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The molecular pathways of ultraviolet-induced carcinogenesis
Mutation Research 428 Ž1999. 5–10 www.elsevier.comrlocatermolmut Community address: www.elsevier.comrlocatermutres
The molecular pathways of ultraviolet-induced carcinogenesis Alain Sarasin
)
Laboratory of Molecular Genetics, UPR 42, CNRS, 94801, Villejuif, France Received 29 November 1998; accepted 11 January 1999
Abstract Cancer development requires the accumulation of numerous genetic changes which are usually believed to occur through the presence of unrepaired DNA lesions. Exogenous or endogenous DNA-damaging agents can lead to mutations in the absence of efficient error-free repair, via replication of DNA damage. Several DNA repair pathways are present in living cells and well-conserved from bacteria to human cells. The nucleotide excision repair ŽNER., the most versatile of these DNA repair systems, recognizes and eliminates a wide variety of DNA lesions and particularly those induced by ultraviolet ŽUV. light. The phenotypic consequences of a NER defect in humans are apparent in rare but dramatic diseases characterized by hypersensitivity to UV and a striking clinical and genetic heterogeneity. The xeroderma pigmentosum ŽXP. syndrome is a human disorder inherited as an autosomal recessive trait. Persistence of unrepaired DNA damage produced by exposure to UV light is associated, in the XP syndrome, with an extremely high level of skin tumors in sun-exposed sites. Several key genes are mutagenized by UV-light and are responsible for skin cancer development. Mutations are found on ras oncogenes, p53 and PTCH tumour suppressor genes in skin cancers from DNA repair proficient as well as XP patients. The typical signature of UV-induced mutations found on these genes allows one to conclude that the uvB part of sunlight is responsible for the initiation of the carcinogenesis process. q 1999 Elsevier Science B.V. All rights reserved. Keywords: DNA damage; Nucleotide excision repair; Ultraviolet; Xeroderma pigmentosum; Skin cancer; p53
1. Introduction Among all human cancers, non-melanoma skin cancers Žessentially Basal Cell Carcinoma and Squamous Cell Carcinoma. are currently estimated to have an annual incidence in USA of roughly 1,000,000, equivalent to the annual incidence of all other human malignancies in USA w1x. Nonmelanoma skin cancers represent the most severe and ultimate response to sun exposure especially in ) Tel.: q33-1-49-58-3420; Fax: q33-1-49-58-3411; E-mail: [email protected]
light-skinned individuals but are usually non-lethal and relatively easily cured. As indicated in Fig. 1, skin cancers are due to a complex of simultaneous and sequential biochemical events, initiated by UV-radiation of different wavelengths ŽuvB, uvA spectra as well as visible light and infrared.. These radiations produce DNA lesions that, in the absence of error-free repair, may give rise to mutation in proto-oncogenes and tumour suppressor genes leading therefore to cancer. During the course of carcinogenesis, other events may, however, strongly influence the appearance of cancers: various immunological responses Žwhich are usually de-
0027-5107r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 4 2 Ž 9 9 . 0 0 0 2 5 - 3
6
A. Sarasinr Mutation Research 428 (1999) 5–10
Fig. 1. Non-melanoma skin cancers are essentially due to mutations produced by unrepaired DNA lesions induced by sun exposure. uvB is directly absorbed by DNA, while uvA gives rise to lesions probably through the production of oxygen-activated species ŽROS.. Other biological partners are also implicated such as the immune response Ždecreased after UV-exposure., virus or genetic predisposition which is very important for regulating DNA repair efficiency.
creased after sun exposure., level of antioxidant defenses in an individual basis, possible infection with specific viruses, genetic predisposition, . . . In this review, I will only present data concerning the role of DNA lesions and DNA repair pathways in the induction of cancers as well as the types of modifications found in specific target genes in skin cancers. Indeed, the general dogma in carcinogenesis is the hypothesis of clonal expansion due to activation or inhibition of crucial genes implicated in the control of cell cycle, maintenance of gene integrity, proliferation and differentiation. Recently, these genes have been classified as ‘gatekeepers’ Žusually tumour suppressor genes which control cellular proliferation and regulate apoptosis, such as Rb, APC, p53 . and ‘caretakers’ Žwhich maintain the integrity
of the genomic information such as XP, MMR, ATM, BRCA1, BRCA2 . w2x. UV-induced carcinogenesis represents a complex sequence of random events following repeated exposure to sun. The uvB spectrum induces predominantly DNA lesions between two adjacent pyrimidines on the same DNA strand which are very mutagenic in the absence of repair. The very high incidence of skin cancers in the DNA repair deficient xeroderma pigmentosum ŽXP. syndrome clearly demonstrates the crucial role of DNA repair pathways in humans in order to protect us from massive tumour development. The fine tuning of repair and its exquisite regulation and interdependence with transcription and cell cycle regulation are absolutely necessary for an efficient resistance to various genotoxic attacks. The partial or total absence of error-free repair will allow mutations to occur at the site of DNA lesions after one or two rounds of cell replication. The random appearance of these mutations on proto-oncogenes, such as the ras family or on tumour suppressor genes, such as p53 or PTCH, will start a cellular process of immortalization followed by cell transformation. The demonstration of the existence of these processes is given by the fact that the molecular UV-signature at the level of mutations is found in these classes of genes in non-melanoma skin cancers. It is clear that once these mutational changes have been introduced in a target cell, cell selection and clonal expansion should follow due to the loss of cell cycle regulation andror the loss of normal differentiation. At the same time, epigenetic process should occur in order to facilitate the promotion step, such as UV-induced immune suppression or UV-induced activation of virus expression. 2. The xeroderma pigmentosum syndrome XP is a rare genetic disorder inherited as autosomal recessive trait. XP patients display pronounced cutaneous manifestations. At early age, most XP children exhibit exaggerated sunburning after minimal sun exposure. Besides acute photosensitivity, progressive dryness, telangiectasia and atrophy arise on sun-exposed sites. All cultured cell lines isolated from XP patients are characterized by hypersensitivity to UV light and UV-like chemicals, that is the
A. Sarasinr Mutation Research 428 (1999) 5–10
result of deficient repair w3–5x. In about 80% of XP cases Žclassical XP., the defect concerns the early steps of nucleotide excision repair ŽNER. mechanism. The other class ŽXP variant. represents a separate category of XP individuals, with a presumed defect in the poorly-defined postreplication repair process, but with apparently normal NER w6x. The most important characteristic of the XP syndrome is the appearance of skin tumors at a median age of around 8 years. This feature is limited to the sun-exposed parts of the body, demonstrating the causative link with the genotoxic consequences of the UV component of solar light. The marked incidence of skin tumors in the XP syndrome is associated with the NER defect and a higher mutation frequency in ras genes than that found in skin cancers from DNA repair-proficient individuals w7x. Kraemer et al. w8x have estimated that XP patients present more than a 2000-fold increment in all forms of skin cancer compared to normal individuals. 3. The nucleotide excision repair system NER is of fundamental importance in all organisms as a mechanism of protection against the effects of a wide variety of structurally unrelated lesions, including various UV-induced DNA damages, bulky chemical adducts and certain types of cross-links. The NER process requires the products of at least 20 genes in eukaryotes and these proteins have been conserved to a remarkable degree throughout eukaryotic evolution, underlining the fundamental importance of this process w9x. Globally, five steps are involved in the NER process: recognition of the DNA lesion, incision of the damaged strand on both sides of the lesion after separation of the two DNA strands by helicases, removal of the damage-containing oligonucleotide, gap-filling by DNA synthesis, and ligation. In principle, the NER pathway is error-free as it utilizes the nucleotide sequence information of the intact complementary DNA strand. For a subset of lesions, two overlapping subpathways have been identified in the NER process: the rapid transcription-coupled repair ŽTCR. of expressed genes, directed to the transcribed strand and the slower genome-overall repair of the bulk DNA, including the non-transcribed strand of active genes. The rapid TCR process allows a wild-
7
type cell to quickly recover RNA transcription and protein synthesis after genotoxic attack while DNA lesions are still present on 90% of its genome. The biological importance of these two pathways is attested by the existence of patients deficient in either one of these two processes w10x. After treatment by DNA-damaging agents, several parameters allow for the detection and evaluation of cellular abnormalities linked to an NER defect, such as reduced levels of DNA repair synthesis Žunscheduled DNA synthesis ŽUDS.., cell survival and recovery of DNA and RNA synthesis rates, and increased mutability. The isolation of NER-deficient mutants in many species including Escherichia coli, yeast, drosophila, rodent cell lines, and humans contributed to the identification and the functional characterization of most of the genes implicated in this pathway. Genetic heterogeneity in human repair disorders is demonstrated by cell complementation assay based on the correction of post-UV unscheduled DNA synthesis in hybrid cells formed by fusion of skin fibroblasts from two different patients. Restoration of normal UV-induced repair synthesis is expected only if the two patients belong to different complementation groups. This assay was useful not only to determine the complementation group of patient in a given type of disease but also to determine how many genes are involved in these syndromes and if overlapping genes may be found among them w11x. Among classical XP patients, seven complementation groups have been determined, designated XPA to XPG. The XP complementation groups differ in frequency and geographic distribution. A large variability is observed among different complementation groups both in terms of clinical features Žnumber of skin tumors, neurological abnormalities, life expectancy. and cellular properties after UV irradiation ŽDNA repair level, cell survival, induction of mutagenesis.. Most XP groups are deficient in both subpathways of NER, in transcription-coupled as well as global genome repair, except in the XPC group, in which the repair defect is limited to the global genome repair w12x. 4. Mutations in skin cancers The majority of DNA lesions induced by sun exposure Žessentially the uvB spectrum. concerns
8
A. Sarasinr Mutation Research 428 (1999) 5–10
adjacent pyrimidines. Cyclobutane-pyrimidine dimers and Ž6–4. pyrimidine–pyrimidones are the major mutagenic photoproducts which give rise to mutations mainly characterized by C to T or CC to TT transitions always located at sites of pyrimidine– pyrimidine sequences. These mutations are now considered as a true signature of sun exposure w13x. These types of mutations have been found in ras oncogenes, p53 and PTCH tumour suppressor genes isolated from skin cancers in sun exposed body sites w7,14–17x. The frequency of mutations varies between 30 to 60% of skin cancers from DNA repairproficient individuals and from 50 to 80% of skin cancers isolated from XP patients. It is interesting that in vivo as well as in vitro XP cells are hypermutagenized after UV-exposure. The CC to TT tandem transitions are absolutely specific of UV-irradiation. Interestingly, the frequency of these mutations is much higher in XP cultured cells and in XP skin cancers. This high level of tandem mutations in DNA repair-deficient patients could be due to the accumulation of unstable photoproducts with time and possible specific chemical modifications leading to these two mutations after replication ŽFig. 2.. The more often mutagenized gene in skin cancers is the tumour suppressor gene p53. Several hot spots are found in this gene which are partly identical to
those found in internal tumours and partly specific of UV-induced ones. It is believed that point mutations on p53 are an early event in skin carcinogenesis since one can find them in pretumoral lesions such as keratoacanthomas and actinic keratosis w15,16x. Even more interesting is the detection of CC to TT transversion in normal sun-exposed skin biopsies by using sophisticated LM-PCR w18x. This result indicates clearly that UV-specific point mutations are detected in normal skin, without any pathological abnormality, after regular sun-exposure. It is therefore easy to understand that these mutated cells in exposed skin can lead to unstable clones, with accumulation of genetic instability on other target genes and clonal expansion leading to tumours. It is also necessary to remember that p53 mutated cells render the cells resistant to apoptosis and survive better to subsequent UV-exposure as well as deteriorations in the regulation of cell cycle and fidelity of genetic maintenance. Recently, the PTCH gene, a new gene isolated in drosophila as part of signal transduction involved in development and cell differentiation has been implicated in the genesis of skin cancer. The PTCH gene encodes a transmembrane protein Ž12 membranespanning domains with two extra-cellular loops. close to the ABC transporter family. This protein is the receptor for the sonic hedgehog protein and interacts
Fig. 2. uvB irradiation induces mainly DNA lesions at py–py sequences. At C–C–G sites containing a methylated CpG sequence, the level of tandem CC to TT transitions is statistically higher than at other sites w16x. The spontaneous or uvBruvA induced-deamination of these sites can lead to C–T lesions or to U–T lesions. The absence of repair in XP cells increased very much the probability to find tandem mutations while unique C to T transitions are found proportionnaly at higher frequency in DNA repair proficient patients than in XP cells w16x.
A. Sarasinr Mutation Research 428 (1999) 5–10
9
Fig. 3. Several ways to transform a normal cell into a tumoural cell by UV-exposure ŽSee Section 5 for explanation..
with the SMO protein implicated in the signal transduction pathway leading to TGF-b and WNT family activations. The PTCH gene has been found to be mutated in patients afflicted with the dominant, autosomal conditions of Gorlin’s syndrome ŽBasal Cell Naevus Syndrome. characterized by skeletal abnormalities, internal cancers Žmedulloblastomas. and numerous BCC w19x. Around 50–60% sporadic BCC from DNA repair proficient and XP patients contain point mutations in the PTCH gene w17,20x. However, SCC neither present mutations on this gene, indicating that the PTCH gene is not expressed similarly in the stem cells responsible for BCC and SCC development.
5. Conclusion The paradigm of the XP disease is characterized by the relationship between unrepaired DNA lesions, induced mutations, and carcinogenesis. This model
was the first demonstration in humans of the somatic mutation theory in the initiation of cancer. It is clear that a multiprocess disease as complicated as cancer cannot be fully-studied by looking only at cultured cells. It is plausible that initiated cells, probably produced constantly in UV-exposed skin, should be either growth-inhibited or destroyed by the immunological defenses of the individuals. Several reports concerning low natural killer activity in XP patients, low interferon production in XP fibroblasts, and high UV-induced ICAM inhibition in XP cells allows one to suggest that immunosurveillance may be impaired in XP patients. This result asks the question of the role of immune response in the development of skin cancers in normal individuals. The very high level of skin cancers observed in patients after renal transplantation is the real demonstration of the fundamental role of immunosurveillance. UV-induced skin cancers as well as other types of tumours are characterized by a very high level of
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A. Sarasinr Mutation Research 428 (1999) 5–10
genetic modifications. As shown in Fig. 3, two main pathways can transform normal skin cells into tumoral cells after UV-irradiation. The first one Žtop of Fig. 3. corresponds to early mutations knocking out a gene involved in the maintenance of the genetic integrity. In such case, the mutation rates will increase at each division and tumoral cells will appear due to the accumulation of genetic changes. The DNA repair genes are the best candidates for this pathway as exemplified by the XP genetic syndrome. The second pathway Žbottom of Fig. 3. corresponds to early mutations knocking out a gene involved in the control of cell proliferation. In such case, chromosomal abnormalities will strongly increase at each cell division leading also to the accumulation of genetic changes. The tumour suppressor genes Ž p53, PTCH . as well as the ras family oncogenes can fulfil this action. Hereditary disease such as the Gorlin’s syndrome is a good example of the involvement of this category of genes in skin cancers.
References w1x H.S. Black, F.R. deGruijl, P.D. Forbes, J.E. Cleaver, H.N. Ananthaswamy, E.C. deFabo, S.E. Ullrich, R.M. Tyrrell, Photocarcinogenesis: an overview, J. Photochem. Photobiol. B 40 Ž1997. 29–47. w2x K.W. Kinzler, B. Vogelstein, Gatekeepers and caretakers, Nature 386 Ž1997. 761–763. w3x A. Sarasin, The paradox of DNA repair-deficient diseases, Cancer J. 4 Ž1991. 233–237. w4x J.E. Cleaver, Defective repair replication of DNA in xeroderma pigmentosum, Nature 218 Ž1968. 652–656. w5x K.H. Kraemer, M.M. Lee, J. Scotto, Xeroderma pigmentosum, cutaneous, ocular and neurological abnormalities in 830 published cases, Arch. Dermatol. 123 Ž1987. 241–250. w6x A.R. Lehmann, S. Kirk-Bell, C.F. Arlett et al., Xeroderma pigmentosum cells with normal levels of excision repair have a defect in DNA synthesis after UV-irradiation, Proc. Natl. Acad. Sci. USA 72 Ž1975. 219–223. w7x L. Daya-Grosjean, C. Robert, C. Drougard, H.G. Suarez, A. Sarasin, High mutation frequency in ras genes of skin tumors isolated from DNA repair deficient xeroderma pigmentosum patients, Cancer Res. 53 Ž1993. 1625–1629.
w8x K.H. Kraemer, D.D. Levy, C.N. Parris et al., Xeroderma pigmentosum and related disorders:examining the linkage between defective DNA repair and cancer, J. Invest. Dermatol. 103 Ž1994. 96–101. w9x L. Ma, J.H.H. Hoeijmakers, A.J. van der Eb, Mammalian nucleotide excision repair, Biochim. Biophys. Acta 1242 Ž1995. 137–164. w10x W. Vermeulen, A.J. van Vuuren, M. Chipoulet et al., Three unusual repair deficiencies associated with transcription factor BTF2 ŽTFIIH.: evidence for the existence of a transcription syndrome, Cold Spring Harbor Symp. Quant. Biol. 59 Ž1994. 317–329. w11x E.A. De Weerd-Kastelein, W. Keijer, D. Bootsma, Genetic heterogeneity of xeroderma pigmentosum demonstrated by somatic cell hybridization, Nature 238 Ž1972. 80–83. w12x D. Bootsma, G. Weeda, W. Vermeulen et al., Nucleotide excision repair syndromes: molecular basis and clinical symptoms, Philos. Trans. R. Soc. London, Ser. B 347 Ž1995. 75–81. w13x L. Daya-Grosjean, N. Dumaz, A. Sarasin, The specificity of p53 mutation spectra in sunlight induced human cancers, Photochim. Photobiol. 28 Ž1995. 115–124. w14x N. Dumaz, A. Stary, T. Soussi, L. Daya-Grosjean, A. Sarasin, Can we predict solar ultraviolet radiation as a causal event in human tumors by analysing the mutation spectra of the p53 gene?, Mutat. Res. 307 Ž1994. 375–386. w15x N. Dumaz, C. Drougard, A. Sarasin, L. Daya-Grosjean, Specific UV-induced mutation spectrum in the P53 gene of skin tumors from DNA repair deficient xeroderma pigmentosum patients, Proc. Natl. Acad. Sci. USA 90 Ž1993. 10529– 10533. w16x G. Giglia, N. Dumaz, C. Drougard, M.F. Avril, L. DayaGrosjean, A. Sarasin, P53 mutations in skin and internal tumors of xeroderma pigmentosum patients belonging to the complementation group C, Cancer Res. 58 Ž1998. 4402–4409. w17x N. Bodak, S. Queille, M.F. Avril, B. Bouadjar, C. Drougard, A. Sarasin, L. Daya-Grosjean, High levels of patched gene mutations in basal cell carcinomas from xeroderma pigmentosum patients, 96 Ž1999. 5117–5122. w18x A. Ouhtit, M. Ueda, H. Nakazawa, M. Ichihashi, N. Dumaz, A. Sarasin, H. Yamasaki, Quantitative detection of ultraviolet-specific p53 mutations in normal skin from Japanese patients, Cancer Epidemiology, Biomarkers and Prevention 6 Ž1997. 433–438. w19x P.W. Ingham, Transducing hedgehog: the story so far, EMBO J. 17 Ž1998. 3505–3511. w20x M.R. Gailani, M. Stahlebackdahl, D.J. Leffell, M. Glynn, P.G. Zaphiropoulos, C. Pressman, A.B. Uden, M. Dean, D.E. Brash, A.E. Bale, R. Toftgard, The role of the human homolog of Drosophila patched in sporadic basal-cell carcinomas, Nat. Genet. 14 Ž1996. 78–81.
The molecular pathways of ultraviolet-induced carcinogenesis Alain Sarasin
)
Laboratory of Molecular Genetics, UPR 42, CNRS, 94801, Villejuif, France Received 29 November 1998; accepted 11 January 1999
Abstract Cancer development requires the accumulation of numerous genetic changes which are usually believed to occur through the presence of unrepaired DNA lesions. Exogenous or endogenous DNA-damaging agents can lead to mutations in the absence of efficient error-free repair, via replication of DNA damage. Several DNA repair pathways are present in living cells and well-conserved from bacteria to human cells. The nucleotide excision repair ŽNER., the most versatile of these DNA repair systems, recognizes and eliminates a wide variety of DNA lesions and particularly those induced by ultraviolet ŽUV. light. The phenotypic consequences of a NER defect in humans are apparent in rare but dramatic diseases characterized by hypersensitivity to UV and a striking clinical and genetic heterogeneity. The xeroderma pigmentosum ŽXP. syndrome is a human disorder inherited as an autosomal recessive trait. Persistence of unrepaired DNA damage produced by exposure to UV light is associated, in the XP syndrome, with an extremely high level of skin tumors in sun-exposed sites. Several key genes are mutagenized by UV-light and are responsible for skin cancer development. Mutations are found on ras oncogenes, p53 and PTCH tumour suppressor genes in skin cancers from DNA repair proficient as well as XP patients. The typical signature of UV-induced mutations found on these genes allows one to conclude that the uvB part of sunlight is responsible for the initiation of the carcinogenesis process. q 1999 Elsevier Science B.V. All rights reserved. Keywords: DNA damage; Nucleotide excision repair; Ultraviolet; Xeroderma pigmentosum; Skin cancer; p53
1. Introduction Among all human cancers, non-melanoma skin cancers Žessentially Basal Cell Carcinoma and Squamous Cell Carcinoma. are currently estimated to have an annual incidence in USA of roughly 1,000,000, equivalent to the annual incidence of all other human malignancies in USA w1x. Nonmelanoma skin cancers represent the most severe and ultimate response to sun exposure especially in ) Tel.: q33-1-49-58-3420; Fax: q33-1-49-58-3411; E-mail: [email protected]
light-skinned individuals but are usually non-lethal and relatively easily cured. As indicated in Fig. 1, skin cancers are due to a complex of simultaneous and sequential biochemical events, initiated by UV-radiation of different wavelengths ŽuvB, uvA spectra as well as visible light and infrared.. These radiations produce DNA lesions that, in the absence of error-free repair, may give rise to mutation in proto-oncogenes and tumour suppressor genes leading therefore to cancer. During the course of carcinogenesis, other events may, however, strongly influence the appearance of cancers: various immunological responses Žwhich are usually de-
0027-5107r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 4 2 Ž 9 9 . 0 0 0 2 5 - 3
6
A. Sarasinr Mutation Research 428 (1999) 5–10
Fig. 1. Non-melanoma skin cancers are essentially due to mutations produced by unrepaired DNA lesions induced by sun exposure. uvB is directly absorbed by DNA, while uvA gives rise to lesions probably through the production of oxygen-activated species ŽROS.. Other biological partners are also implicated such as the immune response Ždecreased after UV-exposure., virus or genetic predisposition which is very important for regulating DNA repair efficiency.
creased after sun exposure., level of antioxidant defenses in an individual basis, possible infection with specific viruses, genetic predisposition, . . . In this review, I will only present data concerning the role of DNA lesions and DNA repair pathways in the induction of cancers as well as the types of modifications found in specific target genes in skin cancers. Indeed, the general dogma in carcinogenesis is the hypothesis of clonal expansion due to activation or inhibition of crucial genes implicated in the control of cell cycle, maintenance of gene integrity, proliferation and differentiation. Recently, these genes have been classified as ‘gatekeepers’ Žusually tumour suppressor genes which control cellular proliferation and regulate apoptosis, such as Rb, APC, p53 . and ‘caretakers’ Žwhich maintain the integrity
of the genomic information such as XP, MMR, ATM, BRCA1, BRCA2 . w2x. UV-induced carcinogenesis represents a complex sequence of random events following repeated exposure to sun. The uvB spectrum induces predominantly DNA lesions between two adjacent pyrimidines on the same DNA strand which are very mutagenic in the absence of repair. The very high incidence of skin cancers in the DNA repair deficient xeroderma pigmentosum ŽXP. syndrome clearly demonstrates the crucial role of DNA repair pathways in humans in order to protect us from massive tumour development. The fine tuning of repair and its exquisite regulation and interdependence with transcription and cell cycle regulation are absolutely necessary for an efficient resistance to various genotoxic attacks. The partial or total absence of error-free repair will allow mutations to occur at the site of DNA lesions after one or two rounds of cell replication. The random appearance of these mutations on proto-oncogenes, such as the ras family or on tumour suppressor genes, such as p53 or PTCH, will start a cellular process of immortalization followed by cell transformation. The demonstration of the existence of these processes is given by the fact that the molecular UV-signature at the level of mutations is found in these classes of genes in non-melanoma skin cancers. It is clear that once these mutational changes have been introduced in a target cell, cell selection and clonal expansion should follow due to the loss of cell cycle regulation andror the loss of normal differentiation. At the same time, epigenetic process should occur in order to facilitate the promotion step, such as UV-induced immune suppression or UV-induced activation of virus expression. 2. The xeroderma pigmentosum syndrome XP is a rare genetic disorder inherited as autosomal recessive trait. XP patients display pronounced cutaneous manifestations. At early age, most XP children exhibit exaggerated sunburning after minimal sun exposure. Besides acute photosensitivity, progressive dryness, telangiectasia and atrophy arise on sun-exposed sites. All cultured cell lines isolated from XP patients are characterized by hypersensitivity to UV light and UV-like chemicals, that is the
A. Sarasinr Mutation Research 428 (1999) 5–10
result of deficient repair w3–5x. In about 80% of XP cases Žclassical XP., the defect concerns the early steps of nucleotide excision repair ŽNER. mechanism. The other class ŽXP variant. represents a separate category of XP individuals, with a presumed defect in the poorly-defined postreplication repair process, but with apparently normal NER w6x. The most important characteristic of the XP syndrome is the appearance of skin tumors at a median age of around 8 years. This feature is limited to the sun-exposed parts of the body, demonstrating the causative link with the genotoxic consequences of the UV component of solar light. The marked incidence of skin tumors in the XP syndrome is associated with the NER defect and a higher mutation frequency in ras genes than that found in skin cancers from DNA repair-proficient individuals w7x. Kraemer et al. w8x have estimated that XP patients present more than a 2000-fold increment in all forms of skin cancer compared to normal individuals. 3. The nucleotide excision repair system NER is of fundamental importance in all organisms as a mechanism of protection against the effects of a wide variety of structurally unrelated lesions, including various UV-induced DNA damages, bulky chemical adducts and certain types of cross-links. The NER process requires the products of at least 20 genes in eukaryotes and these proteins have been conserved to a remarkable degree throughout eukaryotic evolution, underlining the fundamental importance of this process w9x. Globally, five steps are involved in the NER process: recognition of the DNA lesion, incision of the damaged strand on both sides of the lesion after separation of the two DNA strands by helicases, removal of the damage-containing oligonucleotide, gap-filling by DNA synthesis, and ligation. In principle, the NER pathway is error-free as it utilizes the nucleotide sequence information of the intact complementary DNA strand. For a subset of lesions, two overlapping subpathways have been identified in the NER process: the rapid transcription-coupled repair ŽTCR. of expressed genes, directed to the transcribed strand and the slower genome-overall repair of the bulk DNA, including the non-transcribed strand of active genes. The rapid TCR process allows a wild-
7
type cell to quickly recover RNA transcription and protein synthesis after genotoxic attack while DNA lesions are still present on 90% of its genome. The biological importance of these two pathways is attested by the existence of patients deficient in either one of these two processes w10x. After treatment by DNA-damaging agents, several parameters allow for the detection and evaluation of cellular abnormalities linked to an NER defect, such as reduced levels of DNA repair synthesis Žunscheduled DNA synthesis ŽUDS.., cell survival and recovery of DNA and RNA synthesis rates, and increased mutability. The isolation of NER-deficient mutants in many species including Escherichia coli, yeast, drosophila, rodent cell lines, and humans contributed to the identification and the functional characterization of most of the genes implicated in this pathway. Genetic heterogeneity in human repair disorders is demonstrated by cell complementation assay based on the correction of post-UV unscheduled DNA synthesis in hybrid cells formed by fusion of skin fibroblasts from two different patients. Restoration of normal UV-induced repair synthesis is expected only if the two patients belong to different complementation groups. This assay was useful not only to determine the complementation group of patient in a given type of disease but also to determine how many genes are involved in these syndromes and if overlapping genes may be found among them w11x. Among classical XP patients, seven complementation groups have been determined, designated XPA to XPG. The XP complementation groups differ in frequency and geographic distribution. A large variability is observed among different complementation groups both in terms of clinical features Žnumber of skin tumors, neurological abnormalities, life expectancy. and cellular properties after UV irradiation ŽDNA repair level, cell survival, induction of mutagenesis.. Most XP groups are deficient in both subpathways of NER, in transcription-coupled as well as global genome repair, except in the XPC group, in which the repair defect is limited to the global genome repair w12x. 4. Mutations in skin cancers The majority of DNA lesions induced by sun exposure Žessentially the uvB spectrum. concerns
8
A. Sarasinr Mutation Research 428 (1999) 5–10
adjacent pyrimidines. Cyclobutane-pyrimidine dimers and Ž6–4. pyrimidine–pyrimidones are the major mutagenic photoproducts which give rise to mutations mainly characterized by C to T or CC to TT transitions always located at sites of pyrimidine– pyrimidine sequences. These mutations are now considered as a true signature of sun exposure w13x. These types of mutations have been found in ras oncogenes, p53 and PTCH tumour suppressor genes isolated from skin cancers in sun exposed body sites w7,14–17x. The frequency of mutations varies between 30 to 60% of skin cancers from DNA repairproficient individuals and from 50 to 80% of skin cancers isolated from XP patients. It is interesting that in vivo as well as in vitro XP cells are hypermutagenized after UV-exposure. The CC to TT tandem transitions are absolutely specific of UV-irradiation. Interestingly, the frequency of these mutations is much higher in XP cultured cells and in XP skin cancers. This high level of tandem mutations in DNA repair-deficient patients could be due to the accumulation of unstable photoproducts with time and possible specific chemical modifications leading to these two mutations after replication ŽFig. 2.. The more often mutagenized gene in skin cancers is the tumour suppressor gene p53. Several hot spots are found in this gene which are partly identical to
those found in internal tumours and partly specific of UV-induced ones. It is believed that point mutations on p53 are an early event in skin carcinogenesis since one can find them in pretumoral lesions such as keratoacanthomas and actinic keratosis w15,16x. Even more interesting is the detection of CC to TT transversion in normal sun-exposed skin biopsies by using sophisticated LM-PCR w18x. This result indicates clearly that UV-specific point mutations are detected in normal skin, without any pathological abnormality, after regular sun-exposure. It is therefore easy to understand that these mutated cells in exposed skin can lead to unstable clones, with accumulation of genetic instability on other target genes and clonal expansion leading to tumours. It is also necessary to remember that p53 mutated cells render the cells resistant to apoptosis and survive better to subsequent UV-exposure as well as deteriorations in the regulation of cell cycle and fidelity of genetic maintenance. Recently, the PTCH gene, a new gene isolated in drosophila as part of signal transduction involved in development and cell differentiation has been implicated in the genesis of skin cancer. The PTCH gene encodes a transmembrane protein Ž12 membranespanning domains with two extra-cellular loops. close to the ABC transporter family. This protein is the receptor for the sonic hedgehog protein and interacts
Fig. 2. uvB irradiation induces mainly DNA lesions at py–py sequences. At C–C–G sites containing a methylated CpG sequence, the level of tandem CC to TT transitions is statistically higher than at other sites w16x. The spontaneous or uvBruvA induced-deamination of these sites can lead to C–T lesions or to U–T lesions. The absence of repair in XP cells increased very much the probability to find tandem mutations while unique C to T transitions are found proportionnaly at higher frequency in DNA repair proficient patients than in XP cells w16x.
A. Sarasinr Mutation Research 428 (1999) 5–10
9
Fig. 3. Several ways to transform a normal cell into a tumoural cell by UV-exposure ŽSee Section 5 for explanation..
with the SMO protein implicated in the signal transduction pathway leading to TGF-b and WNT family activations. The PTCH gene has been found to be mutated in patients afflicted with the dominant, autosomal conditions of Gorlin’s syndrome ŽBasal Cell Naevus Syndrome. characterized by skeletal abnormalities, internal cancers Žmedulloblastomas. and numerous BCC w19x. Around 50–60% sporadic BCC from DNA repair proficient and XP patients contain point mutations in the PTCH gene w17,20x. However, SCC neither present mutations on this gene, indicating that the PTCH gene is not expressed similarly in the stem cells responsible for BCC and SCC development.
5. Conclusion The paradigm of the XP disease is characterized by the relationship between unrepaired DNA lesions, induced mutations, and carcinogenesis. This model
was the first demonstration in humans of the somatic mutation theory in the initiation of cancer. It is clear that a multiprocess disease as complicated as cancer cannot be fully-studied by looking only at cultured cells. It is plausible that initiated cells, probably produced constantly in UV-exposed skin, should be either growth-inhibited or destroyed by the immunological defenses of the individuals. Several reports concerning low natural killer activity in XP patients, low interferon production in XP fibroblasts, and high UV-induced ICAM inhibition in XP cells allows one to suggest that immunosurveillance may be impaired in XP patients. This result asks the question of the role of immune response in the development of skin cancers in normal individuals. The very high level of skin cancers observed in patients after renal transplantation is the real demonstration of the fundamental role of immunosurveillance. UV-induced skin cancers as well as other types of tumours are characterized by a very high level of
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genetic modifications. As shown in Fig. 3, two main pathways can transform normal skin cells into tumoral cells after UV-irradiation. The first one Žtop of Fig. 3. corresponds to early mutations knocking out a gene involved in the maintenance of the genetic integrity. In such case, the mutation rates will increase at each division and tumoral cells will appear due to the accumulation of genetic changes. The DNA repair genes are the best candidates for this pathway as exemplified by the XP genetic syndrome. The second pathway Žbottom of Fig. 3. corresponds to early mutations knocking out a gene involved in the control of cell proliferation. In such case, chromosomal abnormalities will strongly increase at each cell division leading also to the accumulation of genetic changes. The tumour suppressor genes Ž p53, PTCH . as well as the ras family oncogenes can fulfil this action. Hereditary disease such as the Gorlin’s syndrome is a good example of the involvement of this category of genes in skin cancers.
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