UV-induced DNA damage, repair, mutations and oncogenic pathways in skin cancer

Journal of Photochemistry and Photobiology B: Biology 63 (2001) 19–27 www.elsevier.com / locate / jphotobiol

UV-induced DNA damage, repair, mutations and oncogenic pathways in skin cancer a, c b Frank R. de Gruijl *, Henk J. van Kranen , Leon H.F. Mullenders a

Department of Dermatology, Leiden Univ. Med. Ctr., Leiden, The Netherlands Department of Radiation Genetics, Leiden Univ. Med. Ctr., Leiden, The Netherlands c State Institute of Public Health and the Environment, RIVM, Bilthoven, The Netherlands b

Received 14 August 2001; accepted 14 August 2001

Abstract Repair of UV induced DNA damage is of key importance to UV-induced skin carcinogenesis. Specific signal transduction pathways that regulate cell cycling, differentiation and apoptosis are found to be corrupted in skin cancers, e.g., the epidermal growth-stimulating Hedgehog pathway in basal cell carcinomas (BCCs). Mutations in genes coding for proteins in these pathways lead to persistent disturbances that are passed along to daughter cells, e.g., mutations in the gene for the Patched (PTCH) protein in the Hedgehog pathway. Thus far only the point mutations in the P53 gene from squamous cell carcinomas and BCCs, and in PTCH gene from BCC of xeroderma pigmentosum (XP) patients appear to be unambiguously attributable to solar UV radiation. Solar UVB radiation is most effective in causing these point mutations. Other forms of UV-induced genetic changes (e.g., deletions) may, however, contribute to skin carcinogenesis with different wavelength dependencies.  2001 Elsevier Science B.V. All rights reserved. Keywords: UV radiation; Skin cancer; Oncogenic pathways; DNA damage; DNA repair

1. Introduction

1.1. On cancer Proper physiological balances (‘homeostasis’) are maintained in tissues and among cells in circulation. To this end, normal cells function under the control of their environment and cancer is basically a disease of cells whose growth and function is ‘out of control’. Cells communicate through signal transduction pathways in which cascades of chemical interactions ultimately lead to the activation or de-activation of certain cellular processes. These signaling pathways run between and within cells. Internal cellular signaling pathways exist for further control of functioning of individual cells. Cell proliferation and terminal differentiation are regulated by such internal and external signaling pathways, and cancer appears to result from disturbances in a combination of growthcontrolling pathways. The cells lose their original confine*Corresponding author: Dermatology, LUMC, Sylvius Lab. Room 3038, Wassenaarseweg 72, NL-2333 AL Leiden, The Netherlands. Fax: 131-71-5271-910. E-mail address: f.r.de [email protected] (F.R. de Gruijl). ]

ment and invade and disrupt surrounding tissues. Six essential alterations in cell physiology have recently been proposed that collectively dictate malignant growth: selfsuffiency in growth signals (‘anchorage free growth’ in vitro), insensitivity to growth-inhibitory signals, evasion of programmed cell death (apoptosis), limitless replicative potential (‘immortalization’), sustained angiogenesis, and tissue invasion and metastasis [1]. Many environmental factors may affect signaling pathways, and many are meant to in order to invoke proper cellular responses. Toxic agents may, however, adversely disturb growth-controlling pathways. The damage to proteins involved in the signal transduction will usually only have a temporary effect as proteins are broken down and synthesized in continuous renewal. Even damage to the mRNA from which the proteins are translated will have a temporary effect because the mRNA is also renewed. If these ‘epi-genetic’ interferences occur repeatedly, they may noticeably enhance or inhibit carcinogenic progression (i.e., the agent may act as a ‘promotor’ or ‘anticarcinogen’, respectively). A permanent disturbance in a signaling pathway may be introduced by damaging a gene that codes for a protein in the pathway. If the damage leads to an altered genetic code

1011-1344 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S1011-1344( 01 )00199-3

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(mutation) or a complete loss of the gene, the altered protein or its complete absence can obviously corrupt the signal transduction pathway of a cell (an agent which causes these permanent oncogenic changes could be considered as a classical ‘initiator’). This genetic defect will be passed along to daughter cells, and thus the corresponding defect in signal transduction will propagate. There are two categories of genes with direct relevance to cancer: oncogenes whose proteins contribute to cancer formation through a dominant gain of function, and tumor suppressor genes whose proteins suppress carcinogenic progression. The latter enable cancer growth through a recessive loss of function. Viruses can introduce their own oncogenes into a cell, or genes that encode proteins capable to de-activate tumor suppressor proteins. In addition to the genetic (mutational) mechanisms as described, more recently epigenetic mechanisms, exemplified by promoter hypermethylation, have been demonstrated to contribute significantly to the gene inactivation [2].

1.2. On UV radiation UV radiation is a very prominent environmental toxic agent, but it does not penetrate the human body any deeper than the skin. Conjugated bonds in organic molecules absorb shortwave UV radiation around 200 nm, but in linear repeats or in ring structures the absorption shifts to longer wavelengths [3] up to and over 300 nm, i.e., in the range of the solar spectrum at ground level [4]. Proteins which contain tryptophan or tyrosine can therefore absorb solar UV radiation and start up (photo-) chemical reactions. Thus, UV exposure may cause (‘epigenetic’) disturbances in signaling pathways. The bases in DNA all contain ring structures with an abundance of conjugated bonds, which makes DNA a very prominent absorber of UV radiation in cells. Genes in cells are, therefore, easily damaged upon UV irradiation, and mutations may subsequently occur. This implies that human skin exposed to sunlight is under continuous threat of accumulating oncogenic damage. Skin cancers are not readily induced and mainly occur at old ages, which attests to an impressive adaptation of the human skin to this continuous environmental stress.

2. Three types of skin cancers Skin cancer is a very common form of cancer among white Caucasians, and by far the most frequent form in white Caucasians living in tropical and subtropical areas: e.g., in the USA over 30% of cancer cases concern skin cancer, with more than 1 million cases per year [5]. The three main types are basal cell carcinomas (BCCs), squamous cell carcinomas (SCCs) and cutaneous melanomas (CMs). All these types show a north–south gradient over the USA, i.e., a positive correlation with ambient UV

radiation. BCC is the most common of the three, but the relative north–south increases are most substantial in SCC. Although the incidences are high, the mortality is generally low when compared to internal cancers. The obvious advantage with skin cancers is that they become visible at a very early stage and are cosmetically undesirable, which implies that therapies are applied early with correspondingly more success. People who sunburn easily and never tan run the highest risk of contracting all three types of skin cancer. SCC appears to be most straightforwardly related to the total sun (UV) exposure: these tumors occur on skin areas that are most regularly exposed (face, neck and hands) and the risk goes up with the life-long accumulated UV dose. BCC and CM do not show these simple relations to UV exposure: these tumors appear to be more related to intermittent over-exposure (episodes of sunburn, also on irregularly exposed skin) and sun exposure in childhood [6,7]. Considering the latest developments in understanding carcinogenesis, the question arises how the different types of skin cancers are related to disturbances in specific signaling pathways. Before addressing this question, we will first have a closer look at how UV radiation can damage genes that code for proteins in crucial signaling pathways.

3. UV-induced DNA damage, repair and genetical alterations Sites of neighboring pyrimidine bases in a DNA strand are preferentially damaged by UVC, UVB and UVA2 radiations, forming dimers between these bases: either a cyclobutane pyrimidine dimer (CPD), a 6-4 photoproduct (6-4PP) or its Dewar form. A dominance of pyrimidine dimers is considered to be specific for shortwave UV radiation. Moreover, methylation of cytosine has been shown to strongly enhanced the formation of dimers at pyrimidine bases when cells are exposed to UVB light [8] and those sites have been implicated as the main sites for p53 gene mutations in human skin tumors. Although these lesions are the best studied types of UV-induced DNA damage, it is good to keep in mind that UV radiation induces a much wider range of DNA damage: e.g., protein–DNA crosslinks, oxidative base damage (e.g., 8oxo-7,8-dihydroxyguanine) and single-strand breaks. These types of DNA damage can also be induced by other agents and hence, such damage would not necessarily be recognized as caused by UV radiation, but may contribute to UV carcinogenesis. The predominant solar DNA damage appears to be the pyrimidine dimers, which are repaired enzymatically by nucleotide excision repair (NER). Xeroderma pigmentosum (XP) patients lack this form of repair, and run a dramatically increased risk of all three types of skin

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cancers [9]. This indicates that NER is the main line of defence against UV carcinogenesis, which has been experimentally verified in transgenic animals with deficiencies in NER [10,11]. The increased risk of XP patients with so called XP-variant cellular phenotype shows that besides NER, an other form of cellular response is important. The XP-variant patients are proficient in NER but lack functional DNA polymerase-h [12]. This is a specialized polymerase which can bypass a (UV-) damaged base in DNA synthesis, thereby frequently inserting an adenine opposite the lesion; its activity needs to be controlled by a proofreading exonuclease to prevent mutations [13]. When mutated, the dysfunctional polymerase leads to hypermutability and an increase in sister chromatide exchange by recombinational repair [14]. In this context, it is interesting to note that most plants lack NER, and instead depend on light-activated enzymes (‘photolyases’) to repair the sun-induced pyrimidine dimers (photolyases are not present in human skin). In absence of ‘photo-repair’ genomic recombination increases dramatically [15]. This study indicates that homologous recombinational repair is an important form of repair of UV-induced DNA damage in plants. The importance of recombinational repair of UV-induced DNA damage in human skin remains to be determined, but errors in this repair process may obviously cause a different class of genetic alterations from point mutations caused by pyrimidine dimers. In line with the predominance of pyrimidine dimers it was found that UVB-induced point mutations occur almost exclusively at di-pyrimidine sites, mostly C to T transitions [16] (note that with random point mutations 75% would be expected to be associated with di-pyrimidic sites). Surprisingly, despite the frequent dimer formation at these sites, adjacent thymines did not appear to be associated with mutations; possibly by default insertion of an adenine in a synthesized DNA strand opposite a ‘non-instructive’ (i.e., damaged) base in the template strand; a typical action of polymerase-h, see above (this is referred to as the ‘A-rule’ [17]). As mentioned earlier, UV radiation can generate reactive oxygen species (ROS) which can cause oxidative damage to the DNA bases, e.g., forming 8-oxo-G. The relative importance of DNA damage from ROS will increase with increasing wavelength over the UV band [18]. 8-oxo-G is a miscoding lesion causing G to T transversions [19]. Curiously enough, UVA irradiation was found to yield many T to G transversions in the adenine phosphoribosyltransferase (aprt) gene of CHO cells [20]. An explanation could be the mispairing of A to an 8-oxo-G that was formed in the nucleoside pool instead of the genomic DNA [21]. More recent experimental studies have clearly shown that UV(B) radiation can also cause deletions [22] and chromosomal aberrations in mammalian cells, such as detected by micro-nuclei formation, i.e., UV radiation is clastogenic [23,24]. It is, however, unknown which primary DNA lesions are responsible for these more

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crude genetical effects. As DNA damage other than pyrimidine dimers may cause such genetic alterations – which may be even more carcinogenic – an optimum protection may require more than minimizing pyrimidine dimers. From the above it is clear that pyrimidine dimers are a main form of solar UV-induced DNA damage and that NER is the main line of defence against genetic alterations that these dimers may cause. However, it is also clear that other forms of DNA damage and repair play a role, and may be important under specific circumstances.

4. Archetypal UV mutations in P53 from BCC and SCC The P53 tumor suppressor gene is found to be mutated in a majority of human cancers. The p53 protein is therefore an apparent cellular ‘Achilles’ heel’, it plays a pivotal role in several signaling pathways related to DNA damage and expression of oncogenes [25]. Nuclear p53 expression is elevated after UV irradiation, and following a genotoxic insult p53 is involved in cell cycle arrest (late G1 and G2 / M), apoptosis and NER. In SCC and BCC the P53 gene appears to bear point mutations with the exact features of UVB-induced point mutations, i.e., associated with di-pyrimidinic sites, mostly C to T transitions and 5–10% CC to TT tandem mutations [26,27]. Evidently, the P53 gene is also a target in UV carcinogenesis, which has been extensively confirmed in mouse experiments [28–31]. In line with this finding, it is found that the wavelength dependency of the induction of SCC closely parallels that of the induction of pyrimidine dimers in the skin, especially over the UVB and UVA2 bands [32]. Experiments with hairless mice show that clusters of epidermal cells with mutant p53 occur long before SCC become visible [33]; such clusters of mutant p53 have also been found in human skin [34,35]. Dysfunctional p53 is likely to affect protective responses against DNA damage and oncogenic signaling. Hence, the early occurrence of P53 mutations may cause genomic instability and thus facilitate further carcinogenic progression. BCC and SCC indeed frequently show losses of chromosomal fragments, i.e., loss of heterozygozity (LOH) [36]. LOH is mostly restricted to chromosome arm 9q in BCC, whereas it is more diverse in SCC (3p, 9p, 13q, 17p, 17q). Next to mutant p53, actinic keratoses (AKs), precursors of SCC, also carry extensive LOH [37]. Although one might suspect that LOH is attributable to mutant p53, it was found that LOH also occurs without any detected mutation in the P53 gene. Considering the relation between AKs, SCC and sun exposure, the question then arises whether the observed LOH could also be directly caused by primary DNA damage from UV radiation. A mutation in the P53 gene is clearly not enough to cause BCC or SCC. At the very least some oncogenic

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pathway has to be activated; e.g., a mitogenic pathway which normally starts with the activation (oligomerization) of a receptor tyrosine kinase (RTK), e.g., EGF-R, at the cell membrane, and is further mediated through proteins like RAS into the cytoplasm from which transcription factors are finally activated. Activating RAS mutations have been reported in a minority of SCC and BCC to various percentages [38,39]. These activating mutations are restricted to the codons 12, 13 and 61, and are not specific of UV radiation. The RAS-pathway may be involved in SCC and BCC, but it is not usually effected through genetic changes in the RAS family of genes.

5. Hedgehog and BCC Patients with Gorlin syndrome, or basal cell nevus syndrome (BCNS), suffer from developmental abnormalities, internal cancers and multiple BCC. This genetic trait was traced to locus 9q22 and turned out to be carried by mutations in the Patched (PTCH ) gene [40]. Next to frequent LOH at this locus, many sporadic – non-familial – BCC showed mutations in the (remaining) PTCH allele [41]: 12 out of 37 tumors in SSCP screening, and nine of these tumors showed LOH of PTCH. The SSCP was apparently not sensitive enough as two tumors without variant SSCP or LOH were both found to have inactivating mutations. Seven of 15 mutations occurred at dipyrimidinic sites and were C to T transitions (among which two CC to TT tandem mutations), and could, therefore, have been caused by UV radiation. The mutations also included five deletions and a 300 bp duplication. UV radiation may have caused these other types of mutations, but they could also have been caused by other exogenous or endogenous genotoxic challenges. Seventy to 90% of BCC in XP patients were found to bear PTCH mutations, predominantly UV-specific mutations [42–44]. In a French study with mostly XPC patients, 63% of the mutations were CC to TT tandem mutations, compared to 11% in nonXP BCC [44]. The NER deficiency in the XP patients apparently caused a dominant contribution of pyrimidine dimers to the mutations. The PTCH gene is a human homologue of the Patched (Ptc) gene in Drosophila melanogaster. The Ptc protein stands in the Hedgehog (Hh) signaling pathway which is important in embryonic development, and well studied in Drosophila. This pathway is triggered by extra-cellular ‘Hedgehog’ (Hh) which couples to Ptc, a serpentine-like receptor woven through the cell membrane. Ptc forms a complex with an other serpentine transmembrane protein, ‘Smoothened’ (Smo). Upon coupling to Hh, Ptc relieves suppression of Smo which then activates the intracellular signaling cascade which ultimately releases zinc-finger transcription factors, ‘cubitus interruptus’ (Ci) in Drosophila and its ortholog Gli in humans. The canonical pathway in humans is depicted in Fig. 1. In contrast to

Fig. 1. A schematic representation of the Hedgehog pathway in human cells, and its connections with other oncogenic events (outer circle depicts the cell membrane and the inner one the nuclear membrane).

Drosophila, humans have at least three homologues of Hh (sonic, desert & indian), two of Ptc (PTCH1 & 2), and three of Ci ( GLI1 -3) (for a review see Ref. [45]). In Drosophila Ci is modified by cAMP-dependent protein kinase-A (PKA) from an active to a repressive form by phosphorylation and subsequent proteolysis [46]. In vertebrates a similar process renders Gli3 a potent repressor [47]. Retroviral transduction of Sonic Hedgehog (SHH ) into normal human keratinocytes resulted in expression of SHH and of the known Gli-target BMP-2B, as well as expression of BCL-2 protein [48]. The latter protein is commonly expressed in BCC and is a well-known suppressor of apoptosis. Protein levels of BCL-2 have been reported to be negatively correlated with those of P53 in human BCC [49]. Skin grafts of the SHH-transgenic keratinocytes onto immune-deficient mice show the specific histologic features of BCC [48]. Shh causes epithelial proliferation and breaks a p21(CIP1 / WAF1)-induced growth arrest [50]. GLI1 induces epidermal hyperplasia in transfected frog embryos, and it is expressed in almost all BCC [51]. This indicates that activation of the Hh pathway is essential to the formation of BCC. This has been confirmed in transgenic mouse strains. Although SCCs are readily induced in mice by chemical carcinogens or UV radiation, BCC or BCC-like tumors from epidermal appendages are hardly ever reported [52]. Such tumors do, however, ‘spontaneously’ arise in transgenic mice with an activated Hh pathway, either by

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overexpression of Shh or by mutant Smo [53,54]. Considering the crucial developmental role of the Hh pathway, it is no surprise that such animals either do not survive the perinatal period (with overexpression of Hh) or cannot reproduce (with mutant Smo). Transgenic mice with constitutive expression of Gli1 restricted to basal cells of the epidermis develop BCC and follicle-derived tumors, viz. trichoepitheliomas, trichoblastomas and cylindromas [55]. These BCCs do not carry H-Ras or P53 mutations, but the peripheral cells of a tumor did show an apparent reactive expression of high levels of the wildtype p53 protein. Ptc heterozygous knockout mice have been reported to show BCNS-like developmental abnormalities and to develop medullablastomas and rhabdomyosarcomas, but no BCC [56,57]. These animals do develop microscopically detectable follicular neoplasms resembling human trichoblastomas and 40% develop BCC-like tumors after 9 months. Upon exposure to ionizing or UV radiation the trichoblastomas and BCC occur earlier, and increase in size and numbers [58]. Moreover, these exposures cause a clear shift in histological features toward BCC. UV exposure also induces more SCC and more fibrosarcomas earlier in the Ptc1 / 2 mice when compared to their wildtype littermates, whereas ionizing radiation does not induce these tumors. The exposed wildtype mice did not develop any trichoblastomas nor any BCC. The trichoblastomas and BCC show frequent loss of the wildtype Ptc allele, and all the ones tested (n512) showed expression of Gli1 (SCC, n52, did not). Two BCCs out of five UVinduced trichoblastoma / BCC-like tumors carried p53 mutations (three in total, two C to T and one C to G). These experimental data show that UV radiation can play an important role in causing or enhancing the development of BCC. Next to the induction of p53 mutations, UV radiation could exert a more direct effect on the Shh pathway by enhanced loss of the wildtype Ptc gene and / or possible mutation of this gene or Smo.

6. INK4 a and melanoma Some familial cutaneous melanomas (CMs) are linked to markers on chromosome 9p21, which led to the positional cloning of the ‘multiple tumor suppressor’ (MTS1 ) gene [59]. The locus is designated CDKN2 A in the human genome project. It is also named INK4 a [60] after the original finding that its product p16 INK4a becomes associated with cyclin dependent kinases upon transformation of human fibroblasts by SV40 virus [61], and acts as an inhibitor of CDK4 and CDK6 [62]. CDK4 is thus prevented from phosphorylating pRB and activating the E2F-1 transcription factor, see Fig. 2. INK4 a is one out of a family of CDK inhibitor genes (INK4 a-d or CDKN2 A-D and the CIP/KIP family, p21, p27 and p57 ). At the 9p21 locus it is directly flanked by INK4 b, coding for p15 INK4b . An alternative reading frame in INK4 a codes for the

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Fig. 2. INK4a / CDKN2A transcription and signaling. Ras-induced senescence is associated with increased levels of P16 and P53, and can be blocked by P14-ARF, an alternatively spliced product of the INK4a / CDKN2A locus.

protein p14 ARF (p19 ARF in mice), which does not appear to inhibit any CDK but binds to MDM2 and thus interferes with the degradation of p53 [63]. Interestingly, E2F-1 binds upstream from exon 1b (Fig. 2), and thus leads to increased levels of p14 ARF which through stabilizing p53 [64] may counteract illicit activation of the RB pathway [65]. Conversely, wild type p53 can down regulate transcription of p14 ARF [66], thus closing a regulatory feedback loop [67]. Consequently, the loss of INK4 a disrupts two cell control pathways: one through p16 INK4a / CDK4 / 6 / pRb and the other through p14 ARF / MDM2 / p53. The loss of the 9p21 locus often involves both INK4 a and INK4 b, which implies the disruption of at least three cell cycle control pathways. However, from familial CM it appears that p16 INK4a is more consistently targeted than p14 ARF or p15 INK4b [68]. Chromosomal hotspots have been identified in CM by frequent loss of 6q and 10q and by nonrandom karyotypic rearrangements involving chromosome 1. However, the loss of heterozygosity (LOH) at 9p21 is most compellingly linked to both familial and sporadic CM [69]. Partial or complete homozygous loss of INK4 a is observed in a

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majority (about 60%) of cell lines derived from sporadic CM, and most of the remaining cell lines (e.g., eight out of 11) bear point mutations that are typical of UV radiation, i.e., C to T transitions at dipyrimidine sites [70]. Although 60–70% of sporadic melanomas (n562) show a lack of p16 INK4a expression and all (n55) of the metastases [71], the high number of homozygous losses and mutations of INK4 a found in cell lines is not reproduced in primary CM. Homozygous deletions are found in approximately 10% and reported mutation rates range from 0 to 25% [60]. The reason for this discrepancy is not entirely clear but it could be due to a high selection for a loss or mutation of INK4 a in generating the cell lines [72]. It should, however, also be noted that LOH at 9p21-22 in CM is quite common, and is even frequently observed in microdissected dysplastic nevi (in 75%), potential precursors of CM, as is LOH at 17p13 (locus of P53 ) (in 60%) [73]. De novo methylation in the promoter region of the remaining INK4 a allele could potentially silence transcription, but such methylation has thusfar only been detected in 10% of primary CM [74]. Alternatively, mutations in non-coding (59UTR) regions could play a role, as was recently found in a CM-prone family [75]. Signaling pathways related to p16 INK4a apparently play an important role in the pathogenesis of CM. And, although the INK4 a locus often shows LOH and less frequently mutations in primary CM, it is not clear if and to what extent solar UV radiation is responsible for these pertinent genetic changes.

7. RTK mitogenic pathway and INK4 a in CM Another family of genes that is implicated in CM are the RAS oncogenes, more specifically N-RAS. 25–70% of CM from regularly sun-exposed sites have been reported to carry activating point mutations in N-RAS, whereas none of the CM from irregularly exposed sites carried such mutations [76,77]. In a comparative study the percentage of N-RAS mutated CM from sun-exposed sites was higher in an Australian population (24%) than in a European population (12%) [76]. These mutations occur in the vicinity of dipyrimidine sites, the typical UV targets, but they are not dominated by C to T transitions. As mentioned earlier, the RAS proteins function in mitogenic pathways which start by activation of a RTK at the cell membrane, e.g., EGF-R, see Fig. 2. It is well known that oncogenic RAS will transform most immortal cell lines and make them tumorigenic upon transplantation into nude mice. Surprisingly, Serrano et al. [78] found that expression of oncogenic RAS (producing an activated HRAS G12V ) in primary human or rodent cells results in a state that is phenotypically characterized as ‘senescence’: the cells are viable and metabolically active but remain in the G1-phase of the cell cycle. This oncogenic RAS-

induced arrest in G1 is accompanied by an accumulation of both p16 and p53. The link between these pathways is likely to be mediated by p14 ARF (see previous section and Fig. 2). Inactivation of either p16 or p53 prevented this G1 arrest: the arrest did not occur in p532 / 2 cells, p162 / 2 cells, cells transfected with a dominant negative p53 mutant ( p53 175H ) and cells with mutant Cdk4 R24C insensitive to p16. Thus, cells immortalized by dysfunctional p16 or p53 will not go into senescence upon RAS activation, but may progress to a tumorigenic state. In a fish model (with hybrids of Xiphophorus maculatis and helleri) an RTK gene (Xmrk) of EGF-R family and an Ink4 a homologue (CdknX or DIFF ) appear to be important for hereditary CM [79,80]. This provides experimental evidence for the cooperation of an RTK mitogenic pathway and dysfuntional Ink4 a in melanomagenesis. In further evidence, Chin et al. [81] demonstrated that Ink4 a 2 / 2 mice in which expression of a human mutant H-RAS G12V transgene was restricted to melanocytes, developed melanomas. Although UV irradiation did not (yet) cause CM in this mouse model, UV irradiation of hybrid Xiphophorus fish did cause CM [82]. It is, however, as yet unclear how UV radiation affected CdknX /Ink4 a or the Xmrk /RTK mitogenic pathway.

8. Conclusions Research is rapidly gaining ground in understanding DNA repair pathways and the signaling pathways, or rather signaling networks, that play a role in cancer. Generally, cancer appears to arise from disruption of signaling needed for normal cell proliferation and homeostasis: in SCC it is possibly an activated RTK / RAS pathway in combination with dysfunctional P53 tumor suppression, in BCC the HH pathway with possibly dysfunctional P53, and in CM again possibly an activated RTK / RAS in combination with inactivation of the INK4a locus. These combinations may be required, but not necessarily sufficient for the development of a tumor. Additional oncogenic events may be necessary. Regaining control over the signaling by specific molecular targeting may offer possibilities for refined and specific future therapies against cancer. Although the skin cancers appear to be related to UV radiation, the effect of UV radiation is only unambiguously clear in point mutations of P53 in SCC and BCC, and in PTCH from BCCs of XP patients. The mutations found in the other relevant genes are of a wider variety, which may (in part) be caused by solar UV radiation. Experiments are needed to clarify if and how UVB or UVA radiation can affect other relevant genes. Knowledge on the full range of UV-induced DNA damage and ensuing genetic changes is of obvious importance, especially for a better understanding of the etiology of the most fatal skin cancer, malignant melanoma.

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Acknowledgements The authors would like to thank the Dutch Cancer Society and the European Commission for the financial support of their research groups.

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