Stem Cell Factor 9789155452919, 9155452914

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Stem Cell Factor Jonathan Roy Keller1,* and Diana Marie Linnekin2 1

Intramural Research and Support Program-SAIC, Frederick Cancer Research and Development Center-NCI, Building 560, Room 12-03, PO Box B, Frederick, MD 21702-1201, USA 2 Basic Research Laboratory-DBS, Frederick Cancer Research and Development Center-NCI, Building 567, PO Box B, Frederick, MD 21702-1201, USA * corresponding author tel: 301-846-1461, fax: 301-846-6646, e-mail: [email protected] DOI: 10.1006/rwcy.2000.09003.

SUMMARY Stem cell factor (SCF) and its receptor, c-Kit, are essential for hematopoietic, melanocyte, and germ cell development. In the hematopoietic compartment, SCF has been shown to act on cells at multiple stages of development, including effects on primitive stem cells and their more differentiated progeny. In particular, SCF can function to promote stem/progenitor cell survival, proliferation and differentiation (especially in combination with other growth factors), adhesion, activation, and migration/chemoattraction. While cKit is highly expressed on differentiated mast cells and SCF is a potent mast cell growth factor, c-Kit expression is downregulated during the maturation of all other hematopoietic lineages. SCF can exist in both soluble and membrane forms which may have distinct biological functions in vivo. Further understanding of this important hematopoietic regulator will provide important insights into the biology, biochemistry, and molecular biology of hematopoetic stem cells.

BACKGROUND

Discovery Definitive hematopoiesis is a complex cellular process that is regulated, in part, by hematopoietic growth factors (HGFs) and the bone marrow microenvironment, which act to promote the survival, proliferation, and differentiation of hematopoietic stem cells and their progeny (Spangrude, 1991; Ogawa, 1993; Metcalf, 1993). HGFs are produced by accessory or

stromal cells (macrophages, fibroblasts, endothelial cells, and adipocytes) in the bone marrow and elsewhere, which interact with specific receptors expressed on hematopoietic cells. The story of the discovery of one HGF/receptor pair, stem cell factor (SCF) and SCF receptor (SCFR), illustrates the importance of HGFs in hematopoietic development. This story began with studies of inbred mouse strains that have naturally occurring mutations affecting SCF or its receptor that were initiated 20 years ago. Specifically, homozygote offspring from W mouse strains (white spotting locus on chromosome 5), or Sl mouse strains (steel locus on chromosome 10) showed varying degrees of impaired hematopoietic development (hypoplastic with macrocytic anemia), alterations in coat color or pigmentation (lack of cutaneous melanocytes), and defects in gametogenesis (sterility) (Russell, 1979; Silvers, 1979). Bone marrow transplantation and embryo fusion studies using W or Sl mutant mouse strains led to the seminal observation that the W mutant defects were intrinsic to hematopoietic stem and mast cells, while the Sl mutant defects were extrinsic to hematopoietic cells in that bone marrow stromal cells were required to support hematopoietic development (Russell et al., 1959; Altus et al., 1971; McCulloch et al., 1964, 1965). Because both W and Sl mice showed defects in the same three cell lineages (hematopoietic, melanocyte, and germ cell) Elisabeth Russell speculated that the W locus might encode a growth factor receptor and the Sl locus its complementary ligand (Russell, 1979). This hypothesis was confirmed, in part, by the observation that the W locus encoded the protooncogene c-kit (SCFR), a transmembrane tyrosine kinase, which was largely deleted in one strain of W mice (W/W) mice and results in the most severe

878 Jonathan Roy Keller and Diana Marie Linnekin phenotype in homozygous offspring (prenatal lethality). W mouse strains that produce viable homozygous offspring do not show deleted SCFR, but rather have mutations in the kinase domain that affect the kinase enzymatic activity (Charbot et al., 1988; Geissler et al., 1988). SCFR is structurally related to other transmembrane tyrosine kinases, including c-fms, the receptor for macrophage colony-stimulating factor (M-CSFR), and platelet-derived growth factor receptor (PDGFR) (Qui et al., 1988). The search for the SCFR ligand ended several years later when three groups simultaneously purified it from medium conditioned by fibroblasts (stromal cells) or a rat liver-derived cell by assaying the proliferative response of cell lines or normal bone marrow cells to SCFR ligand (Huang et al., 1990; Zsebo et al., 1990a; Williams et al., 1990). The proteins were purified to homogeneity, sequenced and used to clone the corresponding cDNAs (Anderson et al., 1990; Copeland et al., 1990; Martin et al., 1990).

Figure 1 Soluble and transmembrane forms of SCF. The amino acid sequences encoded by exon 6 are shown in light blue, which contains the major proteolytic site (purple arrow) that results in the production of soluble SCF164. Soluble SCF164 forms nonconvalently linked dimers using the cysteine residues indicated. SCF220 lacks exon 6 and the primary proteolytic site and remains membrane bound. The signal peptide sequence is shown in purple and the transmembrane domain is shown as a green box. (Full colour figure can be viewed online.)

Alternative names SCF is also known as steel factor (SF) because it was mapped to the Sl locus, Kit ligand (KL), and mast cell growth factor (MCGF) (Anderson et al., 1990; Copeland et al., 1990; Huang et al., 1990; Martin et al., 1990; Williams et al., 1990; Zsebo et al., 1990b).

Structure SCF is produced in both soluble and transmembrane forms that are encoded by two distinct mRNAs generated from the same gene by alternate splicing of the primary RNA transcript (Flannagan et al., 1991; Anderson et al., 1990, 1991). Both isoforms of SCF are initially produced as transmembrane proteins that contain a 25 amino acid signal peptide sequence (SCF248 or SCF220). Transmembrane SCF248 can be proteolytically cleaved to give rise to soluble SCF, which contains the first 164 amino acids of SCF248 (Figure 1). The alternatively spliced isoform, SCF220, encodes a shorter 220 amino acid transmembrane protein identical to SCF248 except that it lacks amino acids 147±177, the region containing the proteolytic cleavage site. Little or no soluble SCF is produced from SCF220.

Main activities and pathophysiological roles SCF plays essential roles in the survival, growth, and differentiation of cells responsible for hematopoietic,

germ, and melanocyte cell development during both embryonic development and adult life (Bernstein et al., 1991b; Besmer et al., 1993). In particular, SCF is expressed along the migratory pathways for primordial germ cells and melanocytes as well as sites of active hematopoiesis (fetal liver). SCF is essential for the migration and homing of stem cells to their appropriate developmental sites; moreover, SCF directly promotes the survival of these stem cell populations and greatly enhances their proliferation in response to other growth factors. While SCF is critical for the development of a number of tissues, this review will only focus on hematopoietic cells and will refer to cells from other tissue where appropriate.

GENE AND GENE REGULATION

Accession numbers SCF cDNAs have been cloned and sequenced from a variety of species including mouse (GenBank: U44725) (Anderson et al., 1990; Huang et al., 1990; Zsebo et al., 1990b), human (GenBank: M59964), rat (GenBank: M59964) (Martin et al., 1990), pig (L07786) (Zhang and Anthony, 1994), chicken (D13516) (Zhou et al., 1993), quail (GenBank: U43079) (Pettite and Kulik, 1996), dog (S53329) (Shull et al., 1992), cat (Dunham and Onions, 1995) (DDBJ: D50833), and horse (GenBank: AF053498).

Stem Cell Factor

Chromosome location Mouse SCF has been mapped to chromosome 10 in a region between the peptidase 2 (Pep-2) and phenylhydroxylase (Pah) genes. Comparing the maps of mouse and human chromosomes indicated that the human SCF gene might be located on the distal end of the long arm of human chromosome 12 (Copeland et al., 1990; Huang et al., 1990; Zsebo et al., 1990b). This was later confirmed by in situ hybridization where it was shown that human SCF mapped to chromosome 12q22 (Anderson et al., 1991) and somatic cell hybrids which mapped SCF to 12q14.3-qter (Geissler et al., 1991). The mouse SCF gene spans roughly 50 kb and is composed of nine exons (Martin et al., 1990; Brannan et al., 1992). The locations of the intron and exon boundaries are highly conserved between species including human, rat, and mouse. The first exon of the mouse encodes 198 nucleotides of the 50 UTR and the first five amino acids of the signal peptide. Exons 2±6 encode the extracellular domain of the transmembrane-bound ligand, while exon 7 encodes a portion of the extracellular domain and 23 amino acids of the membrane-spanning region. Exons 8 and 9 encode 23 amino acids and 22 amino acids of the cytoplasmic domain, respectively. There are two major RNA transcripts for SCF detected by northern blot analysis that range in size from 5.5 kb to 6.5 kb for the larger message and from 3 kb to 4.6 kb for the smaller mRNA. The difference in the size of the mRNA transcripts is due to the length of the 30 UTR which is determined by the use of one of two polyadenylation sites (Anderson et al., 1990; Bedell et al., 1996). The largest SCF mRNA transcript predicted by sequence analysis is roughly 5.4 kb and contains 197 nucleotides of 50 UTR, an ORF of 818 nucleotides and a 30 UTR of 4432 nucleotides. The smaller mRNA differs in the length of the 30 UTR region which is 2732 nucleotides. There are 14 ATTA motifs in the 30 UTR of the SCF mRNA which have been shown to affect HGF mRNA stability and thereby could greatly affect protein levels (Shaw and Kamen, 1986; Bedell et al., 1996).

Regulatory sites and corresponding transcription factors A comparison of the 50 flanking sequences of mouse, rat, chicken, pig, and human SCF show a high degree of homology (82±94% identity) within a 110 nucleotide interval upstream of the initiator methionine that includes a GGCGGG motif representing the core

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binding motif for the transcription factors TFIID and SP1 (Bedell et al., 1996; Taylor et al., 1996; Jiang et al., 1997). The human SCF promoter contains a TATA box beginning at ÿ38 bp and a CCAAT box at ÿ71 bp (Figure 2). The major transcription initiation start site for SCF in mouse brain, lung, kidney, heart, ovary, and testes is located 28 nucleotides downstream from the consensus TATA motif in the murine promoter (Bedell et al., 1996). There are also three ATGs located at positions ‡88, ‡123, and ‡193, with the latter encoding the initiator methionine for SCF, however; initiation of translation from ‡88 or ‡123 would produce peptides of 37 and 7 amino acids in length respectively. Other important sites in the human promoter include SP1 sites at ÿ224, ÿ139, and ÿ50, AP-2 sites at ÿ202 and ÿ91, and AP-1 and cAMP-response elements further upstream. In comparison, the mouse promoter contains SP1 sites, a hepatocyte-acute phase factor 1 motif (H-APF-1), a negative regulatory element-box 1 (NRE-1), a reverse TPA responsive element (TGAGTA), TRE/Rev sites, NFB elements, as well as AP-2 and AP-1 sites (Taylor et al., 1996; Jiang et al., 1997). A functional analysis of the rat SCF promoter sequences performed by transfecting a reporter plasmid-containing promoter sequences into SCF-expressing and nonSCF-expressing cell lines showed that sequences from ÿ119 to ‡43 nucleotides contained the SCF core promoter activity (Jiang et al., 1997). Transfection of plasmids containing additional 50 sequence (up to ÿ1461 nucleotides) had no further effect on reporter gene expression. However, SCF core promoter activity could be greatly enhanced by treating cells with cAMP or forskolin (previously shown to increase SCF expression in Sertoli cells), suggesting that cAMPdependent regulatory elements might play a role in SCF gene activation. Human SCF promoter sequences were also cloned into luciferase reporter constructs and analyzed for function by deletion analysis. The shortest promoter construct that encompassed the core promoter activity activated by cAMP or forskolin contained sequences from ÿ167 to ‡109 nucleotides. In comparison, promoter constructs that contained additional 50 sequences (from ÿ853 or ÿ2185 nucleotides) showed the same increase in reporter activity in cells treated with cAMP; however, basal levels of reporter activity were significantly reduced, suggesting the potential for negative regulatory elements.

Cells and tissues that express the gene See section on Cellular sources that produce SCF.

880 Jonathan Roy Keller and Diana Marie Linnekin

Figure 2 Human SCF promoter DNA sequence. The DNA sequence of the upstream region of the SCF gene is shown, including the first five codons and corresponding amino acids of Exon 1 is underlined. DNA sequence elements that are homologous to transcription factor-binding elements are also underlined and indicated above the sequence. TATA and CAT box sequences are underlined and indicated above the sequence. The transcription start site is shown as a red arrow.

PROTEIN

Accession numbers SCF cDNAs isolated from a variety of species encode proteins of roughly 273 amino acids including mouse (SwissProt: P20826) (Anderson et al., 1990; Huang et al., 1990; Zsebo et al., 1990a), human (SwissProt: P21583) (Martin et al., 1990), and rat (SwissProt: P21581) (Martin et al., 1990), pig (SwissProt: Q29030) (Zhang and Anthony, 1994), chicken (SwissProt: Q09108) (Zhou et al., 1993), dog (SwissProt: Q06220) (Shull et al., 1992).

Sequence The amino acid sequences for SCF from human, mouse, pig, cat, and dog have been aligned and show a strong degree of conservation across species (Figure 3). The 25 amino acid signal peptide sequence

and the transmembrane region are shown in the boxed areas. SCF protein sequences that are conserved between species are shown in red shaded letters. Black shaded letters indicate conservative amino acid differences between species while all other amino acid differences are shown as nonshaded black letters. Mouse and human proteins show an overall amino acid identity of 82% with 92% identity in the cytoplasmic domain.

Description of protein SCF protein is produced in both soluble and membrane-bound forms (Anderson et al., 1990, 1991). These isoforms arise from two differentially spliced cDNAs. One cDNA encodes a 273 amino acid precursor that contains a cleavable 25 amino acid Nterminal signal peptide and gives rise to the 248 amino acid transmembrane protein SCF248 also known as KL-1 or SCF-1. SCF248 is comprised of a 189 amino

Stem Cell Factor

Figure 3 Alignment of SCF amino acid sequences. Human, murine, porcine, feline and canine SCF amino acid sequences were aligned. The signal peptide and transmembrane regions of SCF are shown in the boxed areas. Identical SCF amino acids between species are shown in red shaded letters. Black shaded letters indicate conservative amino acid changes between species, while black nonshaded letters indicate amino acid divergence between species. The four cysteines involved in the intrachain disulfide bonds are shown in shaded yellow letters. (Full colour figure can be viewed online.)

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882 Jonathan Roy Keller and Diana Marie Linnekin acid extracellular peptide domain, a 22 amino acid membrane-spanning region (from amino acids 190 to 212 of SCF248) and a short 36 amino acid intracellular cytoplasmic tail (amino acids 212±248) (see Figure 1). SCF248 can be proteolytically cleaved to give rise to soluble SCF, which contains the first 164 amino acids of SCF248. The alternatively spliced form of SCF cDNA encodes a shorter 220 amino acid transmembrane protein SCF220, also known as KL-2 or SCF-2, that is identical to SCF248 except that it lacks amino acids 147±177 (encoded by exon 6), which contains a proteolytic cleavage site important for the production of soluble SCF. This transcript arises from the use of an alternative 30 splice acceptor site in the RNA, which skips the 84 nucleotides contained within exon 6. A second proteolytic cleavage site in mouse SCF has been identified between amino acids 178±181 (12 amino acids in exon 7) that can be used in vitro to generate soluble SCF; however, whether or not this site is used in vivo is unknown (Huang et al., 1992; Toksoz et al., 1992; Majumdar et al., 1994). The soluble form of the SCF protein (164 amino acids) forms noncovalently linked homodimers that have extensive N- and O-linked glycosylation with an apparent molecular mass of 50±60 kDa. SCF dimers are required for SCF receptor dimerization, receptor transphosphorylation, activation, and signal transduction (Lev et al., 1992; Heldin, 1995; Hsu et al., 1997). In this regard, dimerization defective isoforms of SCF show greatly reduced biological activity including the ability to bind SCFR. The majority of soluble SCF exists as a monomer in serum because it is in low concentration; however, it is predicted that transmembrane SCF exists predominantly as dimers because of the enhanced probability for SCF monomers to interact with each other and form dimers in the membrane. The exact physiological role(s) of soluble and membrane-bound SCF are not fully understood. However, analysis of mouse strains with naturally occurring mutations in the SCF gene have provided some insights into the importance of these two isoforms (also described in the knockout and transgene sections below). In particular, a mutation of SCF in one mouse strain, steel-dickie (Sl d ), results in the production of soluble but not transmembrane SCF (Flannagan and Leder, 1990; Brannan et al., 1991). Homozygous Sl d mice are viable but they have complete lack of skin pigmentation, are sterile, show severe anemia, and are mast cell deficient. Thus, it is speculated that the membrane-associated form of SCF is critical for normal mouse development, suggesting that some of the important biological effects of SCF are mediated through juxtacrine stimulation of target cell populations (stimulation through

membrane-bound ligands). While it is not known whether the levels of soluble SCF are significantly reduced in Sl d homozygous mice compared with wildtype mice and might also contribute to the phenotype, administration of purified SCF to Sl d homozygous mice increases the hematocrit, and mast cell numbers at the site of injection, suggesting that a portion of the hematological effects in Sl d homozygous mice can be reversed by administration of soluble SCF (Zsebo et al., 1990b). Furthermore, studies comparing the expression of soluble and membrane-bound isoforms of SCF in vitro have shown that human hematopoietic cell growth can be maintained for longer periods on stromal cells that express transmembrane SCF (SCF220) in comparison with stromal cells that express SCF248 which is cleaved to produce soluble SCF (Toksoz et al., 1992). In addition, the survival and proliferation of factor-dependent hematopoietic cell lines are enhanced when they are co-cultured on stromal cells that express a noncleavable variant of transmembrane SCF (SCFx9/D3) versus SCF248 (Kapur et al., 1998). Thus, transmembrane SCF may be critical for the maintenance and survival of primitive hematopoietic progenitor cells and for mast cell accumulation and proliferation. Finally, the membrane-bound isoform of SCF can also function as an adhesion molecule for mast cells and hematopoietic progenitor cells (Kaneko et al., 1991; Adachi et al., 1992; Avraham et al., 1992; Long et al., 1992; Kodama et al., 1994).

Discussion of crystal structure The structure of SCF has not been directly determined; however, a model has been predicted based on the published structural model for M-CSF that was determined at 2.5 AÊ by X-ray crystallography (Bazan, 1991; Pandit et al., 1992) (Figure 4). There are four antiparallel (helical bundles (I, II, III, and IV) which are connected by two overhand loops. Thus far, all other examples of four  helix proteins have been identified as cytokines including GM-CSF, growth hormone, and IL-4 (Bazan, 1991). The four cysteine residues in SCF are proposed to be involved in intramolecular disulfide bonds, which contribute to the three-dimensional structure of the protein (Figure 4). The intramolecular disulfide pairs are Cys4±Cys89 and Cys43±Cys138, both of which are critical for full biological activity (Nishikawa et al., 1992; Langley et al., 1994; Jones et al., 1996). The same four Cys residues are conserved between other family members, including M-CSF and Flt-3 ligand (Flt-3L). However, M-CSF contains three additional Cys residues, two that are involved in another intramolecular

Stem Cell Factor

Figure 4 A molecular model of human SCF. This model is based on the published structure of human M-CSF (reprinted with permission from Broudy, 1997, copyright 1997, Blood). The SCF four helical bundles are shown (I, II, III, IV) and the location of the intramolecular bonds is indicated with yellow balls. (Full colour figure can be viewed online.)

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that share significant structural homologies as discussed in the crystal structure section above. These include conserved cysteine residues, the four helix bundle structure and the transmembrane domain.

Posttranslational modifications Mouse and human SCF are extensively glycosylated and contain both N- and O-linked sugars (Anderson et al., 1990; Huang et al., 1990; Arakawa et al., 1991). In particular, the predicted molecular mass of monomeric SCF is 18,589 kDa, which is close to the molecular weight of E. coli-derived SCF which migrates at an apparent molecular weight of 18.5 kDa on SDS-PAGE (Arakawa et al., 1991). In comparison, natural and CHO-derived monomeric forms of SCF migrate on SDS-PAGE with an apparent molecular mass of 28±35 kDa, which can be reduced to 18.5 kDa by removal of both O- and N-linked sugars, indicating extensive glycosylation (30% by weight) (Zsebo et al., 1990a; Arakawa et al., 1991; Lu et al., 1992; Langley et al., 1992).

disulfide bond and one that is involved in an intermolecular disulfide bond (between M-CSF monomers) (Bazan, 1991; Pandit et al., 1992). Thus far, no intermolecular bonds between SCF monomers have been identified, however, it is speculated that there is a large area of contact between SCF monomers which is sufficient for stable dimer formation. Truncation mutational analysis of SCF protein indicates that the majority of the extracellular sequences (those present in the soluble form of SCF) are required for biological activity (first 141 of 164 residues) (Nishikawa et al., 1992; Langley et al., 1994). Studies of mouse and human SCF chimeras demonstrate that SCF residues 1±35 and 79±97 located in the first and third  helix are required for SCF's ability to synergize with GM-CSF (Matous et al., 1996). In agreement with this study, the sequences recognized by one neutralizing SCF monoclonal mapped to residues Leu79 and Asn97. Another region between 121 and 128 in the fourth helix is also required for synergistic responses with GM-CSF.

Important homologies SCF, Flt-3 ligand, and M-CSF are all type I transmembrane proteins with short cytoplasmic domains

CELLULAR SOURCES AND TISSUE EXPRESSION

Cellular sources that produce Analysis of SCF gene expression in the developing embryo suggests that this gene has multiple functional roles in development. For example, the SCF gene is expressed along the migratory pathways for stem cells that give rise to hematopoietic, melanocyte, and germ cell lineages as indicated by in situ hybridization and northern blot analysis (Matsui et al., 1990; Motro et al., 1991; Keshet et al., 1991). Specifically, SCF is expressed in the genital ridge of day 10 embryos, while the receptor (SCFR) is expressed in the migrating germ cells. SCF expression in the genital ridge decreases after day 11.5 of gestation but remains high in the developing gonads during sexual differential (both in the testes and ovary). This pattern of expression suggests that SCF is involved in regulating the migration, proliferation, and differentiation of germ cells, which is in agreement with the sterile phenotype of Sl mice. Similarly, SCF is expressed in mesenchymal cells located in the limb bud where melanocyte precursors colonize while SCFR is expressed on the migrating cells. The expression of SCF persists during and after stem cell colonization of the limb buds. SCF

884 Jonathan Roy Keller and Diana Marie Linnekin is also expressed in fetal liver, which is the migratory site for hematopoietic stem cells in the developing embryo between embryonic days 12 and 17. As would be predicted, there is a marked reduction in the number of SCFR-positive stem cells that migrate to the fetal livers of Sl/Sl embryos. SCF mRNA transcripts were also detected in other tissues in the developing embryo, including the spinal cord, forebrain, cerebellum, and olfactory bulbs, suggesting that SCF might play a role in the developing CNS. While there are no gross neurological defects in Sl mutant mice, Sl/Sl d mutant mice show a defect in hippocampal-dependent learning (spatial learning) (Motro et al., 1996). SCF is expressed by stromal or accessory cells in the adult where these cells constitute the hematopoietic microenvironment and include endothelial cells, monocytes, and fibroblasts (Flannagan and Leder, 1990; McNiece et al., 1991a; Aye et al., 1992; Heinrich et al., 1993; Linenberger et al., 1995; Broudy et al., 1996). SCF is also produced by intestinal epithelial cells, Sertoli cells, and follicular cells that surround oocytes in the gonads, in thymic stroma and brain cells, including those that constitute the olfactory bulb, thalamus, cerebellum, and brainstem (Tajima et al., 1991; deCastro et al., 1994; Klimpel et al., 1995). SCF expression has also been detected in human CD34-positive cells and keratinocytes in the skin (Longley et al., 1993; Ratajczak et al., 1995). While mRNA levels for the alternatively spliced variants of SCF, SCF220, and SCF248 vary considerably between tissues, SCF248 expression is the predominant species in fibroblasts, brain, thymus, spleen, and bone marrow, while SCF220 is prominent in placenta, cerebellum, and testes (Huang et al., 1992; Brannan et al., 1991). The mechanisms responsible for regulating the levels of alternatively spliced SCF mRNA are unknown, as are the enzyme(s) responsible for the proteolytic cleavage of SCF248 that produce soluble SCF.

Eliciting and inhibitory stimuli, including exogenous and endogenous modulators Inflammatory stimuli including IL-1, TNF, and bacterial pathogens can induce SCF production in vitro by bone marrow stromal cells including endothelial cells and fibroblasts (Anderson et al., 1990; Koenig et al., 1994; Linenberger et al., 1995). In other tissues, SCF can be induced in Sertoli cells with dibutyryl cAMP, and agents that increase the production of intracellular cAMP levels, including

follicle-stimulating hormone (FSH), growth hormone-releasing hormone (GHRH), forskolin, and cholera toxin. Both TGF and TNF have been shown to directly inhibit SCF-mediated growth and survival effects on hematopoietic cell progenitors in vitro and in vivo (Keller et al., 1992; McNiece et al., 1992; Jacobsen et al., 1994, 1995a, 1995b, 1996). Local administration of glucocorticoids at sites of allergic inflammation in vivo significantly decreases mast cell numbers and inhibits SCF production (Finotto et al., 1997). The effect of glucocorticoids on mast cell numbers was reversed by exogenous SCF, suggesting that SCF is required for the growth and survival of mast cells at local sites of allergy in vivo. SCF mRNA levels are increased in bone marrow and spleen cells from mice that were previously treated with 5-fluorouracil (5-FU), a myeloablative chemotherapeutic agent (Hunt et al., 1992; van Os et al., 1997). SCF expression was also increased in bone marrow cells from mice that had received a sublethal dose of gamma-irradiation, suggesting that increased SCF mRNA and protein levels may be a crucial part of the host response to hematopoietic injury (Limmani et al., 1995). In this regard, administration of antibodies that inhibit SCF's ability to bind to its receptor greatly increases the sensitivity of mice to irradiation, suggesting that SCF may play a crucial role in recovery of hematopoietic cells from radiation-induced hypoplasia. In this regard, administration of SCF alone has been shown to protect mice from the lethal effects of irradiation (Zsebo et al., 1992). Furthermore, it is known that IL-1 or TNF can protect mice from the lethal effects of irradiation, and that IL-1-mediated radioprotection can be prevented by administration of antibodies which block SCF/SCFR binding, indicating that IL-1's effects are mediated, in part, through SCF (Neta et al., 1993). While the levels of SCF mRNA expression have not been determined in cells from patients that have received myeloablative chemotherapy or radiation therapy, the concentration of soluble SCF in normal human serum ranges from roughly 1 to 3 ng/mL and is not increased in the serum from patients with myelodysplasia or aplastic anemia (Langley et al., 1993; Bowen et al., 1993; Testa et al., 1994; Abkowitz et al., 1996). Furthermore, the levels of SCF expression do not significantly differ between stromal cells from aplastic anemic patients and stromal cells from normal donors, suggesting that levels of soluble SCF are not predictive for those hematopoietic disorders examined. In summary, as is the case for many HGFs, the physiological mechanisms and signals responsible for regulating SCF levels in vivo are unknown.

Stem Cell Factor

RECEPTOR UTILIZATION SCF utilizes the SCF receptor, which is member of the type III receptor tyrosine kinase family that includes c-Fms and PDGFR and Flk-2/Flt-3 receptors (Besmer et al., 1986; Yarden et al., 1987; Qiu et al., 1988; Ullrich and Schlessinger, 1990). The SCFR receptor has been given the designation CD117.

IN VITRO ACTIVITIES

In vitro findings SCF alone has little or no direct effect on the proliferation of primitive and more committed human and mouse hematopoietic progenitor cells, or stem cells in other tissues. However; in the presence of other HGFs, SCF stimulates the proliferation of hematopoietic cells in soft agar (size and number of colonies) and liquid cultures (Broxmeyer et al., 1991a,b; de Vries et al., 1991; Lowry et al., 1991; Metcalf and Nicola, 1991; Migliaccio et al., 1991a,b; Tsuji et al., 1991; Williams et al., 1992; Ku et al., 1996; Ramsfjell et al., 1996, 1997). For example, SCF directly synergizes with (a) erythropoietin (EPO) to promote unilineage erythroid colony formation (burst-forming units ± erythroid, BFU-E), (b) IL-3 to promote multilineage colony formation (colonyforming unit ± multipotential, CFU-multi) containing granulocytes, macrophages, mast cells, and megakaryocytes (and erythroid cells in the presence of EPO), (c) GM-CSF to promote granulocyte±macrophage (GM) colony formation, (d) M-CSF or G-CSF to promote unilineage CFU-G and CFU-M colony formation respectively, and (e) thrombopoietin (TPO) to stimulate megakaryocytic colony formation in soft agar. SCF in combination with multiple HGF combinations that include IL-3, IL-6, and G-CSF can support the expansion of progenitor cells in liquid culture including BFU-E, CFU-multi, CFU-G, CFU-M, and CFU-GM (Bernstein et al., 1991a; Migliaccio et al., 1992; Haylock et al., 1992; Brandt et al., 1992). While the combination of HGFs (SCF, IL-3, G-CSF, IL-6 (or IL-11)) can promote the expansion of committed progenitor cells in vitro, it does not significantly expand the number of pluripotential hematopoietic stem cells (PHSCs), which are capable of reconstituting the entire hematopoietic system, including lymphoid cells, myeloid cells, erythroid cells, and megakaryocytes when transplanted in vivo (Peters et al., 1996). In fact, inclusion of IL-3 in these HGF

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combinations consistently reduces the lymphoid repopulating ability of the surviving PHSC population, while combinations of SCF and IL-11 maintain PHSC activity in liquid culture (Hirayama et al., 1992, 1994; Holyoake et al., 1996). SCF by itself is a potent survival factor for primitive PHSCs and can be used to transiently maintain the survival of PHSCs and their more committed progeny in vitro (Leary et al., 1992; Li and Johnson, 1994; Keller et al., 1995). Thus far, SCF does not affect the self-renewal capability of stem cells alone or in combination with other HGFs in vitro. Mouse PHSCs and human PHSCs can be maintained in co-cultures with stromal, adherent, or accessory cell populations for extended periods in vitro (Dexter et al., 1977). The presence of PHSCs in these cultures is monitored by the continued production of progenitor cells [CFU ± spleen (CFU-s) or cells that form macroscopic spleen colonies when transplanted into irradiated mouse recipients, CFU ± culture (CFU-c), and their differentiated progeny (total cell output)] over time in the nonadherent fraction of these cultures. Antibodies that block SCF/SCFR interactions inhibit the production of CFU-s, CFU-c, and total cell number to nearly undetectable levels indicating that SCF is absolutely required to maintain hematopoietic development in these cultures (Kodama et al., 1992; Wineman et al., 1993; Miller et al., 1997). Interestingly, the survival and maintenance of the PHSC population was not impaired in the continued presence of the SCF-blocking antibodies since hematopoiesis could be restored in these cultures when the antibodies were removed. The presence of PHSCs was also confirmed in these cultures by transplanting cells into lethally irradiated mouse recipients in competitive repopulating assays, which are designed to detect PHSC activity. PHSCs can also be maintained on stromal cells that do not produce SCF. Taken together, SCF can promote the survival of PHSCs and is absolutely required to maintain hematopoietic development in vitro and in vivo, however, there are other HGF or microenvironmental signals that can support the survival and development of the SCFRexpressing PHSCs (Kodoma et al., 1992; Wineman et al., 1993; Ortiz et al., 1999). SCF can synergize with IL-7 to stimulate pro-B cell proliferation, however, SCF is not required for pre-B cells to further differentiate and it does not induce the proliferation of B220‡ cytoplasmic ‡ pre-B cells (Rolink et al., 1991; McNiece et al., 1991b; Billips et al., 1992; Funk et al., 1993). Further, while SCF can affect primitive B cell progenitor cell growth in vitro, there is not a strict requirement for SCF in B cell development because, (1) pro-B cell numbers can

886 Jonathan Roy Keller and Diana Marie Linnekin expand on stromal cells that do not produce SCF, and (2) W/W fetal liver cells that lack SCFR expression can differentiate into B cells in RAG2 knockout mice, which lack mature B cells (Rolink et al., 1991; Faust et al., 1993; Takeda et al., 1997). In comparison, there is a critical role for SCF in early T cell development. SCF can synergize with IL-7 to stimulate the proliferation of primitive CD4ÿ CD8ÿ CD3ÿ thymocytes but not more differentiated single CD4‡ or CD8‡ cells (Godfrey et al., 1992; deCastro et al., 1994; Morrissey et al., 1994). Furthermore, T cell development is also defective in W/W and Sl/Sl mice where the genes for SCFR and SCF have been completely deleted respectively. In addition, W/W fetal liver cells do not give rise to thymocytes when transplanted into RAG2 knockout mice (Rodewald et al., 1995; Takeda et al., 1997). The receptor for SCF, SCFR, is expressed on the majority of hematopoietic progenitors but is not expressed on their differentiated progeny including mature myeloid and monocytic cells or lymphoid and erythroid cell populations; however, its expression is maintained at high levels on mast cells. In this regard, SCF promotes the survival, proliferation and differentiation of mast cellsin vitro (Tsai et al., 1991a, 1991b; Iemura et al., 1994). In addition, SCF in combination with IL-3 and IL-4 promotes the proliferation of mast cells and their progenitors (Kirshenbaum et al., 1992; Rennick et al., 1995). SCF can promote the growth and differentiation of natural killer (NK) cells in combination with IL-2, IL-7, or IL-15in vitro (Matos et al., 1993; Silva et al., 1994; Shibuya et al., 1995; Mrozek et al., 1996). SCF can also promote the proliferation of the highly effective antigen-presenting dendritic cell population in response to GM-CSF or GM-CSF plus TNF (Young et al., 1995; Szabolcs et al., 1995). SCF can promote the adhesion of mast cells, hematopoietic progenitor cells and cell lines to fibronectin or vascular cell adhesion molecule 1 (VCAM-1) expressed on endothelial cells by activating VLA-4 and VLA-5 integrin expression on progenitor cells (Kinashi and Springer, 1994; Dastych and Metcalfe, 1994; Kovach et al., 1995; Levesque, 1996). Membrane-bound SCF can also function as an adhesion molecule for mast cells and progenitor cells which express SCFR (Kaneko et al., 1991; Long et al., 1992; Kodama et al., 1994; Broudy et al., 1996). As discussed above, SCF is expressed along the migratory routes for germ cells, melanocytes and hematopoietic cells, allowing these cells to home to proper developmental sites. In this regard, SCF is also a potent chemoattractant for mast cells and progenitor cells (Meininger et al., 1992; Nilsson et al., 1994; Okumura et al., 1996).

Regulatory molecules: Inhibitors and enhancers See section on Eliciting and inhibitory stimuli, including exogenous and endogenous modulators.

Bioassays used The majority of murine factor-dependent cell lines derived from mouse hematopoietic tissues are IL-3dependent and require IL-3 for their continued proliferation in vitro. However, some of these cell lines also proliferate in response to murine SCF, including the mast cell lines MC/9 and H-7 originally used to purify SCF, and some subclones of FDC-P1 that were also used to purify IL-3 (Dexter et al., 1980; Martin et al., 1990). In addition, there are SCFdependent cell lines, including EML (Tsai et al., 1994), that require SCF for their continued growth and proliferation. Human factor-dependent cell lines have been established by culturing patient leukemic cells in the presence of HGF in vitro and include the cell lines MO7e and MB-O2 (Avanzi et al., 1990; Hendrie et al., 1991; Broudy et al., 1993). These cell lines proliferate in response to SCF in a dosedependent manner and represent sensitive and reliable bioassays to determine the specific activity of mouse and human SCF. It should be noted that rodent SCF is active on human cells, while human SCF is 100-fold less active on rodent cell lines, therefore, bioassays require mouse or human cell lines where appropriate (Martin et al., 1990).

IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS

Normal physiological roles A single injection of SCF to normal mice promotes a dose-dependent increase in total number of peripheral blood leukocytes, including neutrophils and immature myeloid cells but not circulating platelets or red cells (Ulich et al., 1991). While total bone marrow cellularity was not significantly altered by this treatment, bone marrow morphology appeared more primitive with a decrease in the number of mature neutrophils and an increase in the number of primitive myeloid and erythroid cells (Molineux et al., 1991; Ulich et al., 1991). Daily injections of

Stem Cell Factor SCF for 2 weeks in rodents and nonhuman primates promoted a mast cell hyperplasia in many tissues including the bone marrow (Tsai, 1991b; Galli et al., 1993). Daily injections of SCF in nonhuman primates resulted in increased numbers of neutrophils and lymphocytes; however, in contrast to the rodents, increased numbers of basophils, eosinophils, and monocytes were also observed (Andrews et al., 1991). Administration of SCF to rodents also increases the number of progenitors, including CFU-s, short-term reconstituting cells (STRCs ± radioprotective when transplanted in vivo), and PHSCs in the bone marrow, peripheral blood, and spleen (increased spleen size and cell number) (Molineux et al., 1991; Fleming et al., 1993; Bodine et al., 1993; Briddell et al., 1993; de Revel et al., 1994; Yan et al., 1995). This is a consequence of SCF-induced progenitor/stem cell mobilization and redistribution as well as effects on proliferation and expansion of progenitor cells including PHSCs (Bodine et al., 1993; Fleming et al., 1993; Harrison et al., 1994). Increased numbers of hematopoietic progenitor cells, including those capable of engrafting lethally irradiated baboons, were observed in the peripheral blood and bone marrow of nonhuman primates treated with SCF and could be increased up to 100± 1000-fold in the circulation (Andrews et al., 1991, 1992). SCF in combination with G-CSF was more effective in mobilizing progenitor/stem cells capable of protecting lethally irradiated baboons; however, whether these progenitors include PHSCs remains to be determined (Andrews et al., 1994, 1995; McNiece and Briddell, 1995). In comparison, the combination of SCF and G-CSF showed a rapid increase in the number of PHSCs in the peripheral blood and bone marrow of mice after 14 days (Bodine et al., 1996). Thus, SCF alone or in combination with G-CSF effectively mobilizes stem cells into the periphery and provides a convenient source of stem cells for transplantation. SCF has also been shown to be critical for maintaining constitutive hematopoiesis in studies where animals were administered antibodies that block SCF function by inhibiting the binding of SCF to its receptor. Treatment of mice with 1 mg of antibody every other day for 2 weeks resulted in the elimination of erythroid cells followed by myeloid cells, while total bone marrow cellularity remained unchanged. However, the majority of cells (90%) found in the bone marrow were B220-positive (normally 30±40%), including pre-B cells that express cytoplasmic  chains, and IgM-bearing B lymphocytes (Ogawa et al., 1991; Rico-Vargas et al., 1994). Myeloid progenitor cell populations including those that respond to IL-3, GM-CSF, and M-CSF declined after a single

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antibody injection while the number of progenitors that respond to IL-7 increased. The majority of the committed CFU-s day 8 and more primitive CFU-s day 12 colonies were also eliminated from the bone marrow by this treatment in vivo, however, CFU-s day 12 progenitors were not completely eliminated. In similar experiments looking at other organ systems, adult spermatogenesis and melanocyte development were also blocked by the administration of these antibodies in vivo (Nishikawa et al., 1991). These antibodies also inhibited SCFR/SCF expression in fetal tissues and specifically blocked fetal thymic colonization by triple negative fetal liver progenitors in vitro (Godfrey et al., 1994). Furthermore, these antibodies also inhibit the development of CD4‡ CD8‡ cells in fetal thymus reconstitution assays (Hozumi et al., 1994). Taken together, these data suggest that SCF is critical for the development of most hematopoietic cell lineages except B cells.

Species differences Although mouse and human SCF are roughly 82% identical at the amino acid level, human SCF is 100-fold less active on mouse cells, while mouse and rat SCF are equally active on both human and rodent cells.

Knockout mouse phenotypes The phenotypes of mice with naturally occurring mutations in SCF or the Sl locus demonstrate that the SCF/SCFR axis is critical for hematopoiesis, melanogenesis, and gametogenesis. Sl mutant mice that have completely deleted the coding sequences for SCF do not produce viable homozygous offspring; embryos only survive to roughly 15 days gestation. In comparison, Sl mutant mice where SCF sequences are present, produce viable homozygous offspring that show defects in hematopoietic development, pigmentation, and germ cell development (sterility) depending on the severity of the mutation. Thus, null alleles are lethal, while viable alleles retain some of the normal function of SCF (summarized in Table 1). For example, Sl gb, Sl J, Sl10H, Sl, Sl 8H, Sl 12H, and Sl 18H have complete deletions of the SCF gene and all homozygous embryos die at roughly 15 days gestation with severe defects in fetal liver hematopoiesis without effects on other organ systems (Russell, 1979; Silvers, 1979; Copeland et al., 1990; Huang et al., 1990; Zsebo et al., 1990b). The smallest complete deletion in SCF coding sequences occurs in Sl gb mice where the total genomic deletion is roughly 120 kb.

888 Jonathan Roy Keller and Diana Marie Linnekin Table 1 Summary of Steel locus mutants Gene symbol

Gene name

Mutation

Consequence of mutation

Phenotype of homozygous mice

Sl gb

Steel-Grizzle Belly

120 kb deletion

Absence of SCF

Prenatal lethality

Steel-J

650 kb deletion

Absence of SCF

Prenatal lethality

Sl

J

Sl

10H

Steel-10H

680 kb deletion

Absence of SCF

Prenatal lethality

Sl

Steel

> 810 kb deletion

Absence of SCF

Prenatal lethality

Sl d

Steel dickie

4 kb intragenic deletion

Lack of membrane-bound SCF

Severely anemic Lack of pigmentation Sterility in both sexes

Sl

17H

Steel-17H

Point mutation in splice acceptor

Absence of normal cytoplasmic SCF domain

Macrocytic anemia White spotting Male sterility

Sl pan

Steel-Panda

Rearrangement/

Decreased SCF expression

inversion >100 kb 50 SCF gene

Mild anemia Decreased pigmentation Female sterility

Sl

con

Steel-contrasted

Rearrangement 0

> 100 kb 5 SCF gene

Decreased SCF expression

Mild anemia Decreased pigmentation Female sterility

This deletion begins in the 30 untranslated region of SCF and includes the entire SCF coding sequences of roughly 50 kb, as well as 60 kb of DNA sequence 50 of the SCF coding region (Bedell et al., 1996). Furthermore, the deletions detected in Sl J and Sl10H (larger than the deletion in Sl gb) are also smaller deletions than those seen in the original Sl allele where lethality occurred at the same time during gestation. In summary, late gestation lethality appears to be the true null phenotype; however, whether other genes are deleted in Sl mice with larger genomic deletions remains to be determined. The best-characterized mouse strain with a naturally occurring viable mutation of SCF is the Sl d/Sl d mouse. These mice have a 4 kb intragenic deletion which removes the exons encoding the transmembrane and cytoplasmic domains of SCF resulting in the Sl d transcript, which encodes the soluble isoform of SCF (Brannan et al., 1991; Flannagan et al., 1991). These mice are viable but have severe macrocytic anemia with mast cell deficiencies, sterility in both sexes, and lack skin pigmentation. The bone marrow of Sld is hypocellular with both myeloid and erythroid progenitor cell deficiencies. Thus, it is speculated that the membrane-associated form of SCF is critical for

the development of three distinct cell lineages, including hematopoietic cells. Another informative viable mutant of the Sl locus is the Sl17H mutation, which is a splice site mutation that causes exon 8 to be skipped, resulting in the splicing of exon 7 with exon 9. The deletion of exon 8 which encodes the cytoplasmic domain of SCF results in a frameshift mutation that replaces the normal SCF cytoplasmic domain with extraneous amino acids before a stop codon is encountered (Brannan et al., 1992). This mutation does not affect the ability of SCF to form dimers but slows the rate of SCF transport to the cell surface and subsequent stability (Tajima et al., 1998a). Total bone marrow cellularity, leukocyte counts, platelets and hematocrit are unaffected in these mice. However, myeloid progenitor cells and tissue mast cells are slightly decreased. longterm bone marrow culture cells (LTBMCs) established from these mice show a greatly reduced ability to support the production of total hematopoietic cell output. Furthermore, these mice have reduced levels of CFU-s progenitor cells. Further, male mice are sterile while female mice are unaffected. Two other Sl mouse strains including Sl pan and Sl con have DNA rearrangements roughly 100 kb 50 of

Stem Cell Factor the SCF coding sequences (Bedell et al., 1995). These mutations affect SCF mRNA expression in the gonads and lead to reduced numbers of primordial germ cells and result in female sterility (male sterility is not observed).

Transgenic overexpression To better understand the physiological roles of soluble versus membrane isoforms of SCF, Sl d mice were engineered to stably express soluble and membrane-restricted SCF transgenes. Both Sl d transgenic mouse strains showed increases in the number of multipotential and more committed myeloid progenitor cells in the bone marrow and increased numbers of peripheral blood leukocytes (mainly neutrophils) (Kapur et al., 1998). However, only Sl d mice that expressed the membrane-restricted isoform of SCF showed (1) a reversal in the severe runting phenotype, (2) an increase in bone marrow cellularity, and (3) an increase in the hematocrit and red cell count. However, neither transgene could rescue the defects in germ cells, melanocytes, or mast cells in Sl d mice. The lack of effect of either SCF transgene on other organ systems may be due to the choice of promoter in these constructs which could have restricted the transgene expression to specific cell types (Kapur et al., 1998). In other experiments, transgenic mouse strains were engineered to exclusively produce the membrane-restricted form of SCF (SCF220 or KL-2) by homologous recombination in embryonic stem cells. No effect on hematopoiesis including peripheral blood counts or bone marrow cellularity or progenitor numbers were observed in these mice (Tajima et al., 1998b). Furthermore, there were no observable effects on gametogenesis since male and female mice produced normal numbers of offspring. However, these mice showed defects in the production of dermal mast cells, and were also sensitive to sublethal doses of gamma-irradiation, suggesting a role for soluble SCF in hematopoietic recovery following radiation injury. Because human SCF can act as an antagonist of mouse SCF, transgenic mice were produced that express human membrane-restricted SCF (SCF220) to separate the effects of soluble and transmembrane SCF (Kapur et al., 1997, 1998). No significant effect on hematopoiesis was observed in these mice, including total leukocyte counts and red cell numbers in the peripheral blood, or bone marrow cellularity and progenitor numbers. However, these mice have a coat color defect, have greatly reduced numbers of dermal mast cells, and show abnormal thymocyte

889

maturation, suggesting that melanoblasts, dermal mast cells, and thymocyte progenitors are not able to migrate to the appropriate sites, or are unable to survive and proliferate once they arrive. Transgenic mouse strains were constructed which restricted the expression of SCF248 or SCF220 transgenes to keratinocytes in normal mice (Kunisada et al., 1998). These studies found that expression of SCF248 in keratinocytes results in cutaneous mastocytosis and epidermal hyperpigmentation due to the maintenance of melanocytes and melanin production in the epidermis. In comparison, expression of the membranerestricted form of SCF, SCF220, in keratinocytes promoted epidermal melanocytosis and melanin production without mastocytosis. This suggests that soluble SCF is required for dermal mast cell migration and/or proliferation while membrane-associated SCF is required for melanocyte growth and differentiation. In other studies, expression of SCF248 in keratinocytes also promoted pigmentation in sites which normally do not contain melanocytes or their precursors, suggesting that SCF can also stimulate the migration of melanocytes in vivo (Kunisada et al., 1998).

Pharmacological effects None reported for mice.

Interactions with cytokine network None reported in vivo.

Endogenous inhibitors and enhancers See section on Eliciting and inhibitory stimuli, including exogenous and endogenous modulators.

PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY

Normal levels and effects Normal plasma levels of human SCF range from 1 to 5 ng/mL (Langley et al., 1993). In addition, SCF levels do not significantly vary in patients with a

890 Jonathan Roy Keller and Diana Marie Linnekin variety of hematological disorders including aplastic anemia and myelodysplastic syndromes (McNiece and Briddell, 1995; Kumar and Alter, 1998).

Role in experiments of nature and disease states While no abnormalities involving the SCF genetic loci have been reported, locally high concentrations of soluble SCF have been found in lesions of human cutaneous mastocytosis (Longley et al., 1993, 1995). This disease is characterized by accumulations of mast cells as well as increases in the production of epidermal melanin similar to that observed in transgenic animals that expressed SCF transgenes in keratinocytes (described above). This has led to the hypothesis that locally produced SCF can promote mast cell hyperplasia.

IN THERAPY

Preclinical ± How does it affect disease models in animals? SCF administration to Sl/Sl d mice reverses the macrocytic anemia resulting in increased mean red blood cell volume and red blood cell counts that are within 70±85% of their littermate control levels. This effect was reversed when SCF treatment was stopped (Hunt et al., 1992; Galli et al., 1994). Increased numbers of lymphocytes and platelets were also observed in Sl/Sl d mice treated with SCF, which were also observed in normal mice treated with SCF. Injection of SCF into Sl/Sl d mice caused a two-fold and 20-fold increase in the number of primitive progenitors (CFU-s day 12) found in the bone marrow and spleen, respectively (Bodine et al., 1992). Sl/Sl d mice do not experience a rebound in platelet counts after 5-FU treatment, while normal mice show an overshoot between 10 and 20 days post 5-FU treatment (Hunt et al., 1992). To determine whether SCF was critical for rebound (overshoot) thrombocytosis after 5-FU treatment in Sl/Sl d mice, mice were treated with SCF after 5-FU. Administration of SCF to 5-FU-treated Sl/Sl d mice promoted the same overshoot as that seen in control 5-FU-treated mice, demonstrating the importance of SCF in megakaryocyte growth and development. Megakaryocyte progenitor (CFU-MK) numbers are increased in the bone marrow and spleen of normal mice treated with SCF, while platelet counts are not increased in the peripheral

blood. Therefore, it is speculated that the major effect of SCF treatment in Sl/Sl d mice is to promote the proliferation of immature megakaryocytes and their precursors, while other factors such as thrombopoietin are critical for the final stages of platelet maturation.

Pharmacokinetics Baseline SCF serum levels in patients that were to receive SCF treatment in a phase I clinical trial were roughly 1 ng/mL (Glaspy, 1996). SCF serum levels reached a maximum 13±14 hours after the first dose of SCF. Maximum serum SCF levels were observed between 10 and 14 hours after subsequent doses and SCF. Adsorption of SCF was relatively low after the first dose of SCF.

Toxicity The use of SCF in vivo has been limited due to adverse side effects. These included dermatological reactions such as pruritic wheals and edema at the site of injection as well as increased melanization (pigmentation) of the epidermis at doses above 25 mg/kg body weight over a 14-day period (Demetri et al., 1993; Costa et al., 1996; Weaver et al., 1996; Moskowitz et al., 1997). In addition, 10±20% of the patients treated with SCF developed allergic-like reactions characterized by urticaria with respiratory symptoms that required discontinuation of treatment (Grichnik et al., 1995; Costa et al., 1996). Because mast cell hyperplasia was suspected in these cases, a prophylactic antihistamine treatment was incorporated into subsequent protocols (McNiece and Briddell, 1995).

Clinical results A phase I clinical trial of SCF was conducted in lung cancer and breast cancer patients who received 10± 50 mg/kg/day SCF by subcutaneous injection for 14 days (Glaspy, 1996; Demitri et al., 1993). An increase in the total number of peripheral blood leukocytes was observed prior to chemotherapy and some acceleration in platelet and leukocyte numbers was seen after chemotherapy. Daily administration of SCF to patients at dosages of up to 50 mg/kg for 14 days did not affect the number of peripheral blood CD34‡ cells; however, increased numbers of CD34‡ cells, that included both committed and more primitive hematopoietic progenitor cells, were detected in the bone marrow (Tong et al., 1993). SCF therapy also promoted an increase in bone marrow

Stem Cell Factor cellularity with significant increases in promyelocytes without apparent effects on other marrow hematopoietic cells (Orazi et al., 1995). Because SCF was shown to potently mobilize progenitor cells in mice and nonhuman primates, subsequent clinical trials have focused on the ability of SCF (in lower doses) in combination with G-CSF to mobilize peripheral blood progenitor cells for transplantation. A phase I/II trial in non-Hodgkin's lymphoma patients was performed to compare mobilized peripheral blood progenitor cells for autologous transplantation from patients treated with the combination of SCF plus G-CSF or GCSF alone (Moskowitz et al., 1997). Patients transplanted with G-CSF plus SCF-mobilized peripheral blood cells showed a decreased time to normal platelet levels following chemotherapy compared with transplanted G-CSF-mobilized peripheral blood. In another phase II trial, fewer apheresis procedures were required to collect sufficient numbers of peripheral blood progenitor cells from breast cancer patients for transplantation studies (Kumar and Alter, 1998). In a phase I study of patients with refractory aplastic anemia, SCF was well tolerated at all doses and patients that received SCF showed neutrophil improvements that were improved by the combination with G-CSF (Kumar and Alter, 1998).

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ACKNOWLEDGEMENTS The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. We are grateful for the critical review of this manuscript by Drs Sally Spence, Jon Dermott, and J.J. Oppenheim. The publisher or recipient acknowledges right of the U.S. Government to retain a nonexclusive, royalty-free license in and to any copyright covering the article. This project has been funded in whole or in part with Federal Funds from the national Cancer Institute, National Institutes of Health, under contract number NO1-CO-56000.