DNA detection. Science 265:2085-2088. Nishimura EK, Jordan SA, Oshima H, Moriyama M, Jackson IJ, Barrandon Y, Miyachi Y and Nishikawa S. (2002) Dominant role of the niche in melanocyte stem-cell fate determination. Nature 416:854-860. Noguchi S, Motomura K, Inaji H, Imaoka S and Koyama H. (1993) Clonal analysis of fibroadenoma and phyllodes tumor of the breast. Cancer Res 53:4071-4074. Nordstrom T, Gharizadeh B, Pourmand N, Nyren P and Ronaghi M. (2001) Method enabling fast partial sequencing of cDNA clones. Anal Biochem 292:266-271. Nordstrom T, Ronaghi M, Forsberg L, de Faire U, Morgenstern R and Nyren P. (2000) Direct analysis of single-nucleotide polymorphism on double-stranded DNA by pyrosequencing. Biotechnol Appl Biochem 31:107-112. Nylén P, Bergqvist U, Fischer T, Glansholm A, Hansson J, Surakka J, Söderberg P and Wester U. (2002) Ultraviolett strålning och hälsa - ett kunskapsunderlag. Arbete och Hälsa Nyren P. (1987) Enzymatic method for continuous monitoring of DNA polymerase activity. Anal Biochem 167:235-238. Nyren P, Pettersson B and Uhlen M. (1993) Solid phase DNA minisequencing by an enzymatic luminometric inorganic pyrophosphate detection assay. Anal Biochem 208:171-175. O'Meara D, Ahmadian A, Odeberg J and Lundeberg J. (2002) SNP typing by apyrase-mediated allelespecific primer extension on DNA microarrays. Nucleic Acids Res 30:e75.

62

Å Sivertsson

O'Meara D, Wilbe K, Leitner T, Hejdeman B, Albert J and Lundeberg J. (2001) Monitoring resistance to human immunodeficiency virus type 1 protease inhibitors by pyrosequencing. J Clin Microbiol 39:464-473. Oefner PJ. (2000) Allelic discrimination by denaturing high-performance liquid chromatography. J Chromatogr B Biomed Sci Appl 739:345-355. Okayama H, Curiel DT, Brantly ML, Holmes MD and Crystal RG. (1989) Rapid, nonradioactive detection of mutations in the human genome by allele-specific amplification. J Lab Clin Med 114:105113. Omholt K, Karsberg S, Platz A, Ringborg U and Hansson J. (2002) Screening of N-ras codon 61 mutations in paired primary and metastatic cutaneous melanomas: mutations occur early and persist throughout tumor progression. Submitted Orita M, Iwahana H, Kanazawa K, Hayashi K and Sekiya T. (1989) Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc Natl Acad Sci USA 86:2766-2770. Ortonne JP. (2002) Photobiology and genetics of malignant melanoma. Br J Dermatol 146 Suppl 61:11-16. Orum H, Jakobsen MH, Koch T, Vuust J and Borre MB. (1999) Detection of the factor V Leiden mutation by direct allele-specific hybridization of PCR amplicons to photoimmobilized locked nucleic acids. Clin Chem 45:1898-1905. Ory K, Legros Y, Auguin C and Soussi T. (1994) Analysis of the most representative tumour-derived p53 mutants reveals that changes in protein conformation are not correlated with loss of transactivation or inhibition of cell proliferation. Embo J 13:3496-3504. Oshima H, Rochat A, Kedzia C, Kobayashi K and Barrandon Y. (2001) Morphogenesis and renewal of hair follicles from adult multipotent stem cells. Cell 104:233-245. Pastinen T, Kurg A, Metspalu A, Peltonen L and Syvänen AC. (1997) Minisequencing: a specific tool for DNA analysis and diagnostics on oligonucleotide arrays. Genome Res 7:606-614. Pastinen T, Partanen J and Syvanen AC. (1996) Multiplex, fluorescent, solid-phase minisequencing for efficient screening of DNA sequence variation. Clin Chem 42:1391-1397. Perry DJ and Carrell RW. (1992) Hydrolink gels: a rapid and simple approach to the detection of DNA mutations in thromboembolic disease. J Clin Pathol 45:158-160. Platz A, Ringborg U, Brahme EM and Lagerlof B. (1994) Melanoma metastases from patients with hereditary cutaneous malignant melanoma contain a high frequency of N-ras activating mutations. Melanoma Res 4:169-177. Polakis P and McCormick F. (1993) Structural requirements for the interaction of p21ras with GAP, exchange factors, and its biological effector target. J Biol Chem 268:9157-9160. Pollock PM, Yu F, Qiu L, Parsons PG and Hayward NK. (1995) Evidence for u.v. induction of CDKN2 mutations in melanoma cell lines. Oncogene 11:663-668. Polyak K, Xia Y, Zweier JL, Kinzler KW and Vogelstein B. (1997) A model for p53-induced apoptosis. Nature 389:300-305. Pontén F, Berg C, Ahmadian A, Ren ZP, Nistér M, Lundeberg J, Uhlén M and Pontén J. (1997) Molecular pathology in basal cell cancer with p53 as a genetic marker. Oncogene 15:1059-1067.

63

Detection and analysis of genetic alterations in normal skin and skin tumours Pontén F, Berne B, Ren ZP, Nistér M and Pontén J. (1995) Ultraviolet light induces expression of p53 and p21 in human skin: effect of sunscreen and constitutive p21 expression in skin appendages. J Invest Dermatol 105:402-406. Pontén F, Lindman H, Bostrom A, Berne B and Bergh J. (2001) Induction of p53 expression in skin by radiotherapy and UV radiation: a randomized study. J Natl Cancer Inst 93:128-133. Pontén F, Williams C, Ling G, Ahmadian A, Nistér M, Lundeberg J, Pontén J and Uhlén M. (1997) Genomic analysis of single cells from human basal cell cancer using laser-assisted capture microscopy. Mut Res 382:45-55. Potten CS. (1981) Cell replacement in epidermis (keratopoiesis) via discrete units of proliferation. Int Rev Cytol 69:271-318. Potten CS and Morris RJ. (1988) Epithelial stem cells in vivo. J Cell Sci Suppl 10:45-62. Prince JA, Feuk L, Howell WM, Jobs M, Emahazion T, Blennow K and Brookes AJ. (2001) Robust and accurate single nucleotide polymorphism genotyping by dynamic allele-specific hybridization (DASH): design criteria and assay validation. Genome Res 11:152-162. Prives C and Manley JL. (2001) Why is p53 acetylated? Cell 107:815-818. Prober JM, Trainor GL, Dam RJ, Hobbs FW, Robertson CW, Zagursky RJ, Cocuzza AJ, Jensen MA and Baumeister K. (1987) A system for rapid DNA sequencing with fluorescent chain-terminating dideoxynucleotides. Science 238:336-341. Qi X, Bakht S, Devos KM, Gale MD and Osbourn A. (2001) L-RCA (ligation-rolling circle amplification): a general method for genotyping of single nucleotide polymorphisms (SNPs). Nucleic Acids Res 29:E116. Ramus SJ and Cotton RG. (1996) Single-tube chemical cleavage of mismatch: successive treatment with hydroxylamine and osmium tetroxide. Biotechniques 21:216-218, 220. Ravnik-Glavac M, Glavac D, Chernick M, di Sant'Agnese P and Dean M. (1994) Screening for CF mutations in adult cystic fibrosis patients with a directed and optimized SSCP strategy. Hum Mutat 3:231-238. Ravnik-Glavac M, Glavac D and Dean M. (1994) Sensitivity of single-strand conformation polymorphism and heteroduplex method for mutation detection in the cystic fibrosis gene. Hum Mol Genet 3:801-807. Rebel H, Mosnier LO, Berg RJ, Westerman-de Vries A, van Steeg H, van Kranen HJ and de Gruijl FR. (2001) Early p53-positive foci as indicators of tumor risk in ultraviolet- exposed hairless mice: kinetics of induction, effects of DNA repair deficiency, and p53 heterozygosity. Cancer Res 61:977-983. Rechitsky S, Strom C, Verlinsky O, Amet T, Ivakhnenko V, Kukharenko V, Kuliev A and Verlinsky Y. (1998) Allele dropout in polar bodies and blastomeres. J Assist Reprod Genet 15:253-257. Reeve MA and Fuller CW. (1995) A novel thermostable polymerase for DNA sequencing. Nature 376:796-797. Ren ZP, Hedrum A, Pontén F, Nistér M, Ahmadian A, Lundeberg J, Uhlén M and Pontén J. (1996) Human epidermial cancer and accompanying precursors have identical p53 mutations different from p53 mutations in adjacent areas of clonally expanded non-neoplastic keratinocytes. Oncogene 12:765773. Ren ZP, Pontén F, Nistér M and Pontén J. (1996) Two distinct p53 immunohistochemical patterns in human squamous-cell skin cancer, precursors and normal epidermis. Int J Cancer 69:174-179.

64

Å Sivertsson Ririe KM, Rasmussen RP and Wittwer CT. (1997) Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem 245:154-160. Ronaghi M. (2001) Pyrosequencing sheds light on DNA sequencing. Genome Res 11:3-11. Ronaghi M, Karamohamed S, Pettersson B, Uhlen M and Nyren P. (1996) Real-time DNA sequencing using detection of pyrophosphate release. Anal Biochem 242:84-89. Ronaghi M, Uhlen M and Nyren P. (1998) A sequencing method based on real-time pyrophosphate. Science 281:363, 365. Ross P, Hall L, Smirnov I and Haff L. (1998) High level multiplex genotyping by MALDI-TOF mass spectrometry. Nat Biotechnol 16:1347-1351. Rosso S, Zanetti R, Martinez C, Tormo MJ, Schraub S, Sancho-Garnier H, Franceschi S, Gafa L, Perea E, Navarro C, Laurent R, Schrameck C, Talamini R, Tumino R and Wechsler J. (1996) The multicentre south European study 'Helios'. II: Different sun exposure patterns in the aetiology of basal cell and squamous cell carcinomas of the skin. Br J Cancer 73:1447-1454. Ruas M and Peters G. (1998) The p16INK4a/CDKN2A tumor suppressor and its relatives. Biochim Biophys Acta 1378:F115-177. Saiki RK, Chang CA, Levenson CH, Warren TC, Boehm CD, Kazazian HH, Jr. and Erlich HA. (1988) Diagnosis of sickle cell anemia and beta-thalassemia with enzymatically amplified DNA and nonradioactive allele-specific oligonucleotide probes. N Engl J Med 319:537-541. Saiki RK, Walsh PS, Levenson CH and Erlich HA. (1989) Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes. Proc Natl Acad Sci U S A 86:6230-6234. Samiotaki M, Kwiatkowski M, Parik J and Landegren U. (1994) Dual-color detection of DNA sequence variants by ligase-mediated analysis. Genomics 20:238-242. Sanger F, Nicklen S and Coulson AR. (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A 74:5463-5467. Sarkar G, Yoon HS and Sommer SS. (1992) Dideoxy fingerprinting (ddF): a rapid and efficient screen for the presence of mutations. Genomics 13:441-443. Schutze K and Clement-Sengewald A. (1994) Catch and move--cut or fuse. Nature 368:667-669. Schutze K and Lahr G. (1998) Identification of expressed genes by laser-mediated manipulation of single cells. Nat Biotechnol 16:737-742. Seite S, Moyal D, Verdier MP, Hourseau C and Fourtanier A. (2000) Accumulated p53 protein and UVA protection level of sunscreens. Photodermatol Photoimmunol Photomed 16:3-9. Sheffield VC, Beck JS, Kwitek AE, Sandstrom DW and Stone EM. (1993) The sensitivity of singlestrand conformation polymorphism analysis for the detection of single base substitutions. Genomics 16:325-332. Sheffield VC, Cox DR, Lerman LS and Myers RM. (1989) Attachment of a 40-base-pair G + C-rich sequence (GC-clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes. Proc Natl Acad Sci U S A 86:232-236. Shieh SY, Ikeda M, Taya Y and Prives C. (1997) DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2. Cell 91:325-334. Siegel J, Fritsche M, Mai S, Brandner G and Hess RD. (1995) Enhanced p53 activity and accumulation in response to DNA damage upon DNA transfection. Oncogene 11:1363-1370.

65

Detection and analysis of genetic alterations in normal skin and skin tumours Siliciano JD, Canman CE, Taya Y, Sakaguchi K, Appella E and Kastan MB. (1997) DNA damage induces phosphorylation of the amino terminus of p53. Genes Dev 11:3471-3481. Smit ML, Giesendorf BA, Vet JA, Trijbels FJ and Blom HJ. (2001) Semiautomated DNA mutation analysis using a robotic workstation and molecular beacons. Clin Chem 47:739-744. Smith J and Modrich P. (1996) Mutation detection with MutH, MutL, and MutS mismatch repair proteins. Proc Natl Acad Sci U S A 93:4374-4379. Smith LM. (1991) High-speed DNA sequencing by capillary gel electrophoresis. Nature 349:812-813. Smith LM, Sanders JZ, Kaiser RJ, Hughes P, Dodd C, Connell CR, Heiner C, Kent SB and Hood LE. (1986) Fluorescence detection in automated DNA sequence analysis. Nature 321:674-679. Soehnge H, Ouhtit A and Ananthaswamy ON. (1997) Mechanisms of induction of skin cancer by UV radiation. Front Biosci 2:D538-D551. Sommer SS, Groszbach AR and Bottema CD. (1992) PCR amplification of specific alleles (PASA) is a general method for rapidly detecting known single-base changes. BioTechniques 12:82-87. Stern RS and Nichols KT. (1996) Therapy with orally administered methoxsalen and ultraviolet A radiation during childhood increases the risk of basal cell carcinoma. The PUVA Follow-up Study. J Pediatr 129:915-917. Suarez-Quian CA, Goldstein SR, Pohida T, Smith PD, Peterson JI, Wellner E, Ghany M and Bonner RF. (1999) Laser capture microdissection of single cells from complex tissues. Biotechniques 26:328335. Sun X, Ding H, Hung K and Guo B. (2000) A new MALDI-TOF based mini-sequencing assay for genotyping of SNPS. Nucleic Acids Res 28:E68. Sweet RW, Yokoyama S, Kamata T, Feramisco JR, Rosenberg M and Gross M. (1984) The product of ras is a GTPase and the T24 oncogenic mutant is deficient in this activity. Nature 311:273-275. Swerdlow AJ, English JS, MacKie RM, O'Doherty CJ, Hunter JA, Clark J and Hole DJ. (1988) Fluorescent lights, ultraviolet lamps, and risk of cutaneous melanoma. Bmj 297:647-650. Syvänen AC, K. A-S, Harju L, Kontula K and Söderlund H. (1990) A primer-guided nucleotide incorporation assay in the genotyping of apolipoprotein E. Genomics 8:684-692. Tabata H, Nagano T, Ray AJ, Flanagan N, Birch-MacHin MA and Rees JL. (1999) Low frequency of genetic change in p53 immunopositive clones in human epidermis. J Invest Dermatol 113:972-976. Tabor S and Richardson CC. (1987) DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci U S A 84:4767-4771. Tabor S and Richardson CC. (1989) Effect of manganese ions on the incorporation of dideoxynucleotides by bacteriophage T7 DNA polymerase and Escherichia coli DNA polymerase I. Proc Natl Acad Sci U S A 86:4076-4080. Tabor S and Richardson CC. (1995) A single residue in DNA polymerases of the Escherichia coli DNA polymerase I family is critical for distinguishing between deoxy- and dideoxyribonucleotides. Proc Natl Acad Sci U S A 92:6339-6343. Tang K, Fu DJ, Julien D, Braun A, Cantor CR and Koster H. (1999) Chip-based genotyping by mass spectrometry. Proc Natl Acad Sci U S A 96:10016-10020. Taranenko NI, Allman SL, Golovlev VV, Taranenko NV, Isola NR and Chen CH. (1998) Sequencing DNA using mass spectrometry for ladder detection. Nucleic Acids Res 26:2488-2490.

66

Å Sivertsson Taylor G, Lehrer MS, Jensen PJ, Sun TT and Lavker RM. (2000) Involvement of follicular stem cells in forming not only the follicle but also the epidermis. Cell 102:451-461. Tormanen VT and Pfeifer GP. (1992) Mapping of UV photoproducts within ras proto-oncogenes in UV-irradiated cells: correlation with mutations in human skin cancer. Oncogene 7:1729-1736. Tornaletti S and Pfeifer GP. (1996) UV damage and repair mechanisms in mammalian cells. Bioessays 18:221-228. Tully G, Sullivan KM, Nixon P, Stones RE and Gill P. (1996) Rapid detection of mitochondrial sequence polymorphisms using multiplex solid-phase fluorescent minisequencing. Genomics 34:107113. Tyagi S and Kramer FR. (1996) Molecular beacons: probes that flourescens upon hybridization. Nature Biotechnology 14:303-308. Tyrrell RM. (1996) Activation of mammalian gene expression by the UV component of sunlight- -from models to reality. Bioessays 18:139-148. Unsal-Kacmaz K, Makhov AM, Griffith JD and Sancar A. (2002) Preferential binding of ATR protein to UV-damaged DNA. Proc Natl Acad Sci U S A 99:6673-6678. Urano Y, Asano T, Yoshimoto K, Iwahana H, Kubo Y, Kato S, Sasaki S, Takeuchi N, Uchida N and Nakanishi H. (1995) Frequent p53 accumulation in the chronically sun-exposed epidermis and clonal expansion of p53 mutant cells in the epidermis adjacent to basal cell carcinoma. J Invest Dermatol 104:928-932. van 't Veer LJ, Burgering BM, Versteeg R, Boot AJ, Ruiter DJ, Osanto S, Schrier PI and Bos JL. (1989) N-ras mutations in human cutaneous melanoma from sun-exposed body sites. Mol Cell Biol 9:3114-3116. van Scott EJ and Reinertson RP. (1961) the modulating influence of stromal environment on epithelial cells studied in human autotransplants. Journal of Investigative Dermatology 36:109-117. Verpy E, Biasotto M, Meo T and Tosi M. (1994) Efficient detection of point mutations on color-coded strands of target DNA. Proc Natl Acad Sci U S A 91:1873-1877. Vet JA, Majithia AR, Marras SA, Tyagi S, Dube S, Poiesz BJ and Kramer FR. (1999) Multiplex detection of four pathogenic retroviruses using molecular beacons. Proc Natl Acad Sci U S A 96:63946399. Vidal-Puig A and Moller DE. (1994) Comparative sensitivity of alternative single-strand conformation polymorphism (SSCP) methods. Biotechniques 17:490-492, 494, 496. Vogelstein B and Kinzler KW. (1993) The multistep nature of cancer. Trends Genet 9:138-141. Wagner T, Stoppa-Lyonnet D, Fleischmann E, Muhr D, Pages S, Sandberg T, Caux V, Moeslinger R, Langbauer G, Borg A and Oefner P. (1999) Denaturing high-performance liquid chromatography detects reliably BRCA1 and BRCA2 mutations. Genomics 62:369-376. Wallace RB, Shaffer J, Murphy RF, Bonner J, Hirose T and Itakura K. (1979) Hybridization of synthetic oligodeoxyribonucleotides to phi chi 174 DNA: the effect of single base pair mismatch. Nucleic Acids Res 6:3543-3557. Walter SD, Marrett LD, From L, Hertzman C, Shannon HS and Roy P. (1990) The association of cutaneous malignant melanoma with the use of sunbeds and sunlamps. Am J Epidemiol 131:232-243. Wang D, Kreutzer DA and Essigmann JM. (1998) Mutagenicity and repair of oxidative DNA damage: insights from studies using defined lesions. Mutat Res 400:99-115.

67

Detection and analysis of genetic alterations in normal skin and skin tumours Wang DG, Fan JB, Siao CJ, Berno A, Young P, Sapolsky R, Ghandour G, Perkins N, Winchester E, Spencer J, Kruglyak L, Stein L, Hsie L, Topaloglou T, Hubbell E, Robinson E, Mittmann M, Morris MS, Shen N, Kilburn D, Rioux J, Nusbaum C, Rozen S, Hudson TJ, Lander ES and et al. (1998) Large-scale identification, mapping, and genotyping of single- nucleotide polymorphisms in the human genome. Science 280:1077-1082. Wartell RM, Hosseini SH and Moran CPJ. (1990) Detecting base pair substitutions in DNA fragments by temperature-gradient gel electrophoresis. Nucleic Acids Res 18:2699-2705. Wasson J, Skolnick G, Love-Gregory L and Permutt MA. (2002) Assessing allele frequencies of single nucleotide polymorphisms in DNA pools by pyrosequencing technology. Biotechniques 32:1144-1146, 1148, 1150 passim. Weghorst CM and Buzard GS. (1993) Enhanced single-strand conformation polymorphism (SSCP) detection of point mutations utilizing methylmercury hydroxide. Biotechniques 15:396-398, 400. Weitzman JB and Yaniv M. (1999) Rebuilding the road to cancer. Nature 400:401-402. Westerdahl J, Olsson H, Masback A, Ingvar C and Jonsson N. (1995) Is the use of sunscreens a risk factor for malignant melanoma? Melanoma Res 5:59-65. Westerdahl J, Olsson H, Masback A, Ingvar C, Jonsson N, Brandt L, Jonsson PE and Moller T. (1994) Use of sunbeds or sunlamps and malignant melanoma in southern Sweden. Am J Epidemiol 140:691699. Wilkins RJ. (1985) Site-specific analysis of drug interactions and damage in DNA using sequencing techniques. Anal Biochem 147:267-279. Williams C, Ponten F, Moberg C, Soderkvist P, Uhlen M, Ponten J, Sitbon G and Lundeberg J. (1999) A high frequency of sequence alterations is due to formalin fixation of archival specimens. Am J Pathol 155:1467-1471. Wolf P, Quehenberger F, Mullegger R, Stranz B and Kerl H. (1998) Phenotypic markers, sunlightrelated factors and sunscreen use in patients with cutaneous melanoma: an Austrian case-control study. Melanoma Res 8:370-378. Wu DY, Ugozzoli L, Pal BK and Wallace RB. (1989) Allele-specific enzymatic amplification of betaglobin genomic DNA for diagnosis of sickle cell anemia. Proc Natl Acad Sci U S A 86:2757-2760. Wu KJ, Shaler TA and Becker CH. (1994) Time-of-flight mass spectrometry of underivatized singlestranded DNA oligomers by matrix-assisted laser desorption. Anal Chem 66:1637-1645. Xiao W, Stern D, Jain M, Huber CG and Oefner PJ. (2001) Multiplex capillary denaturing highperformance liquid chromatography with laser-induced fluorescence detection. Biotechniques 30:13321338. Xie T, Ho SL and Ma OC. (1997) High resolution single strand conformation polymorphism analysis using formamide and ethidium bromide staining. Mol Pathol 50:276-278. Yagi N, Satonaka K, Horio M, Shimogaki H, Tokuda Y and Maeda S. (1996) The role of DNase and EDTA on DNA degradation in formaldehyde fixed tissues. Biotech Histochem 71:123-129. Youil R, Kemper B and Cotton RG. (1996) Detection of 81 of 81 known mouse beta-globin promoter mutations with T4 endonuclease VII--the EMC method. Genomics 32:431-435. Youil R, Kemper BW and Cotton RG. (1995) Screening for mutations by enzyme mismatch cleavage with T4 endonuclease VII. Proc Natl Acad Sci U S A 92:87-91.

68

Å Sivertsson Young AR, Potten CS, Nikaido O, Parsons PG, Boenders J, Ramsden JM and Chadwick CA. (1998) Human melanocytes and keratinocytes exposed to UVB or UVA in vivo show comparable levels of thymine dimers. J Invest Dermatol 111:936-940. Zanetti R, Rosso S, Martinez C, Navarro C, Schraub S, Sancho-Garnier H, Franceschi S, Gafa L, Perea E, Tormo MJ, Laurent R, Schrameck C, Cristofolini M, Tumino R and Wechsler J. (1996) The multicentre south European study 'Helios'. I: Skin characteristics and sunburns in basal cell and squamous cell carcinomas of the skin. Br J Cancer 73:1440-1446. Zhang W, Remenyik E, Zelterman D, Brash DE and Wikonkal NM. (2001) Escaping the stem cell compartment: sustained UVB exposure allows p53- mutant keratinocytes to colonize adjacent epidermal proliferating units without incurring additional mutations. Proc Natl Acad Sci U S A 98:13948-13953. Ziegler A, Jonason AS, Leffell DJ, Simon JA, Sharma HW, Kimmelman J, Remington L, Jacks T and Brash DE. (1994) Sunburn and p53 in the onset of skin cancer. Nature 372:773-776.

69