Photochemistry and Photobiology, 1996, 63(4) 375

References

1. Brash, D. E., J. A. Rudolph, J. A. Simon, A. Lin, G. J. Mc- Kenna, H. P. Baden, A. J. Halparin and J. Ponten (1991) A role for sunlight in skin cancer: UV-induced p53 mutations in squa- mous cell carcinomas. Proc. Natl. Acad. Sci. USA 88, 10124- 10128.

2. Van Kranen, H. J., F. R. de Gruijl, A. de Vries, Y. Sontag, P. W. Wester, H. C. M. Senden, E. Rozenmuller and C. F. van Kreijl ( 1 995) Frequent p53 alterations but low incidence of ras mutations in UV-B-induced skin tumors of hairless mice. Car- cinogenesis 16, 1141-1 147.

3. Li, G., V. C. Ho, K. Berean and V. A. Tron (1995) Ultraviolet radiation induction of squamous cell carcinomas in p53 trans- genic mice. Cancer Res. 55, 2070-2074.

4. De Vries A., C. Th. M. van Oostrom, F. M. A. Hofhuis, P. M. Dortant, R. J. W. Berg, F. R. de Gruijl, P. W. Wester, C. F. van Kreijl, P. J. A. Capel, H. van Steeg and S. J. Verbeek (1995) Increased susceptibility to ultraviolet-B and carcinogens of mice lacking the DNA excision repair gene XPA. Nature 377, 169- 173.

5. Nagazawa, H., D. English, P. L. Randell, K. Nakazawa, N. Mar- tel, B. K. Armstrong and H. Yamasaki (1994) UV and skin cancer specific p53 gene mutation in normal skin as a biological relevant exposure measurement. Proc. Natl. Acad. Sci. USA 91, 637-642.

6. Armstrong, B. K. and A. Kricker (1995) Skin cancer. Derma- toepidemiology 13, 583-994.

7. De Gruijl, F. R. and P. D. Forbes (1995) UV-induced skin can- cer in a hairless mouse model. BioEssays 17, 65 1-660.

8. De Gruijl, F. R. and J. C. van der Leun (1991) Development of skin tumors in hairless mice after discontinuation of ultraviolet irradiation. Cancer Res. 51, 979-984.

9. Coebergh, J. W. W., H. A. M. Neumann, L. W. Vrints, L. van der Heijden, W. J. Meijer and M. Th. Verhagen-Teulings (1991) Trends in the incidence of nonmelanoma skin cancer in SE Netherlands 1975-1988: a registry based study. Br. J . Dermatol. 125, 353-359.

10. Scotto, J. and T. R. Fears (1982) incidence of Nonmelanoma Skin Cancer in the United States. U.S. Dept. of Health Publ. no. NIH 82-2433, National Health Institutes, Bethesda, MD.

11. De Gruijl, F. R. and J. C. van der Leun (1994) Estimate of the wavelength dependency of ultraviolet carcinogenesis in humans and its relevance to the risk assessment of a stratospheric ozone depletion. Health Phys. 67, 314-325.

12. De Gruijl, F. R. and J. C. van der Leun (1993) Influence of ozone depletion on the incidence of skin cancer; quantitative prediction. In Environmental UV Photobiology (Edited by A. R. Young, J. Moan and L. Bjom), pp. 89-1 12. Plenum Press, New York.

13. Kelfiens, G., F. R. de Gruijl and J. C. van der Leun (1991) Tumorigenesis by short-wave ultraviolet A: papillomas versus squamous cell carcinomas. Carcinogenesis 12, 1377-1382.

14. Berg, R. J. W., F. R. de Gruijl and J. C. van der Leun (1993) Interaction between ultraviolet-A and ultraviolet-B radiations in skin cancer induction in hairless mice. Cancer Res. 53, 4212- 4217.

15. De Gruijl, F. R., H. J. C. M. Sterenborg, P. D. Forbes, R. E. Davies, C. Cole, G. Kelfkens, H. van Weelden, H. Slaper and J. C. van der Leun (1993) Wavelength dependence of skin can- cer induction by ultraviolet irradiation of albino hairless mice. Cuncer Res. 53, 53-60.

16. Berg, R. J. W., A. de Laat, L. Roza, J. C. van der Leun and F. R. de Gruijl (1995) Substitution of equally carcinogenic UV-A for UV-B irradiations lowers epidermal thymine dimer levels during skin cancer induction in hairless mice. Carcinogenesis

17. Ley, R. D. (1985) Photoreactivation of UV-induced pyrimidine dimers and eruthema in marsupial Monodelphis domestica. Proc. Natl. Acad. Sci. USA 82, 2409-241 1.

18. Ziegler, A., D. J. Leffel, S. Kunala, H. W. Sharma, M. Gailani, J. A. Simon, A. J. Halperin, P. E. Shapiro, A. E. Bale and D. E. Brash (1993) Mutation hotspots due to sunlight in the p53

16, 2455-2459.

gene of nonmelanoma skin cancers. Proc. Nutl. Acad. Sci. USA 90, 42 164220.

19. Berg, R. J. W., H. J. van Kranen, H. G. Rebbel, A. de Vries, W. A. van Vloten, C. F. van Kreijl, J. C. van der Leun and F. R. de Gruijl (1996) Early p53 alterations in mouse skin carcin- ogenesis by UVB radiation: immunohistochemical detection of mutant p53 in clusters of preneoplastic cells. Proc. Natl. Acad. Sci. USA. 93, 274-278.

20. Ruven, H. J. T., C. J. M. Seelen, P. H. M. Lohman, H. van Kranen, A. A. van Zeeland and L. H. F. Mullenders (1995) Strand-specific removal of cyclobutane pyrimidine dimers from the p53 gene in the epidermis of UVB-irradiated hairless mice. Oncogene 9, 3427-3434.

21. Dumaz, N., C. Drougard, A. Sarasin and L. Daya-Grosjean (1993) Specific UV-induced mutation spectrum in the p53 gene of skin tumors from DNA-repair-deficient xeroderma pigmen- tosum patients. Proc. Natl. Acad. Sci. USA 90, 10529-10533.

Mutagenic Lesions in Carcinogenesis: Induction and Repair of Pyrimidine Dimers

Betsy M. Sutherland

Biology Department, Brookhaven National Laboratory, Upton, NY, USA

Sunlight impinging on human skin produces a panoply of damages that can induce erythema, premature aging and can- cers. Because of the central role of DNA in cellular metab- olism and replication, sunlight-induced damage to DNA is now known to be a critical step in production of many types of biological damage. The UV components in sunlight pro- duce most of the alterations of DNA, although recent evi- dence indicates that visible light may also play a role in induction of melanoma ( 1 ) . Among the types of UV-induced DNA damage, cyclobutyl pyrimidine dimers (CPD),** formed between adjacent pyrimidines on the same DNA strand, are the most numerous. Although non-CPD photo- products clearly have biological impact, current evidence in- dicates that CPD are lesions that are associated with solar oncogenesis (2).

Photorepair of UV-induced DNA damage

Plants and animals, simple and complex organisms, eukary- otes and prokaryotes have several repair systems for coping with damage to DNA. Nucleotide excision repair (NER), a multienzyme system that removes damaged DNA regions from DNA and replaces them by new synthesis, was found to be effective for dealing with CPD in bacterial cells (3,4). However, when cultured rodent cells were examined for NER, it was not found, and thus excision was thought to be absent from mammalian cells (5). Shortly thereafter, excision repair was demonstrated in human cells (6). It is now known to be

**Abbreviations: CPD, cyclobutyl pyrimidine dimer; MED, minimal erythema1 dose; NER, nucleotide excision repair; PR, photorepair; PRE, photoreactivating enzyme (photolyase); WBC, white blood cell.

376 Hasan Mukhtar et al.

a major mechanism that is used to cope with CPD and other bulky lesions. In addition, preferential repair of such lesions in transcribed genes lends further efficiency to the strategy human cells use for dealing with such damage (7).

Photorepair (PR) is a single-enzyme repair system in which a photolyase or photoreactivating enzyme (PRE) re- verses CPD to their parental monomers using visible or near UV light as an energy source. It was found first in a lower eukaryote (8) and from a prokaryote (9) but was then re- ported to be absent in mammalian cells (10,ll). Then several investigators-Sutherland, using biochemical assays (12); Harm, using bacterial transformation assays (1 3,14); Wagner ( I 5) and Henderson (1 6), who measured viral reactivation- found PR activity in human tissues, cells or their extracts. In addition, PR of CPD in human skin in situ was measured by two independent groups using different methods (1 7-1 9). However, Roza and his colleagues were able to detect PR in human skin only after preirradiation with visible light (20). This suggested that genotype or local or systemic sun- light exposure might affect PR activity levels, or that exper- imental conditions, for example, light intensity and spectral distribution, could determine the relative contributions of various repair paths in humans.

Against this background of differing results, the failure of Li and colleagues (21) to find PR enzyme activity in human WBC (white blood cells) using CPD-specific assays sug- gested that previous results might have reflected repair of nondimer lesions. However, recent results of Sutherland and Bennett (22), obtained using three defined DNA substrates and two CPD-specific endonucleases, showed that active photolyase was present in extracts of human WBC and that the enzyme could also carry out cellular photorepair of CPD in genomic DNA.

UV-induced lesions in DNA that produce oncogenic mu tations

If human cells have multiple, efficient repair pathways for removing or reversing pyrimidine dimers induced by UV, what lesions actually produce mutations leading to skin can- cer? A priori one might think that unrepaired lesions might be a random subset of the total induced lesions, perhaps in proportion to the incident frequency. However, chromato- graphic measurement of dimer frequencies in isolated DNA (23) and sequence-level analysis of defined sequence oli- gonucleotide (24) indicate that T-T dimers are produced in highest numbers, whereas they are rather rare as mutagenic lesions in sunlight-induced skin cancers (25,26). Another hy- pothesis might be that the base composition of individual dimers (T-T, C-T, T-C, C-C) might determine the repair rate of all dimers of that composition. The tendency of poly- merases to insert an A opposite a noncoding lesion, there- fore, could lessen the mutagenicity of T-T dimers. Exami- nation of critical sequences in the p53 gene sequences of individual squamous cell and basal cell carcinomas, how- ever, indicates that dimers of the same base composition at different sites can be quite different in mutagenic potential. Light on this perplexing question was provided by the work of Tornaletti et al., who showed that cultured human cells repaired different regions of the p53 gene at different rates, and that sites for mutations observed in human skin cancers

were repaired poorly compared to several other regions of the gene (27). They suggested that differences in local chro- matin structure could be responsible for the differential re- pair. However, the recent work of Sutherland and Bennett (22) on isolated defined sequence oligonucleotides indicates differential repair rates at different sequence sites, even in the absence of chromatin scaffolding on the substrate DNA.

Potential oncogenic target

What properties make a particular site in DNA a potential oncogenic target? First, the bases must have a finite to high probability of alteration by the oncogene. For the case of sunlight, the quantum yield or probablility of altering adja- cent pyrimidines is much higher than a pyrimidine surround- ed by purines, or than for purines. Second, there must be a significant biological consequence of alteration of DNA se- quence at that site. Thus mutations will remain silent if the region is located in a nonexpressed region, if posttranscrip- tional modifications make the information in that region non- informational, if coding ambiguity allows the specification in the resulting protein of the same amino acid or if the mutation leads to a codon for an amino acid that does not significantly alter the function of the protein. Finally, there must be poor repair at that site by all cellular repair systems, all constitutive and inducible pathways, including excision and photorepair.

Conclusions

Normal human recreational, occupational, cosmetic or medi- cal exposure to natural sunlight or artificial UV sources in- duces high frequencies of CPD in exposed skin. One minimal erythema1 dose (MED) produces from 30 to - 150 CPDklb (Mb, one million DNA bases) in humans of varying skin type, and exposures up to 4-5 MED are not uncommon. Thus hu- man skin cells may be confronted with as many as 750 CPD/ Mb. This may be compared with the -40 produced at the D,, of cultured human skin fibroblasts by 254 nm UV. Consid- ering the high frequency of initial damage that human skin may meet, the multiple redundancies in genetic information, repair systems and protein structure combine to provide re- markably efficient protection against solar oncogenesis.

Acknowledgement-This research was supported by the Office of Health and Environmental Research of the U.S. Department of En- ergy.

References

1. Setlow, R. B., E. Grist, K. Thompson and A. D. Woodhead (1993) Wavelengths effective in induction of malignant mela- noma. Proc. Natl. Acad. Sci. USA 90, 6666-6670.

2. Brash, D. E., S. Seetharam, K. H. Kraemer, M. M. Seidman and A. Bredberg (1987) Photoproduct frequency is not the major determinant of UV base substitution hot spots or cold spots in human cells. Proc. Natl. Acad. Sci. USA 84, 3782-3786.

3. Setlow, R. B. and W. L. Carrier (1964) The disappearance of thymine dimers from DNA: an error-correcting mechanism. Proc. Natl. Acad. Sci. USA 51, 226-231.

4. Boyce, R. P. and P. Howard-Flanders (1964) Release of ultra- violet light-induced thymine dimers from DNA in E. coli K12. Proc. Natl. Acad. Sci. USA 51, 293-300.

5. Trosko, J. E., E. H. Y. Chu and W. L. Carrier (1965) The in- duction of thymine dimers in ultraviolet-irradiated mammalian cells. Radiat. Res. 24. 667-672.

Photochemistry and Photobiology, 1996, 63(4) 377

6. Regan, J. D., J. E. Trosko and W. L. Carrier (1968) Evidence for excision of ultraviolet-induced pyrimidine dimers from the DNA of human cells in vitro. Biophys. J. 8, 319-325.

7. Bohr, V. A., C. A. Smith, D. S . Okumoto and P. C. Hanawalt (1985) DNA repair in an active gene: removal of pyrimidine dimers from the DHFR gene of CHO cells is much more effi- cient than in the genome overall. Cell 40, 359-369.

8. Kelner, A. (1949) Effect of visible light on the recovery of Streptomyces griseus conidia from ultraviolet irradiation injury. Proc. Natl. Acad. Sci. USA 35, 73-79.

9. Dulbecco, R. (1 950) Experiments on photoreactivation of bac- teriophages inactivated with ultraviolet radiation. J. Bacteriol. 59, 329-347.

10. Cook, 3 . S. and J. R. McGrath (1967) Photoreactivating-enzyme activity in metazoa. Proc. Natl. Acad. Sci. USA 58, 1359-1365.

1 1. Cleaver, J. E. (1 966) Photoreactivation: a radiation repair mech- anism absent from mammalian cells. Biochem. Biophys. Res. Commun. 4, 569-576.

12. Sutherland, B. M., P. Runge and J. C. Sutherland (1974) DNA photoreactivating enzyme from placental mammals: origin and characteristics. Biochemistry 13, 47104715.

13. Harm, H. (1980) Damage and repair in mammalian cells after exposure to non-ionizing radiations 111. Ultraviolet and visible light irradiation of cells of placental mammals, including hu- mans, and determination of photorepairable damage in vitro. Mutat. Res. 69, 167-176.

14. Harm, H. (1976) Damage and repair in mammalian cells after ultraviolet and/or visible light treatment. In Symposium on Bi- ological Effects and Measurement of Light Sources (Edited by D. G . Hazzard), pp. 175-193. HEW, Rockville, MD.

15. Wagner, E. K., M. Rice and B. M. Sutherland (1975) Photo- reactivation of herpes simplex virus in human fibroblasts. Na- ture 254, 627-628.

16. Henderson, E. E. (1978) Host cell reactivation of Epstein-Barr virus in normal and repair-defective leukocytes. Cancer Rex 38, 3256-3263.

17. Sutherland, B. M., L. C. Harber and I. E. Kochevar (1980) Py- rimidine dimer formation and repair in human skin. Cancer Res. 40, 3181-3185.

18. D’Ambrosio, S . M., J. W. Whetstone, L. Slazinski and E. Low- ney (1981) Photorepair of pyrimidine dimers in human skin in vivo. Photochem. Photobiol. 34, 461464.

19. D’Ambrosio, S . M., E. Bisaccia, J. W. Whetstone, D. A. Scar- borough and E. Lowney (1983) DNA repair in skin of lupus erythematosus following in vivo exposure to ultraviolet radia- tion. J . Invest. Dermutol. 81, 452454.

20. Roza, L., F. R. de Gruijl, J . B. A. Bergen Henegouwen, K. Guikers, H. van Weelden, G. P. van der Schans and R. A. Baan (1991) Detection of photorepair of UV-induced thymine dimers in human epidermis by immunofluorescence microscopy. 1. In- vest. Dermatol. 96, 903-907.

21. Li, Y. F., S.-T. Kim and A. Sancar (1993) Evidence for lack of DNA photoreactivating enzyme in humans. Proc. Natl. Acad. Sci. USA 90, 43894393.

22. Sutherland, B. M. and P. V. Bennett (1995) Human white blood cells contain cyclobutyl pyrimidine dimer photolyase. Proc. Natl. Acad. Sci. USA 92, 9732-9736.

23. Setlow, R. B. and W. L. Carrier (1966) Pyrimidine dimers in ultraviolet-irradiated DNA’s. J. Mol. Bid. 17, 237-254.

24. Brash, D. E. and W. A. Haseltine (1982) UV-induced mutation hotspots occur at DNA damage hotspots. Nature 298, 189-192.

25. Brash, D. E., J. A. Rudolph, J. A. Simon, A. Lin, G. J. Mc- Kenna, H. P. Baden, A. J. Halperin and J . Ponten (1991) A role for sunlight in skin cancer: UV-induced p53 mutations in squa- mous cell carcinoma. Proc. Natl. Acad. Sci. USA 88, 10124- 10128.

26. Ziegler, A,, D. J. Leffell, S. Kunala, H. W. Sharma, M. Gailani, J. A. Simon, A. J. Halperin, H. P. Baden, P. E. Shapiro, A. E. Bale and D. E. Brash (1993) Mutation hotspots due to sunlight in the p53 gene of nonmelanoma skin cancers. Proc. Natl. Acad. Sci. USA 90, 42 164220.

27. Tornaletti, S . and G. Pfeifer (1994) Slow repair of pyrimidine dimers at pS3 mutation hotspots in skin cancer. Science 263, 1436-1438.

Mutagenic Lesions in Photocarcinogenesis: The Fate of Pyrimidine Photoproducts in Repair- Deficient Disorders

James E. Cleaver

Laboratory of Radiobiology & Environmental Health, University of California, San Francisco, CA, USA

The DNA nucleotide excision repair in human cells is a fine- ly controlled, complex system involving recognition proteins that bind to damaged D N A (xeroderma pigmentosum [XP] A) , t t single-strand DNA binding proteins (human single- strand binding protein [HSSB], XPC), components of tran- scription factor (TF)IIH (helicases) that deliver repair gene products t o actively transcribed genes and to other genomic regions and several nucleases (XPG, excision repair cross- complementing group 1 [ERCC 11, XPF), polymerases and ligases. Up to 30 proteins can be involved in the overall process (1,2). Repair acts o n a large variety of different kinds of DNA damage but with rates that can differ widely according to the nature of the damage and its location. Py- rimidine-pyrimidinone (6-4) photoproducts are excised from D N A with a half-life of less than 1 h, compared to over 15 h for the more common cyclobutane pyrimidine dimers. Pho- toproducts in actively transcribed regions of the genome are excised more rapidly than from the rest of the genome, and the transcribed strand is repaired more rapidly than the non- transcribed strand (3).

DNA repair deficiencies and UV-induced cancer

Repair is altered in characteristic ways in different repair deficient disorders (4). In most cases of XP there is a general reduction of repair for both classes of photoproducts. In tri- chothiodystrophy, a disease of photosensitivity, development and hair structure, there appears to be a defect in repair of cyclobutane dimers in the overall genome, but repair of (6- 4) photoproducts is normal (5). In Cockayne syndrome, a disease of photosensitivity development and dysmyelination, there is a more restricted defect in repair of cyclobutane dimers in transcriptionally active regions of the genome, but (6-4) repair may again be normal (6). Conversely, XP group C involves a specific defect in the repair of dimers and (6- 4) photoproducts in the nontranscribed regions of the ge- nome (7). These three diseases and their repair deficiencies give the impression of a correlation between UV-induced cancer in XP and the failure to repair the (6-4) photoproduct.

Control of DNA repair

Associated with these complex controls for D N A repair and their deficiencies in single gene disorders, there are also con- siderable variations in the precise level of D N A repair, which can be related to cancer susceptibility in cells f rom

VtAbbreviations: ERCCI, excision repair cross-complementing group 1; HSSB, human single-strand binding protein; TFIIH, tran- scription factor IIH; XP, xeroderma pigmentosum.