Phorochemistry and Phofobiology Vol. 52, No. 6, pp. 111F1136, 1990

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YEARLY REVIEW

MOLECULAR MECHANISMS OF ULTRAVIOLET RADIATION CARCINOGENESIS

Introduction Skin cancers are the most common type of human cancer. Recent surveys indicate that around 500 000 new cases of skin cancer are diagnosed each year in the United States (Scotto et al., 1983), most of which are thought to result from repeated exposure of the skin to sunlight. The importance of sunlight in the etiology of human skin cancer was recognized as early as 1894 (Unna, 1894). Since then, studies with mice and rats have shown that ultraviolet (UV) radiation present in the sunlight is responsible for this effect (Findlay, 1928; Roffo, 1934). Studies using laboratory animals have shown that wavelengths in the UV-B (280-320 nm) region of the solar spectrum are responsible for the induction of skin cancer (Blum, 1959; Urbach, 1969; Epstein, 1978; Forbes, 1981). Wavelengths in the UV-A (320-400 nm) region have also been shown to cause skin cancer in animals when given in high doses over a long period of time (Zigman et al., 1976; Forbes et al., 1982; Van Weelden et al., 1983; Strickland, 1986). In contrast, wavelengths in the UVC (200-280 nm) region are not present in natural sunlight because they are filtered out by the ozone in the atmosphere. In the past, research on UV carcinogenesis has focused on three major themes: attempts to define the relationship between exposure to UV radiation and induction of skin cancer in man and in laboratory animals, description of the pathogenesis of skin cancer, and investigation into the mechanism by which UV radiation causes neoplastic transformation. In addition, studies performed in the last 15 years have opened up a new area of research on the immunobiology of UV-induced skin cancers.

*Abbreviudons: BCC, basal cell carcinoma; BCNS, basal cell nevus syndrome; CHO, Chinese hamster ovary; DHFR, dihydrofolate reductase; DMBA, dimethyl benzanthracene; KA, keratoacanthoma; NMU, nitrosomethylurea; PCR, polymerase chain reaction; PKC, protein kinase C; RFLP, restriction fragment length polymorphism; RRE, rus-responsive element; SCC, squamous cell carcinoma; TPA, 12-0-tetradecanoyl phorbol 13-acetate; XP, xeroderma pigmentosum.

These studies indicate that UV radiation not only results in the appearance of skin cancers, but also interferes with an immune defense mechanism that normally protects against the development of skin cancers (Kripke, 1986). Carcinogenesis by UV radiation is a complex process that involves initiation, promotion, and progression. In UV carcinogenesis, as in chemical carcinogenesis, there is a long latent period between first exposure and the appearance of primary skin tumors. The cellular and molecular processes that occur during the initial carcinogen-cell interaction and the onset of tumor growth are largely unknown. However, the carcinogenic process begins when a carcinogenic agent, radiation or chemical, damages the cellular DNA, which then leads to a cascade of events including DNA repair, mutation, and transformation. Although many aspects of UV carcinogenesis have been studied extensively, the molecular mechanisms by which UV radiation transforms normal cells into cancer cells has not been fully elucidated. However, within the past few years substantial progress has been made in this area, with evidence that inappropriate expression of protooncogenes due to point mutation, gene amplification, deletion or rearrangement may be involved in UV carcinogenesis. In addition, attempts have been made to establish a direct relationship at the molecular level between UV radiation and activation of oncogenes and to identify the premutagenic lesions in UV carcinogenesis. This review presents some background information on UV-induced DNA damage and repair and their relationship to the process of mutation and transformation, and summarizes the recent advances on the role of oncogenes and tumor suppressor genes in UV carcinogenesis.

Correlation of DNA photoproducts to cell lethality, mutation and transformation Several lines of evidence implicate DNA as the target for most of the biological effects of UV, such as lethality, mutation, and transformation. First, individuals with the genetic disorder xeroderma pigmentosum (XP)* are extremely sensitive to sunlight, frequently developing skin cancers as a result of 1119

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exposure. In addition, their somatic cells are hypersensitive to killing by UV radiation (Cleaver and Bootsma, 1975). This extreme sensitivity of somatic cells to UV radiation has been attributed to a defect in the repair of UV-induced pyrimidine dimers (Cleaver and Bootsma, 1975). Second, the action spectra for cell killing, mutation, and transformation of Syrian golden hamster embryo fibroblasts (Doniger et al., 1981) and human cells (Sutherland et al., 1981) are similar to the DNA absorption spectrum as well as the action spectrum for pyrimidine dimer induction. Third, the reversal of UVinduced pyrimidine dimers in the DNA by enzymatic photorepair reduces the incidence of tumors in the Amazon molly, Poecilia formosa (Hart et al., 1977), and in the South American opossum, Monodelphis domestica (Ley et al., 1988), and reduces the frequency of UV-induced transformation of cultured human fibroblasts (Sutherland et al., 1980). These studies also suggest that pyrimidine dimers, the major UV-induced photoproduct in DNA, are responsible for the above-mentioned UVinduced biological effects. A number of studies have indicated that there is a causal relationship between induction of pyrimidine dimers and UV carcinogenesis. Hart et al. (1977) have shown that injection of UV-irradiated tissue homogenates of Poecilia forrnosa into isogenic recipients resulted in thyroid carcinoma. However, when UV-irradiated tissue homogenates were exposed to photoreactivating light prior to injection, the tumor incidence was reduced significantly, suggesting the involvement of pyrimidine dimers in UV-induced carcinogenesis. Setlow et al. (1989) have shown that a platyfish-swordtail hybrid is prone to the development of melanoma upon UV irradiation, but exposure to visible light following irradiation reduces the prevalence to background levels. Recent studies by Ley et al. (1988) have shown that Monodelphis domestica, which exhibits an efficient enzymatic photoreactivation of UVinduced pyrimidine dimers, is highly susceptible to induction of skin and corneal tumors by UV radiation, and that exposure to photoreactivating light immediately after UV-irradiation results in a substantial decrease in the number of UV-induced corneal tumors. Sutherland er al. (1981) demonstrated that UV-induced transformation of cells to anchorage-independent growth was susceptible to photoenzymatic repair, again implicating pyrimidine dimers in UV-induced transformation. However, Suzuki et al. (1981) reported that although the induction of mutation in V79 Chinese hamster cells and induction of transformation in C3H mouse 10T112 cells by germicidal lamp (254 nm) and unfiltered sunlamp (290-345 nm) matched the DNA absorption spectrum, such was apparently not the case for mutation and transformation induced by a filtered sunlamp (300-345 nm). They found that wavelengths greater than 300 nm yielded appreci-

ably more mutation and transformation than can be accounted for by pyrimidine dimer formation. They have suggested that a chromophore other than, or in addition to, DNA may be involved in induction of mutation and transformation at longer wavelengths. Similarly, Tyrrell and Pidoux (1987) have shown that only about 40% of the total cytotoxic effectiveness present in solar light is due to UV-B alone, the remainder being due to UV-A working in unison with UV-B or another co-carcinogen. Studies by Wood (1985) have also shown that pyrimidine dimers are not the principal premutagenic lesions induced in A phage by UV radiation. Recent studies indicate that UV radiation also induces other non-dimer photoproducts such as (6-4) photoproducts (Haseltine, 1983; Mitchell and Nairn, 1989), cytosine photohydrates (Weiss and Duker, 1987; Doetsch et al., 1988), purine photoproducts (Gallagher and Duker, 1989). and singlestrand breaks in the DNA (Peak et al., 1987). Various data suggest that (6-4) photoproducts may play a significant role in UV-induced lethality and mutagenesis (for review see Brash, 1988). The relative rates of induction of (6-4) photoproducts and cyclobutane dimers have been shown to vary considerably and depended highly on the (A+T)/(G+C) DNA content ratio (Mitchell and Nairn, 1989). The action spectra for the formation of dimers and (6-4) photoproducts decline sharply with increasing wavelengths above 280 nm (Rosenstein and Mitchell, 1987), whereas the action spectra for cell killing (Jones et al., 1987), mutation induction (Tyrrell, 1980; Zelle et al., 1980) and tumorigenesis (Sterenborg et al., 1988) show a prominent shoulder above 300 nm. It is suggested that lesions other than pyrimidine dimers and (6-4) photoproducts may be more important at UV-B and UV-A region of the solar spectrum (Coohill et al., 1987). It is reported that cytosine photohydrates and purine photoproducts, while sparsely formed by UV-C radiation, are maximally formed by UVB radiation. Peak et al. (1987) have shown that a significant amount of DNA damage due to near-UV occurs by single-stand breaks. Near-UV radiation is also known to produce reactive oxygen species such as superoxide anion, singlet oxygen and hydrogen peroxide in bacteria (Webb, 1977; Hartman, 1986) and mammalian cells (Danpure and Tyrrell, 1976). However, the role of these photoproducts in UV carcinogenesis is unknown. Several studies indicate that the ( 6 4 ) photoproduct or its isomer, the Dewar’s complex (Taylor and Cohrs, 1987) may be biologically more relevant at UV-B wavelengths of the solar spectrum (for review see Mitchell and Nairn, 1989). Cleaver et al. (1988a) have isolated a XP group A revertant that selectively repairs (6-4) photoproducts without showing marked reduction in the overall numbers of pyrimidine dimers. These revertant cells exhibited near wild-type levels of cell killing, sister chro-

Yearly Review matid exchange, mutagenesis, and inhibition and recovery of DNA and RNA synthesis after irradiation of cells in culture (Cleaver et al., 1988b). Thus, repair of pyrimidine dimers does not appear to be as important as repair of (64)photoproducts in maintaining a normal phenotype in the face of UV radiation. In addition, Cleaver (1989) has shown that excision repair of pyrimidine dimers from the entire genome is not necessary for the survival of human cells as revealed by the ability of cells to survive up to 48 h after UV irradiation having not removed the dimer. Rather, repair of non-dimer photoproducts such as (64) photoproducts in the genome as a whole or selected key pyrimidine dimers may be more significant (Evans et al., 1990). Studies of trichothiodystrophy, which stems from repair enzyme deficiencies, have also shown that repair synthesis is reduced by 50% in cell cultures taken from those who have it and that this is associated with a marked decrease in the repair of (64)photoproducts from cellular DNA (Broughton et al., 1990). Although these results indicate that (6-4) photoproduct formation may play a greater role than cyclobutane dimer formation in UV-induced lethality and mutagenesis, recent studies demonstrate that preferential DNA repair in specific genomic sequences is probably more important than repair of the overall genome (Bohr, 1987; Bohr and Wassermann, 1988; Bohr et al., 1985, 1986a. 1986b; Cleaver, 1989; Hanawalt, 1989; Mayne et al., 1988; Mellon et al., 1986, 1987). Bohr et al. (1985) found that efficient removal of pyrimidine dimers in the dihydrofolate reductase (DHFR) gene in Chinese hamster ovary (CHO) cells correlated with survival, indicating that repair in essential genes is perhaps the ultimate criterion for cell survival following UV irradiation. Mayne et al. (1988) have shown that fibroblasts from Cockayne's syndrome patients exhibit normal repair of genomic DNA lesions but are deficient in the repair of specific DNA lesions in actively transcribing regions of DNA. Lending further support to the idea that actively transcribed genes are repaired preferentially, Islas and Hanawalt (1990) have shown that when the myc proto-oncogene is actively transcribed in undifferentiated promyelocytes, 56% of the dimers forming in this gene during UV irradiation are removed in the 18-h time period following exposure. However, when myc is transcriptionally down-regulated through the induced differentiation of these cells, dimer removal in this gene in the 18-h following UV irradiation is reduced to 13%. This has an additional point of interest in that many of the tumors resulting from translocations involve breakpoints in the 5' region of the myc gene. Although no such myc activation has been discovered in UVinduced skin carcinogenesis to this date, UV irradiation and treatment with other DNA damaging agents increases the frequency of homologous

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recombination, providing a vehicle by which translocation activation may take place. This increase in the frequency of homologous recombination is especially pronounced when a transcriptionally active gene is involved (Chang et al., 1990). Considerable differences in the repair efficiency of transcriptionally active and inactive proto-oncogenes have been reported. The UV-induced pyrimidine dimers were repaired more efficiently in the constitutively transcribed c-abl proto-oncogene than the transcriptionally inactive c-mos proto-oncogene (Madhani et al., 1986). Analogous to the preferential repair of UVinduced pyrimidine dimers, preferential repair of UV-induced (6-4) photoproducts has also been reported. Thomas et al. (1989) found that the DHFR gene in CHO cells was preferentially repaired compared with the downstream, non-coding sequences, and that the efficiency of repair of UVinduced (6-4) photoproducts was more efficient than pyrimidine dimer repair. However, it remains to be determined whether the preferential repair of (6-4) photoproducts in active genes plays a role in mutation and transformation. Several lines of evidence support the hypothesis that somatic mutations are involved in the process of neoplasia. Studies utilizing skin fibroblasts from excision repair-deficient human XP patients have shown that they exhibit a higher frequency of UVinduced mutation and transformation than normal human skin fibroblasts (Maher et al., 1976, 1982). These results suggest that defects in DNA repair predispose XP cells to UV radiation-induced skin cancer because UV radiation induces a much higher frequency of mutation in XP cells than in normal cells, and some of these mutations may be involved in cancer induction.

DNA repair deficiency and UV carcinogenesis Efficient removal of DNA lesions by cellular repair processes appears to be a critical step in the prevention of tumor formation. A number of human hereditary diseases such as XP, Fanconi's anemia, and ataxia telangiectasia have been shown to be unusual in their processing of damaged DNA (Setlow, 1978; Kraemer, 1983; Friedberg, 1985; Hanawalt and Sarasin, 1986; Lehmann and Norris, 1989). Xeroderma pigmentosum patients are defective in their ability to repair pyrimidine dimers and are hypersensitive to sunlight and prone to the development of skin cancers (Cleaver and Bootsma, 1975). Although these patients are highly susceptible to the development of skin cancers, DNA repair-proficient revertant cells have been isolated from repair-deficient cells, suggesting that a particular patient's condition may stem from a limited number of enzyme deficiencies (Royer-Pokora and Haseltine, 1984; Cleaver et al., 1988a). However,

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as exhibited in cell fusion studies, several separate enzyme deficiencies from individual patients may lead to the same repair-deficient state. Thus, complementation groups among XP patients are established based upon a particular enzyme that is lacking (Kraemer et al., 1987). Moreover, reversal of the UV radiation hypersensitive phenotype has been demonstrated through cell fusions of CHO and XP cells, allowing the possibility to identify and clone genes from revertants that appear to be essential for DNA repair and increased UV resistance (Karentz. and Cleaver, 1986) and through electroporation of chromatin-associated endonucleases (Lambert et al., 1990b). One such enzyme deficiency is lack of a nuclear DNA binding factor from cells of a patient with XP complementation group E (Chu and Chang, 1988). This factor, simply known as XBE, is a homolog of the yeast enzyme photolyase, which participates in both photoreactivation and excision repair processes in Saccharomyces cerevisiae (Patterson and Chu, 1989). Quite possibly, DNA-protein interactions may be mediated between a leucine zipper motif in this protein, facilitating binding to a damaged stretch of DNA by dimerization with another as yet unidentified protein. Additionally, Parrish and Lambert (1990) showed that DNA endonucleases from XP patients group A and D have diminished ability to associate with chromatin and it is at this level that a defect exists in repair enzymes. This enzyme has been isolated and found to remove pyrimidine dimers created by 254 nm UV radiation over 2-fold more efficiently in normal cells than in XP cells (Lambert et al., 1990a). Interestingly, Kantor et al. (1990) have proposed that separate chromatin-specific endonuclease mechanisms exist for transcribed vs non-transcribed regions based upon studies on the ability of cells from XP group C patients to repair active genes preferentially. In these XP patients DNA repair defects may be circumvented through chromosome 9 transfer (Ishizaki et al., 1990) or electroporation of normal human endonucleases into cultured XP group A cells (Tsongalis et al., 1990). Seetharam et al. (1990) have shown that upon in vitro UV irradiation, a shuttle vector, pZ189, exhibits a predominant G-C to A-T transition when transfected into lymphoblasts derived from XP cells from group A patients. Because XP-A derived fibroblasts did not exhibit the same mutagenic properties, the possibility that cellular factors can influence the probability of base substitutions at particular modified DNA sites is implied. Analogous to XP, individuals with the genetic disorder basal cell nevus syndrome (BCNS) are predisposed to sunlight-induced skin cancers. Whereas XP is an autosomal recessive disorder, BCNS is an autosomal dominant disorder with high penetrance (Gorlin and Goltz, 1960; Kraemer, 1983). The cellular and molecular basis for BCNS is unknown. Pre-

vious attempts to demonstrate an increased sensitivity of somatic BCNS cells to killing by UV radiation have been unsuccessful. Studies by Little et al. (1989) and Lehmann et al. (1977) have shown that fibroblasts from BCNS patients exhibit normal survival following UV-irradiation. However, Nagasawa et al. (1988) have reported that BCNS fibroblasts were slightly more sensitive to UV radiation than normal fibroblasts. Recent studies by Applegate et al. (1990) indicate that skin fibroblasts from BCNS patients were hypersensitive to killing by UV-B but not UV-C radiation as compared to skin fibroblasts from normal individuals. In contrast, the skin fibroblasts from BCNS patients exhibited a sensitivity to UV-A radiation similar to that of normal skin fibroblasts (Ananthaswamy, unpublished). DNA repair studies indicated that the increased sensitivity of BCNS skin fibroblasts to killing by UV-B radiation was not due to a defect in the excision repair of pyrimidine dimers. These results indicate that there is an association between hypersensitivity of somatic cells to killing by UV-B radiation and the genetic predisposition to skin cancer in BCNS patients. In addition, these results suggest that DNA lesions (and repair processes) other than the pyrimidine dimer are also involved in the pathogenesis of sunlight-induced skin cancers in BCNS patients. A decreased excision repair of UV-induced pyrimidine dimers has been reported in sporadic basal cell carcinoma (BCC) patients. Alcalay et al. (1990) analyzed skin biopsies from normal individuals and patients with BCC for their ability to remove UV-induced pyrimidine dimers by excision repair. They found that at low doses both BCC patients and normal individuals exhibited similar repair levels. However, with increasing UV dose, the BCC patients exhibited decreased levels of excision repair of UV-induced pyrimidine dimers compared to normal controls. In addition to BCC patients, individuals with actinic keratoses have slightly decreased levels of unscheduled DNA synthesis (Lambert et al., 1976). However, it is not clear whether there is any cause and effect relation between decreased levels of DNA repair and development of BCC or actinic keratosis. Genetic alterations in UV carcinogenesis

Cancer is known to result from mutations that perturb the normal cellular growth control mechanisms. The mutations include not only point mutations but also deletions that remove entire genes or disrupt the normal regulatory sequences, producing translocations, amplifications and gene rearrangements. These mutations occur in two classes of interacting genes; those that facilitate cell growth and tumor formation (oncogenes) in which mutation or overexpression is oncogenic (Bishop, 1983; Land et al., 1983a), and those that inhibit

Yearly Review these processes (the tumor suppressor genes) whose loss is oncogenic (Knudson, 1985; Cavenee et al., 1989). Role of oncogenes in UV carcinogenesis Cellular oncogenes are reported to be activated in a variety of human and rodent tumors (Bishop, 1983; Land et al., 1983a, b; Zarbl et al., 1985; Quintanilla et al., 1986; Balmain and Brown 1988; Bos 1988, 1989; Suarez, 1989). Many normal cells also express these oncogenes (termed proto-oncogenes), which indicates that these genes may play a role in the growth and differentiation of normal embryonic and adult tissues. However, exposure of cells to carcinogenic agents may convert the normal genes into pathologic genes by causing them to produce higher levels of their normal gene product or by inducing structurally aberrant gene products. There is evidence that both these phenomena occur (Balmain et al., 1988). Cellular proto-oncogenes can be activated by both point mutations and chromosomal translocations, suggesting that there may be a direct link between exposure to agents that damage DNA and genetic change leading to malignancy (Bishop, 1983; Land et al., 1983a, b; Zarbl et al., 1985; Quintanilla et al., 1986; Balmain and Brown 1988; Bos, 1988, 1989; Suarez, 1989). Zarbl et al. (1985) have shown that in rat tumors induced by nitrosomethylurea (NMU), the point mutation (G-A transition) responsible for the activation of the c-Ha-ras-1 proto-oncogene was always located at the 12th codon, whereas in rat tumors induced by dimethyl benzanthracene (DMBA), the point mutation was at the 61st codon of the c-Ha-ras locus. Similarly, Quintanilla et al. (1986) have reported that over 90% of tumors, including premalignant papillomas, induced by DMBA and 12-0-tetradecanoyl phorb01-13-acetate (TPA) contained a specific A-T transversion at the second nucleotide of codon 61 of the c-Ha-ras oncogene. Vousden et al. (1986) have shown that treatment of the c-Ha-ras-1 proto-oncogene in vitro with DMBA can transform NIH 3T3 cells upon transfection. These transformants contained mutations at codon 61; 6 at the first base position (C-G), 5 at the second base position (AT), and 5 at the third base position (G-C). One hypothesis suggested by these observations is that a particular class of carcinogens may activate specific oncogenes leading to the development of tumors. In addition, it is suggested that different mutations induced in identical genes by the same carcinogen may result in cell transformation, providing a basis for the hypothesis that one gene may be involved in several tumor induction mechanisms (Brown et al., 1990). Since the DNA damage induced by UV radiation is unique and differs from the lesions induced by any other carcinogen, it is quite possible that specific alterations in oncogene structure can lead to the induction of tumors.

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Activation of oncogenes in human skin cancers As an experimental approach to address this question, several investigators have analyzed human skin cancers for the presence of activated oncogenes. Ananthaswamy et al. (1988) found that genomic DNAs from 3 of 8 non-melanoma human skin cancers occurring on sun-exposed body sites contained activated c-Ha-ras oncogenes capable of inducing tumorigenic transformation when introduced into NIH 3T3 cells by DNA-mediated gene transfer. Analysis of additional human squamous cell carcinomas (SCC) revealed that some of them (3 of 12) contained highly amplified N-ras sequences (Ananthaswamy, unpublished results). However, it is not yet known whether these skin cancers contain specific mutations in Ha-ras or N-ras oncogene. If ras gene mutations are detected at specific codons that are rich in pyrimidine bases, one can hypothesize that these sites could serve as targets for UVinduced premutagenic lesions such as pyrimidine dimers or (6-4) photoproducts. In fact recent studies have provided experimental evidence to support this hypothesis. An important tool in the analysis of tumor samples for oncogene activations has been the polymerase chain reaction (PCR) of human tumor DNA (Saiki et al., 1985; Verlaan-de Vries et al., 1986). Synthetic oligonucleotide probes can then be used to identify the presence of point mutations in amplified DNA immobilized on nylon membranes. Analysis of human skin cancers (non-melanoma and melanoma) by these techniques have revealed point mutations in all three members of the ras gene family (Table 1). Suarez et al. (1989) have reported that 2 of 8 nonmelanoma skin tumors from XP patients contained A-T point mutations at codon 61 of the N-ras oncogene and this mutation occurred opposite a T-T sequence in the anti-sense strand. Most recently, van der Schroeff et al. (1990) analyzed DNA from 30 BCC and 12 SCC and found mutations in ras oncogenes in 4 BCC and 1 SCC. Three BCC and the one SCC contained mutations in codon 12 (GGT to TGT or GAT changing glycine to cysteine or aspartic acid) of K-ras and one BCC contained a mutation in codon 61 (CAG to CAT changing glutatmine to histidine) of the Ha-ras oncogene. Also, White and Balmain (1988) have detected a G-T point mutation at the second nucleotide of codon 12, opposite a potential pyrimidine dimer site residing in the anti-sense strand of the Ha-ras oncogene. These results suggest that UV radiation is actively involved in the induction of mutations in all ras oncogene family members of nonmelanoma skin cancers. Additionally, these results imply that UVinduced pyrimidine dimers or (6-4) photoproducts or both may be involved in the activation of ras oncogenes in these tumors. Mutational activation of ras oncogenes has also been detected in human melanomas. Padua et al. (1985) reported that DNA from a human melanoma

HONNAVARA N. ANANTHASWAMY and WILLIAME. PIERCEALL

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Table 1. Ras gene mutations in human skin cancers

Tumor type BCC BCC BCC BCC SCC SCC SCC SCC

scc* KA

KA KA KA KA Melanoma: Melanoma Melanoma Melanoma Melanoma Melanoma Melanoma Melanoma

Genelcodon No. K-rasll2 K-rasll2 Ha-rasM1 Ha-rasll2 K-rasll2 Ha-rasll2 Ha-rash2 Ha-rasl61 N-rasl6l Ha-rasll2 Ha-rash2 Ha-rasll2 Ha-rasl61 Ha-rasI61 N-rasl61 N-rasl61 N-rasl61 N-rasl61 N-rasIl2t N-rasll3t N-rasll3g N-rasll3S

Nucleotide change GGT + TGT GGT + GAT CAG -+ CAT GGC -+ GTC GGT + TGT GGC + TGC GGC + GAC CAG -+ CTG CAA + CAT GGC + AGC GGC -+ TGC GGC + GAC CAG -+ AAG CAG -+ CTG CAA -+ CATICAC CAA + AAA CAA -+ AAA CAA + CGA GGT -+ GAT GGT + GTT GGT -+ GITT GGT + GAT

Amino acid change glY CYS glY asp gln + his gly + Val @Y CYS +

+

+

glY

dY

-+

CYS

asp gln + leu gln -+ his gly + ser

dY

+

+

CY5

gly asp gln + lys gln -+ leu gln + his gln -+ lys gln + lys gln + arg asp glY gly -+ Val gly + val glY -+ asp +

+

Reference van der Schroeff et al. (1990) van der Schroeff et al. (1990) van der Schroeff et al. (1990) White and Balmain (1988) van der Schroeff ef al. (1990) Corominas ef al. (1989) Corominas et al. (1989) Corominas et al. (1989) Suarez et al. (1989) Corominas et al. (1989) Corominas et al. (1989) Corominas ef al. (1989) Corominas et al. (1989) Corominas et al. (1989) Keijzer et al. (1989) Padua ef al. (1985) van’t Veer ef al. (1989) van’t Veer et al. (1989) van? Veer et al. (1989) van’t Veer ef al. (1989) van’t Veer et al. (1989) van’t Veer et al. (1989)

*From XP patients. ?Same tumor contained mutations in both 12th and 13th codons. $Same tumor had two different mutations at the second position of codon 13.

cell line was able to transform NIH 3T3 cells and that these transformed cells contained a C-A base change at position 1 of the N-rus 61st codon, an interesting result in that this base is next to but not directly opposite a potential pyrimidine site in the anti-sense DNA strand. Keijzer et ul. (1989) have reported that a melanoma cell line from an XP patient contained a mutated N-rus oncogene and the mutation occurred within a short stretch of pyrimidine bases in codon 61. Similarly, van’t Veer et ul. (1989) have reported that 7 of 37 cutaneous melanomas from sun-exposed body sites contained mutations in codon 12, 13 or 61 of the N-rus oncogene and these mutations were all at or near dipyrimidine sites. Interestingly, these investigators found that two different N-rus mutations in the same tumor in two patients (patient number 4 and 10). In patient 4,the two mutations were present in the same codon (codon 13), and in patient 10, the two mutations were in adjacent codons (12 and 13). In addition, two different metastases in patient 4 also contained two different N-ras mutations (valine and aspargine). On the other hand, only one particular type of mutation (N-rus aspargine) was found in two different metastases in patient 10. These results indicate that the two primary melanomas were made up of two different cell populations, each with different N-rus mutations. Although both tumors were able to metastasize, there appeared to be some preference for tumors with N-rus 12 aspargine mutation. In addition, these results strongly suggest that UV radiation present in sunlight plays an active role in the induction of cutaneous melanomas in

humans. Previous correlation between exposure to sunlight and development of cutaneous melanoma was based only on circumstantial evidence. Further investigations in human skin cancers have identified codon 61 as the site of specific mutations in the c-Hams oncogene. Corominas et ul. (1989) reported that 30% of human keratoacanthomas (KA), a benign skin cancer that undergoes periodic regression, and 13% of SCC contained specific mutations (A:T to T:A transversions) in the second position of codon 61 of the c-Ha-ras oncogene. The high percentage of c-Ha-ras mutations found in KA as compared to SCC suggests that activated c-Harus genes may play a role in initiation but not in progression. Similarly, Shukla et ul. (1989) found rus gene mutations in 2 of 4 benign atypical nevi, 4 of 22 primary melanomas, and 4 of 14 secondary tumors demonstrating that rus activation may be an early event in melanoma development. To ascertain the timing of rus oncogene activation in the process of carcinogenesis, Kumar et al. (1990) devised an animal model system that involved induction of mammary carcinomas in rats exposed at birth to the carcinogen NMU. Restriction fragment length polymorphism (RFLP) analysis of PCR-amplified rus sequences revealed that mutated H-rus and K-rus oncogenes were present two weeks after carcinogen treatment and two months before the onset of neoplasia. These results suggest that ras activation is an extremely early event in the onset of mammary carcinogenesis. These results are further substantiated by the work of Greenhalgh et ul. (1989), in which cells possessing a c-Hams mutated at

Yearly Review codon 61 produced benign tumors when grafted into nude mice, but cells transfected with a v-Ha-ras produced papillomas that developed rapidly into SCC. Cells containing both a mutated c-Ha-ras and a v-Ha-ras produced undifferentiated, rapidly lethal carcinomas. This suggests that different mutations and gene dosages of Ha-ras could contribute to initiation, malignant conversion, and malignant progression in skin carcinogenesis of cultured primary murine keratinocytes. The fact that most of the ras gene mutations in human skin cancers occurred at pyrimidine-rich sequences implies that these sites were probably the targets for UV-induced damage that eventually led to mutation and transformation. To determine whether UV radiation can activate the N-ras protooncogene in vitro, Van der Lubbe et al. (1988) exposed the naked N-ras proto-oncogene DNA to UV radiation in vitro and then transfected into Rat2 cells. Their results indicated that UV-irradiated N-ras proto-oncogene DNA, but not unirradiated DNA, led to the transformation of Rat-2 cells. Interestingly, most of these transformants contained mutations at the 61st codon which harbors a T-T doublet. These mutations were similar to those found in skin tumors produced in vivo. A few mutations were also found at the 12th codon of Nras, which harbors a C-C doublet. In addition, these investigators found that treatment of the UVirradiated N-ras proto-oncogene DNA with photoreactivating enzyme prior to transfection reduced the transformation frequency several-fold. These results suggest that DNA damage induced by in vitro UV irradiation can lead to the activation of the N-ras proto-oncogene and that pyrimidine dimers are probably the premutagenic lesions responsible for this activation of N-ras proto-oncogene. In addition to the activation of the N-ras protooncogene by in vitro UV-irradiation, activation of the human c-Ha-ras proto-oncogene by in vitro UVirradiation has also been demonstrated by Pierceall et al. (1989). Thus, these in vitro studies may help to identify the UV-induced lesions responsible for the activation of specific oncogenes. Oncogene activation in UV-induced mouse tumors In contrast to the abundance of studies on chemically-induced rodent tumors, there is little information available regarding oncogene activation in UV-induced murine skin cancers. There is a suggestion, not yet confirmed, that UV-induced mouse skin tumors express an activated Ki-ras oncogene (Strickland et al., 1985). Recently, Husain et al. (1990) have found that UV-B induced Sencar mouse papillomas and carcinomas exhibited 3- to 5-fold higher levels of c-Ha-ras. However, only carcinoma, and not papilloma, DNA was able to induce foci upon transfection into NIH 3T3 cells, which suggests

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that additional genetic changes are necessary for malignant progression. It is not known whether these UV-induced Sencar mouse tumors contain specific mutations in the Ha-ras oncogene, although the presence of an A:T mutation at the 61st codon of H a m s oncogene has been ruled out. An unusual mechanism involving a defect in protein kinase C (PKC) has been reported in UVinduced mouse tumors (Megidish and Mazurek, 1989). Most PKC activity in normal fibroblasts is found in the soluble cytosol fraction that can be extracted with a divalent cation-chelator (particulate/soluble ratio of 0.34.5) (Halsey et al., 1987). By contrast, transformed fibroblasts usually show reversed PKC distribution, with a particulate/soluble ratio between 1 and 2. Although the UV-induced fibrosarcoma cell lines exhibited a PKC distribution pattern like that of most transformed cells, they showed a dramatic variation (particulate/soluble ratio of 7). In particular, the UV-2240 cell line had 87% of the PKC activity associated with the membrane. Subsequent cloning and sequencing of the cDNA for a-PKC (the predominant isoform in UV-2240) revealed that it contained four point mutations, three in the highly conserved regulatory domain and one in the conserved region of the catalytic domain. Transfection of this mutant a-PKC gene into normal BALB/c 3T3 fibroblasts resulted in transformation, as revealed by anchorage-independent growth and tumorigenicity in nude mice. In addition, transformed BALB/c 3T3 cells expressing the mutant aPKC showed a membrane localization similar to that of UV-2240 cells. These results indicate that point mutations in the primary structure of PKC modulate enzyme function and are responsible for the oncogenic behavior of UV-2240 tumor cells. This implies that the PKC gene can be activated through point mutations by UV radiation. Activation of ras oncogenes in mouse tumors induced by y and neutron radiation has been reported (Guerrero et al., 1984; Newcomb et al., 1988; Diamond et al., 1988; Sloan et al., 1990). Guerrero et al. (1984) found that a c-Ki-ras oncogene was activated in mouse lymphomas induced by y-radiation. Diamond et al. (1988) found that the spectrum of ras gene mutations in neutron radiationinduced thymic lymphomas was different from that seen in thymic lymphomas induced by y-radiation in the same strain (RF/J) of mice. In a majority of y-ray-induced lymphomas, the mutations occurred predominantly at the 12th codon of the Ki-ras oncogene (substitution of GAT for GGT). In contrast, neutron radiation-induced thymic lymphomas contained mutations in Ki-ras and N-ras oncogenes. Two neutron radiation-induced lymphomas contained different mutations in codon 12 of Ki-ras (GGT + TGT and GGT -+GTT), one lymphoma contained a mutation in codon 61 of N-ras (CAA + AAA), and very interestingly, one lymphoma

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contained a mutation in codon 146 of Ki-ras (GCA

+ ACA) (Sloan et al., 1990). Activating ras mutations at codon 146 have not been detected in any known human or mouse tumors and thus appears to be unique to neutron radiation-induced mouse tumors. Concurrent activation of multiple oncogenes has been demonstrated in rat tumors induced by ionizing radiation. Sawey et al. (1987) found that c-myc and c-K-ras oncogenes were activated in a panel of ionizing radiation-induced rat tumors. Mutations in codon 12 of N-ras oncogene have also been found in y-ray-induced canine leukemia (Gumerlock et al., 1989). In addition to ras oncogenes, activation of unique non-ras transforming genes has also been reported in x-ray-transformed mammalian cells (Borek et al., 1987) and in y-radiation-induced mouse lymphomas (Newcomb et al., 1988). These results once again support the hypothesis that different DNA damaging agents induce different activating mutations in ras oncogenes in a carcinogenspecific manner. Cooperation between oncogenes Several investigators have shown that oncogenes can cooperate to induce the neoplastic state and/or enhance the transformation frequency. For example, the ras and myc family of genes are able to transform primary or secondary rat embryo fibroblasts when introduced together by DNA-mediated gene transfer (Land et al., 1983b; Ruley, 1983; Newbold and Overell, 1983; Spandidos and Lang, 1989). These studies indicate that myc or a myc-like gene (e.g. E1A) may be required for establishment or immortilization of cells and the ras gene may confer the transformed phenotype. Similarly, ras oncogenes can operate in concert with other oncogenes or genetic changes to produce the transformed state in human skin cancers also. Popescu et al. (1986) have shown that upon in vitro irradiation of fibroblasts, transformed cells arise that exhibit abnormalities on chromosome 11 (on which the human Ha-ras gene is located) and a duplication of the region of chromosome 22 in which the c-sis (plateletderived growth factor-receptor) proto-oncogene resides. Also, Compere et al. (1989) have shown the ability of ras and myc Oncogenes to cooperate in tumor induction when introduced into midgestation mouse embryos by replication-defective retroviral vectors, but because tumor incidence was only 27%, other genetic alterations must take place in addition to the presence of activated myc and ras oncogenes. Multiple oncogene alterations have also been detected in human skin cancers. Suarez et al. (1987) found that two skin tumors from the same XP patient contained an amplified c-myc gene and an amplified and rearranged c-Ha-rus gene, in addition to an activated N-ras oncogene. The activation of Nras oncogene was accompanied by an increase in the level of N-ras mRNA. Shukla et al. (1989) found

that one of the human primary melanomas had concurrent mutations in the 12th codon of Ha-ras and Ki-ras oncogenes, and two melanoma patients with secondary tumors had concurrent mutations in the 12th codon of K-ras and 61st codon of N-ras and two mutations in the 12th codon of Ki-ras (aspartic acid and valine), respectively. These observations lend credence to the hypothesis that a number of genetic and epigenetic alterations must occur for normal cells to become cancerous and ultimately invasive and metastatic. A number of genetic alterations have also been detected in human melanomas. Southern blotting with nick-translated probes of chromosomal breakpoint regions revealed that a structural alteration existed in the c-myb proto-oncogene on chromsome 6 from a cutaneous melanoma (Linnenbach et af., 1988). Subsequent molecular cloning of this locus revealed that the 3’-end of the c-myb locus is deleted before concomitant translocation of a portion of chromosome 12 to chromosome 6 (Dasgupta et al., 1989). Other genetic alterations in addition to the c-myb rearrangement in human melanoma include the appearance of polymorphic alleles for the epidermal growth factor receptor and a-PKC. These results suggest that multiple genetic alterations occur frequently in melanomas, and these alterations may not be the same in all cancers. Oncogene amplifications associated with UV carcinogenesis Oncogenic point mutation may not be the onIy means through which UV radiation induces neoplastic transformation. Numerous changes in the organization of the genomic sequences by amplification and rearrangement of specific cellular sequences may also be a critical step in UV carcinogenesis. Suarez et al. (1989) have shown that skin tumors from XP patients contained amplified Ha-ras, Nras and c-myc mRNA transcripts, in addition to a mutated N-ras and a rearranged Ha-ras oncogene. The presence of several oncogene alterations in the same tumor may have resulted from the massive accumulation of unrepaired UV-induced DNA lesions in XP skin cells. Analogous to XP tumors, human melanoma cell lines also express abnormally high copy numbers of c-Ha-ras-1 (Sekiya et al., 1985) and K-ras p21 protein products (Funato ei al., 1989). Adelaide et al. (1988) examined a human melanoma and found that the hst and int.2 oncogenes of the fibroblast growth factor family appear to be co-amplified. Shin et al. (1987) examined uncultured human melanomas by RFLP analysis and found that c-K-rus, n-myc, c-sis and c-myc are aberrantly or overexpressed in these cancers. In contrast, Ogiso et al. (1988) have found no differential oncogene expression between normal and tumor tissues of the skin, but they do imply that ratios between c-fos and c-myc may be important. Amplification of c-Ha-ras proto-oncogene has also been

Yearly Review reported in UV-induced mouse skin cancers. Husain et al. (1990) have found that UV-B induced Sencar mouse skin papillomas and carcinomas exhibit 3- to 5- fold higher levels of c-Ha-ras. Taken together, these results indicate that gene amplification may also play a role in skin carcinogenesis. UV radiation is known to activate viral DNA sequences in transformed cells. Exposure of simian virus 40 (SV40)-transformed CHO cells to UV radiation resulted in amplification of integrated SV40 sequences (Lavi and Etkin, 1981). Lucke-Huhle et al. (1986) showed that UV-irradiation of SV-40transformed CHO cells resulted in a 15- to 25-fold amplification of SV40 sequences without producing intact virus. This amplification was selective in that other genes, such as DHFR, K-ras, Ha-ras and aactin, were not expressed in abnormally high copy numbers. Tilbrook et al. (1989) have found that 16 of 24 papillomas, 5 of 5 carcinomas in situ, and 6 of 38 SCC induced by UV radiation in the hairless mouse strain Mus musculus contained papilloma virus sequences in their tumor DNA. In addition, inoculation of cell-free extracts of these UV-induced tumors, known to contain papilloma virus DNA sequences, into the skin of the hairless mouse increased the susceptibility of these mice to tumor development by subsequent UV irradiation (Reeve et al., 1989). These studies suggest a mechanism by which carcinogenesis may result from UV activation of the papilloma virus DNA sequences, or cooperation between the effects of the papilloma virus DNA gene products and a separate change induced by UV radiation to induce the neoplastic state. It is interesting to note that human papilloma viral DNA sequences have been detected in hyperkeratotic lesions from sun-damaged skin (Spradbrow et al., 1983), in KA and malignant melanoma (Scheurlen et al., 1986), in patients with epidermodysplasia verruciformis (Lutzner et al., 1984; Orth et al., 1979) and in immunosuppressed patients (Lutzner et al., 1980). Further, progression to malignancy in epidermodysplasia patients occurred only in the lesions on sun-exposed body sites. These results indicate the involvement of both papilloma virus and UV radiation in the process of neoplastic transformation. Loss of growth control and alteration in signal transduction pathways

In the development of human skin cancers, one of the effects involved in ras-mediated tumor initiation may be the suppression of melanogenesis (Tsukamoto et al., 1990). A v-Ha-ras was transfected into normal murine melanocytes and the resulting cell line lost pigmentation and produced amelanotic melanomas upon injection into nude mice. A major difference between the resulting transfected cell line and the parental cell line was that v-Ha-ras-transfected cells were devoid of tyrosine kinase activity. Thus, activated ras may be

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involved in blocking phosphorylation of a protein involved in the regulation of melanogenesis by inhibiting tyrosine kinase activity. Indeed, lack of tyrosine kinase activity may be a key step in neoplastic transformation, as the p62 yes proto-oncogene, possessing a tyrosine kinase domain and carrying homology to src, is impaired in its kinase activity in BCC and reduced 20-fold in SCC in comparison to basal keratinocytes (Krueger et al., 1990). Because no single oncogene activation can result in the onset of neoplasia, oncogenes must act in concert to direct changes in either gene expression at the nuclear level or post-translational modifications of preexisting proteins, resulting in altered function. Tofilon and Meyn (1988) have shown that cell differentiation is accompanied by a post-incision process defect to remove UV damaged stretches of DNA, thus providing a means for enhanced gene mutation. Proliferation control by genes under the influence of specific enhancer sequences may be essential as well (Lang and Spandidos, 1986). A short sequence in the polyoma virus (Py) enhancer has been identified that mediates Ha-rus activity and this sequence also mediates activation by serum and phorbol esters. This ras-responsive element (RRE) is a specific binding site for the mouse transcription factor PEA 1 and for jun. These results suggest a role for p21 in signal transduction from outside the cell to a transcription factor inside the nucleus (Imler et al., 1988). The similarity between RRE and other DNA sequences present in promotor regions of several transformation-related genes suggests that deregulated activation of RRE is a critical event in transformation. The activity of the mouse transcription element VL30 is increased by 20-fold in those cell lines expressing a mutant ras (Owen et al., 1990). The cis element did not respond to ras by neoplastic transformation in a revertant cell line, suggesting that ras-dependent alterations in transcription and transformation are related. Deletion analysis showed that a minimal 53 base pair segment is required in cis for ras transcriptional activation. Site directed deletion of the 5’ proximal binding site, defined by footprinting assays as TGACTCT, abolished ras responsiveness. In a related study, the differentiation of PC12 cells, normally requiring neuronal growth factor, can be bypassed by transfection with ras or src transforming proteins (Sassoni-Corsi et al., 1989). All ras mutants that activated differentiation also activated c-fos transcription through the dyad symmetry element. Transforming p21 activated a signal pathway that led to phorbol ester-responsive element induction. Nuclear extracts of p21-transfected cells showed an increase in AP-1 DNA-binding activity. Thus, fos and jun may play an active role in ras-induced transformation. Signal transduction appears to be mediated, at least in part, through UV radiation. UV radiation has been shown to induce plasminogen activator

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(Miskin and Ben-Ishai, 1981; Rotem et al., 1987), metallothionein and other genes (Angel et al., 1986) in human cells. Additionally, a number of biological effects such as stimulation of arachidonic acid release (DeLeo et al., 1985), up-regulation of histamine-stimulated prostaglandin E2 production, possibly through an increase in PKC levels (Steuer and Pentland, 1990), inhibition of epidermal growth factor binding (Furstenberger et al., 1981), and increased levels of the p53 cellular tumor antigen (Maltzman and Czyzyk, 1984) have been shown to occur in UV-treated cells. Since elevated levels of the p53 antigen have been correlated with some transformation events, it is hypothesized that p53 functions in the regulation of the transition of mammalian cells to active proliferation. Near-UV irradiation of human fibroblasts is known to induce a 32 kDa protein (Keysee and Tyrrell, 1987). This protein, identified as a heme oxygenase, may provide a means through which the oxidative component of UV stress may be alleviated (Keysee and Tyrrell, 1989). More recently, Glazer et al. (1989) identified several UV-induced proteins in human cells that bind to both double-stranded and singlestranded DNA. UV irradiation has also been shown to induce at least 8 proteins to abundant levels in normal, and to a lesser extent in XP cells (Schorpp et al., 1984). Moreover, a 10-kDa factor released into the cell culture supernatants elicited induction of the identical 8 proteins in unirradiated cell cultures. This suggests that a mutagen-hit cell may propogate a signal to other non-treated cells. This evidence can be juxtaposed with a study from Steinberg et al. (1990) in which UV-B irradiation inhibited dye-mediated transfer through gap junctions in human epidermal keratinocytes in a dosedependent fashion, suggesting that UV-B blocks cell-cell communication. One of the major consequences of UV radiation appears to be increased transcriptional activity. Ronai et al. (1988) have shown that RNAs for the cellular oncogenes c-Hams, c-myc, and c-fos as well as RNAs homologous to the endogenous rat leukemia virus, are induced to a great extent by UV-C radiation, and to a lesser extent by UV-B, in rat fibroblast and human keratinocyte cells in culture. Irradiation of cultured mouse keratinocytes increases the level of mRNAs for several keratins, the proteinase inhibitor cystatin A and glyceraldehyde-3-phosphate dehydrogenase (Kartasova et al., 1987). Peak et al. (1989) have shown that upon UV exposure in vitro, mRNAs for p- and y-actin and a-tubulin were induced in quiescent confluent human P3 teratocarcinoma cell lines, a response similar to that induced by tumor-promoting agents. Rosen et al. (1990) have shown recently that mRNA levels of ornithine decarboxylase are induced 4-fold in murine skin upon UV-B irradiation, while the enzyme activity is induced 20- to 30-fold. This suggests a role for UV-B in altering both transcriptional

and translational or post-translational modification efficiencies. Additionally, Chatterjee et af. (1990) have shown that in vivo chronic UV-B irradiation of skin of hairless mice induced a number of biochemical changes, including increases in elastin and glycosaminoglycan content, but that collagen was not induced, consistent with the inability of the skin to support the increasing mass of the tissue. In a related study, Stein et al. (1989) have shown that UV induced activation of genes is mediated by three different enhancer elements: the HIV-1 between positions -105 and -79, which binds a member of the NFkB family; between positions -72 and -65 of the collagenase gene, which binds a heterodimer of jun and fos (AP-1); and between positions -320 and -299 of fos. These elements share no apparent sequence motif and bind separate trans-acting factors. While AP-1 resides in the nucleus and must be modulated there, NFkB is activated in the cytoplasm, indicating that a cytoplasmic signal transduction pathway is triggered by UV-induced DNA damage. Recently, Matsui and DeLeo (1990) have shown that UV-A, like tumor promoting agents, can induce PKC, suggesting that UV-A and tumor promotor agents share a common pathway for the induction of epidermal tumors. Relatedly, UV radiation and phorbol esters cause activation of the cfos gene (Buscher et al., 1988). When the serum responsive element from -319 to -300 is destroyed, activation of c-fos, normally induced in response to both UV and phorbol ester, is impaired severely. Thus, it is not an unlikely possibility that different signal transduction pathways converge onto the same enhancer element and that one of the major effects in signal transduction is for the up-regulation of transcriptional activity through c-fos and fos responsive elements. Further support for the involvement of fos comes from the studies of Greenhalgh et al. (1990). These investigators have shown that co-infection of v-Haras and v-fos in replication-defective retroviral vectors into normal keratinocytes cooperated to produce SCC. Husain and Wick (1990) have presented evidence suggesting that melanogenesis is reduced in cells expressing high levels of fos and that c-fos transcriptional activation is correlated with proliferative growth. These studies clearly link the malignant phenotype with fos expression and, because fos is a transcriptional regulator of other genes, suggest that progression may occur as an indirect result of aberrant expression of the fos protein which changes the expression of fos-controlled cellular genes. Interestingly, Ledwith et al. (1990) have shown that anti-sense fos RNA can cause partial reversion of the transformed phenotypes including, density-dependent growth arrest, reversion to a flat cell morphology, inhibition of anchorage-independent growth, and inhibition of tumorigenicity, induced by a mutant H a m s oncogene in NIH 3T3

Yearly Review

cells. Thus, fos appears to mediate downstream effects of ras-stimulated signal transduction, although fos may amplify the proliferative signal by looping back and binding to tumor promoter responsiveIAP-1 binding consensus sequence elements, thus up-regulating ras transcriptional levels (Spandidos et al., 1988). Tumor suppressor genes in UV carcinogenesis Oncogenes such as ras act as positive growth regulators. In contrast, tumor suppressor genes act as negative growth regulators, blocking transformation and driving cells toward normality. In cells, these genes may act recessively, so that both maternal and paternal copies of the gene product must be inactivated in order for the suppressor function to be eliminated (Knudson, 1985). Two recently cloned genes, Rb and p53, are the first examples of tumor suppressors (Friend et al., 1986; Lee et al., 1987; Fung et al., 1987; Baker et al., 1989; Finlay et al., 1989; Eliyahu et al., 1989). The p53 gene is unique in that the wild-type acts as a suppressor, whereas some mutant alleles act as promoters. The genes that regulate DNA repair may also act as tumor suppressor genes, though this hypothesis is yet to be tested. Since loss of or defects in DNA repair enzymes are hallmarks of many inherited genetic disorders such as XP, ataxia telengietasia, Bloom’s syndrome, and Fanconi’s anemia, it is likely that the wild-type DNA repair enzymes play a key role in suppression of cancer through effective repair of damaged DNA. Molecular analysis of human colorectal carcinomas (Fearon et al., 1990; Fearon and Vogelstein, 1990) has revealed that tumorigenesis proceeds through a series of genetic alterations, including ras gene activation and loss of putative tumor suppressor genes on chromosomes 5,17 and 18 (Fearon et al., 1990; Fearon and Vogelstein, 1990). This type of genetic alteration may also be involved in skin tumors. Cytogenetic analysis has shown that aberrations of chromosomes 1, 2 , 3, 6 , 7, and 9 are common among human malignant melanomas (Pedersen et al., 1986) and, recently, that chromosome l q is lost in BCC (Bare et al., 1990). Loss of genetic material at several loci on eight different chromosomes were found in human malignant melanoma (Dracopoli et al., 1985). Similarly, Krontiris et al. (1985) have reported that a disproportionate number of rare Ha-ras alleles, as revealed by BamHI RFLP, were present in the genomic DNA of familial melanoma patients. In contrast, Sutherland et al. (1986) did not find any linkage between dysplastic nevus syndrome or hereditary melanoma and particular Ha-ras alleles. Although MspIlHpaII digestion of genomic DNA from human tumor samples did not show any correlation between melanoma and rare H a m s alleles, TaqI digestion of the DNA resulted in a frequency of allelic variants that

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was higher in melanoma patients than in normal controls (Radice et al., 1987). In addition to ras, loss of other genes on chromosome 3, 5 , 11, 13, 17, 18, and 22 has been reported in melanoma, retinoblastoma, Wilms’ tumor, bladder, breast, lung, and colorectal cancers (Cavenee et al., 1989; Dracopoli et al., 1985; Hayward et al., 1988, 1989; Krontiris et al., 1985; Vogelstein et al., 1989). Studies by Ananthaswamy et al. (1989) have shown that the loss of a c-Ha-ras-1 allele also occurs frequently in human BCC and SCC developing on sun-exposed body sites. Of 35 skin cancer patients (25 BCC patients and 10 SCC patients) analyzed, 17 (16 BCC patients and 1 SCC patient) were heterozygous for the c-Ha-ras gene. Of these 17 patients, 5 (4BCC and 1 SCC) showed loss of one of the c-Ha-ras alleles in their tumor DNA. Interestingly, 90% (9 of 10) of the patients with SCC contained only one of the c-Ha-ras alleles in their normal skin DNA as compared to 36% (9 of 25) of the patients with BCC. Thus there may be an association between constitutional homozygosity of the c-Ha-ras gene and genetic susceptibility to SCC. However, this remains to be established. In addition to c-Hams gene (located on the short arm of human chromosome ll:pl5), loss of other genes on chromosome 11 or on other chromosomes may occur in human nonmelanoma skin cancers. This remains to be determined. In any case, the loss of Ha-ras and other genes on chromosome 11 in a number of disparate human tumors suggests that some of the events leading to the transformation of normal cells into tumor cells may be common to a variety of human cancers. Since loss of chromosomal sequences in human and mouse tumors have been regarded as evidence that the affected regions contain tumor suppressor genes, it is quite possible that the normal c-Ha-ras allele acts as a tumor suppressor gene and loss of this allele unmasks recessive mutations leading to tumor formation. Although this possibility has not yet been tested conclusively, Bremner and Balmain (1990) have shown that chromosome 7, in which the mouse c-Ha-ras gene resides, exhibits a RFLP in skin papillomas induced by chemical carcinogens and have suggested that loss of a putative tumor suppressor gene at this locus may lead to or be a result of tumor formation. In contrast, Gerhard et al. (1987) have reported that the presence of rare Ha-ras-1 alleles is no more frequent among sporadic melanoma patients than in normal populations, suggesting that other genes must be associated with hereditary predisposition to melanoma. Spandidos and Wilkie (1988) have shown that the normal cH a m s gene can suppress transformation induced by the mutated ras gene. Kitayama et al. (1989) have reported that overexpression of a ras-related gene induced reversion to a normal phenotype in cells transformed with a mutant K-ras allele. Thus, these results indicate that the normal ras allele can

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E. PIERCEALL HONNAVARA N. ANANTHASWAMY and WILLIAM

suppress the effect of a mutant allele, such that the cells gain a selective advantage through loss of the normal allele o r duplication of the mutant allele. Summary U V radiation is a potent D N A damaging agent and a known inducer of skin cancer in experimental animals. There is excellent scientific evidence to indicate that most non-melanoma human skin cancers are induced by repeated exposure to sunlight. U V radiation is unique in that it induces D N A damage that differs from the lesions induced by any other carcinogen. The prevalence of skin cancer on sun-exposed body sites in individuals with the inherited disorder XP suggests that defective repair of UV-induced D N A damage can lead to cancer induction. Carcinogenesis in the skin, as elsewhere, is a multistep process in which a series of genetic and epigenetic events leads to the emergence of a clone of cells that have escaped normal growth control mechanisms. The principal candidates that are involved in these events are oncogenes and tumor suppressor genes. Oncogenes display a positive effect on transformation, whereas tumor suppressor genes have an essentially negative effect, blocking transformation. Activated ras oncogenes have been identified in human skin cancers. In most cases, the mutations in the ras oncogenes have been localized to pyrimidine-rich sequences, which indicates that these sites are probably the targets for UV-induced D N A damage and subsequent mutation and transformation. The finding that activation of ras oncogenes in benign and self-regressing keratoacanthomas in both humans and in animals indicates that they play a role in the early stages of carcinogenesis (Corominas et al., 1989; Kumar et al., 1990). Since cancers d o not arise immediately after exposure to physical or chemical carcinogens, rus oncogenes must remain latent for long periods of time. Tumor growth and progression into the more malignant stages may require additional events involving activation of other oncogenes or deletion of growth suppressor genes. In addition, amplification of proto-oncogenes or other genes may also be involved in tumor induction or progression. In contrast to the few studies that implicate the involvement of oncogenes in U V carcinogenesis, the role of tumor suppressor genes in U V carcinogenesis is unknown. Since cancer-prone individuals, particularly XP patients, lack one or more repair pathways, one can speculate that D N A repair genes could function as tumor suppressors. If so, the loss or defect in D N A repair enzymes would confer susceptibility to both spontaneous and environmentally induced cancers. Another potential candidate *To whom correspondence should be addressed.

that can function as a tumor suppressor gene is the normal c-Ha-ras gene. Spandidos and Wilkie (1988) have shown that the normal c-Ha-ras gene can suppress transformation induced by the mutated ras gene. Kitayama et al. (1989) have reported that overexpression of a ras-related gene induced reversion to a normal phenotype in cells transformed with a mutant K-ras allele. Our studies have indicated that homozygosity of the c-Ha-ras gene may be a predisposing factor in the development of SCC (Ananthaswamy et al., 1989). O n the other hand, the loss of c-Ha-rus allele may be coincidental with the loss of a closely linked suppressor gene. This remains to be determined. Studies addressing these questions should provide new insights into the molecular mechanisms of U V carcinogenesis. Acknowledgements-This work was supported by grant R01-CA-46523 from the National Institutes of Health. William Pierceall is the recipient of a Predoctoral Fellowship from the National Institutes of Health Training Grant T32-CA-09598.

HONNAVARA N. ANANTHASWAMY* and WILLIAME. PIERCEALL Department of Immunology University of Texas M D Anderson Cancer Center 1515 Holcombe Boulevard Houston, TX 77030 USA REFERENCES

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