Methoxsalen

High Mutation Frequency at Ha-ras Exons 1-4 in Squamous Cell Carcinomas from PUVA-treated Psoriasis Patients

ABSTRACT

Clinical follow-up studies have revealed that PUVA-- treated patients are at increased risk of skin cancer, particularly squamous cell carcinoma (SCC). However, since psoralen and UVA (PUVA) is not only a potent mutagen and carcinogen but also an immunosuppressor, and since other (co)carcinogenic factors often exist in psoriasis patients, the exact causes and mechanisms of PUVA-associated SCC are still not completely understood. In order to fill this gap the tools of molecular epidemiology are being used to study the SCC mutational spectra of p53 and Ha-ras, two of the most commonly mutated genes in human cancers. A previous mutation analysis revealed that SCC in PUVA-treated patients often carried mutated p53 genes and that many of the mutations had the UV fingerprint (ie. C-->T or CC-->TT transitions at dipyrimidine sites). In the present study DNA-sequencing analysis revealed a total of 18 Ha-ras missense or nonsense mutations at exons 1-4 in 13 of 17 SCC (76%) from 8 of 11 (73%) PUVA-treated psoriasis patients. Six of the 18 mutations (33%) were of UV-fingerprint type (C->T transitions), five (28%) were at 5'-TpG sites (ie. potential psoralen-binding sites and thus potentially caused by PUVA) and seven were of other type (39%), including six G:C-->T:A transversions at hotspot codon 12. In addition, in the case of 6 of the 11 subjects (55%) both tumor and normal skin samples contained a T:A-4C:G base change at codon 27 (a 5'-ATT site), a change previously hypothesized to be a possible silent Ha-ras polymorphism at one allele. When we compared the present Ha-ras mutation spectrum with the p53 mutation spectrum from a previous study of the samples, we found that approximately half of the tumors harbored mutations in both Ha-ras and p53. Together, our results indicate that Ha-ras mutations are present in a large proportion of PUVA-associated SCC and that UVB, PUVA and other agents may induce Ha-ras mutations and act together with p53 in the formation of SCC in psoriasis patients.

^Abbreviations: GDP, guanosine diphosphate; GTP, guanosine triphosphate; HPV, human papillomavirus; MOP, methoxypsoralen; PCR, polymerase chain reaction; PUVA, psoralen and UVA; SCC, squamous cell carcinoma; SSCP, single-strand conformation polymorphism.

REFERENCES

1. Griffin, A. (1959) Methoxsalen in ultraviolet carcinogenesis in the mouse. J. Invest. Dermatol. 32, 367-372.

2. Pathak, M., F. Daniels, C. Hopkins and T. Fitzpatrick (1959) Ultraviolet carcinogenesis in albino and pigmented mice receiving furocoumarins, psoralen and 8-methoxypsoralen. Nature 183, 728-739.

3. Kripke, M., W. Morison and J. Parrish (1982) Induction and transplantation of routine skin cancers induced by methoxsalen plus ultraviolet (320-400 nm) radiation. J. Natl. Cancer Inst. 68, 685-690.

4. Ananthaswamy, H. (1985) Neoplastic transformation of C3H mouse embryo 10T1/2 cells by 8-methoxypsoralen plus UVA radiation. J. Invest. Dermatol. 85, 102-106.

5. Yang, S., J. Lin, C. Chiou, L. Chen and J. Yang (1994) Mutation specificity of 8-methoxypsoralen plus two doses of UVA irradiation in the hprt gene in diploid human fibroblasts. Carcinogen 15, 201-207.

6. Gunther, Y., F. Gasparro and P. Glazer (1995) Mutagenesis by 8-methoxypsoralen and 5-methylangelicin photoadducts in mouse fibroblasts: mutations at cross-linkable sites induced by adducts as well as cross-links. Cancer Res. 55, 1283-1288.

7. Stem, R. and N. Laird (1994) The carcinogenic risk of treatments for severe psoriasis. Photochemotherapy follow-up study. Cancer 73, 2759-2764.

8. Strauss, G., B. Bridges, M. Greaves, P. Hall-Smith, M. Price and D. Vella-Briffa (1980) Inhibition of delayed hypersensitivity reaction in skin (DNCB test) by 8-methoxypsoralen photochemotherapy: possible basis for pseudo-promoting action in skin carcinogenesis? Lancet II, 556-559.

9. Kripke, M., W. Morison and J. Parrish (1983) Systemic sup

pression of contact hypersensitivity in mice by psoralen plus UVA radiation (PUVA). J. Invest. Dermatol. 81, 87-92.

10. Maier, H., M. Schemper, B. Ortel, M. Binder, A. Tanew and H. Honigsmann (1996) Skin tumors in photochemotherapy for psoriasis: a single-center follow up of 496 patients. Dermatology 193. 185-191.

11. Brash, D., J. Rudolph, J. Simon, A. Lin, G. McKenna, H. Baden, A. Halperin and J. Ponten (1991) A role for sunlight in skin cancer: UV-induced p53 mutations in squamous cell carcinoma. Proc. Natl. Acad. Sci. USA 88, 10 124-10 128.

12. Kress, S., C. Sutter, P. Strickland, H. Mukhtar, J. Schweizer and M. Schwartz (1992) Carcinogen-specific mutational pattern in the p53 gene in ultraviolet B radiation-induced squamous cell carcinomas of mouse skin. Cancer Res. 52, 6400-6403.

13. Rady, P., F. Scinicariello, R. Wagner and S. Tyring (1992) p53 mutations in basal cell carcinomas. Cancer Res. 52, 3804-3806. 14. Campbell, C., A. Quinn and J. Rees (1992) Codon 12 Harvey

ras mutations are rare events in non-melanoma human skin cancer. Br. J. Dermatol. 128, 111-114.

15. Kanjilal, S., W. Pierceall, K. Cummings, M. Kripke and H. Ananthaswamy (1993) High frequency of p53 mutations in ultraviolet radiation-induced murine skin tumors: evidence for strand bias and tumor heterogeneity. Cancer Res. 53, 2961-2964.

16. Moles, J., C. Moyret, B. Guillot, P. Jeanteur, J. Guilhou, C. Theillet and N. Basset-Seguin (1993) p53 gene mutations in human epithelial skin cancers. Oncogene 8, 583-588.

17. Ziegler, A., D. 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, 4216-4220.

18. Ziegler, A., A. Jonason, D. Lefell, J. Simon, H. Sharma, J. Kimmelmann, L. Remington, T. Jacks and D. Brash (1994) Sunburn and p53 in the onset of skin cancer. Nature 372, 773-776.

19. Kubo, Y., Y. Urano, K. Yoshimoto, H. Iwahana, K. Fukuhara, S. Arase and M. Itakura (1994) p53 gene mutations in human skin cancers and precancerous lesions: comparison with immunohistochemical analysis. J. Invest. Dermatol. 102, 440-444.

20. Nakazawa, H., D. English, P. Randell, K. Nakazawa, N. Martel, B. Armstrong and H. Yamasaki (1994) UV and skin cancer: specific p53 gene mutation in normal skin as a biologically relevant exposure measurement. Proc. Natl. Acad. Sci. USA 91, 360-364.

21. Nelson, M., J. Einspahr, D. Alberts and C. Balfour (1994) Analysis of p53 gene in human precancerous actinic keratosis lesions and squamous cell cancers. Cancer Lett. 85, 23-29.

22. Ren, Z., A. Hedrum, F. Ponten, M. Nister, A. Ahmadian, J. Lundeberg, M. Uhlen and H. Ponten (1996) Human epidermal cancer and accompanying precursors have identical p53 mutations different from p53 mutations in adjacent areas of clonally expanded non-neoplastic keratinocytes. Oncogene 12, 765-773.

23. Nataraj, A., P. Wolf, L. Cerroni and H. Ananthaswamy (1997) p53 mutation in squamous cell carcinomas from psoriasis patients treated with psoralen + UVA (PUVA). J. Invest. Dermatol. 109. 238-243.

24. Wang, X., J. McNiff, V. Klump, M. Asgari and F. Gasparro (1997) An unexpected spectrum of p53 mutations from squamous cell carcinomas in psoriasis patients treated with PUVA. Photochem. Photobiol. 66, 294-299.

25. Nataraj, A., H. Black and H. Ananthaswamy (1996) Signature p53 mutation at DNA crosslinking sites in methoxypsoralen and ultraviolet A (PUVA)-induced murine skin cancer. Proc. Natl. Acad. Sci. USA 93, 7961-7965.

26. Sage, E. and A. Bredberg (1991) Damage distribution and mutation spectrum: the case of 8-methoxypsoralen plus UV-A in mammalian cells. Mutat. Res. 263, 217-222.

27. Sage, E.. E. Dobretsky and E. Moustacchi (1993) 8-Methoxypsoralen induced mutations are highly targeted at crosslinkable sites of photoaddition on the non-transcribed strand of a mammalian chromosomal gene. EMBO J. 12, 397-402.

28. Chiou, C. and J. Yang (1995) Mutagenicity and specific mutation spectrum induced by 8-methoxypsoralen plus a low dose of UVA in the hprt gene in diploid human fibroblasts. Carcinogen 16, 1357-1362.

29. Cimino, G., H. Gamper, S. Isaacs and J. Hearst (1985) Psoralens as photoactive probes of nucleic acid structure and function: organic chemistry, photochemistry, and biochemistry. Annu. Rev. Biochem. 54, 1151-1193.

30. Yun, M., S. Choi and S. Shim (1992) A novel photoadduct of 4,5',8-trimethylpsoralen and adenosine. Photochem. Photobiol. 55, 457-460.

31. Sage, E. and M. Moustacchi (1987) Sequence context on 8methoxypsoralen photobinding to defined DNA fragments. Biochemistry 26, 3307-3314.

32. Boyer, V., E. Moustacchi and E. Sage (1988) Sequence specificity in photoreaction of various psoralen derivatives with DNA: role in biological activity. Biochemistry 27, 3011-3018.

33. Aronheim, A., D. Engelbert, N. Li, N. Al-Alawi, J. Schlessinger and M. Karin (1994) Membrane targeting of the nucleotide exchange factor Sos is sufficient for activating the Ras signaling pathway. Cell 78, 949-961.

34. Bar, D. (1989) RAS proteins: biological effects and biochemical targets (review). Anticancer Res. 9, 1427-1438.

35. Hall, A. (1990) The cellular functions of small GTP proteins. Science 249, 635-640.

36. Medema, R. and J. Bos (1993) The role of p21 RAS in receptor tyrosine kinase signaling. Crit. Rev. Oncol. 4, 615-661.

37. Trahey, M. and F. McCormick (1987) A cytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants. Science 238, 542-545.

38. Barbacid, M. (1987) Ras genes. Annu. Rev. Biochem. 56, 779827

39. Van der Schroeff, E., A. Boot and J. Bos (1990) Ras oncogene mutations in basal cell carcinomas and squamous cell carcinomas of human skin. J. Invest. Dermatol. 94, 423-425.

40. Saranath, D., S. Chang, L. Bhoite, R. Panchal, I. Kerr, A. Mehta, N. Johnson and M. Deo (1991) High frequency mutation in codons 12 and 61 of H-ras oncogene in chewing tobacco-related human oral carcinoma in India. Br. J. Cancer 63, 573-578.

41. Anderson, J., J. Irish and B. Ngan (1992) Prevalence of RAS oncogene mutation in head and neck carcinomas. J. Otolaryngol. 21, 321-326.

42. Clark, L., K. Edington, I. Swan, K. McLay, W. Newlands, L. Wills, H. Young, P. Johnston, R. Mitchell and G. Robertson (1993) The absence of Harvey ras mutations during development and progression of squamous cell carcinomas of the head and neck. Br. J. Cancer 68, 617-620.

43. Pellison, L, C. Soler, Y. Chardonnet, S. Euvrard and D. Schmitt (1996) A possible role for human papillomaviruses and c-myc,cH-ras, and p53 gene alterations in malignant cutaneous lesions from renal transplant recipients. Cancer Detect. Prev. 20, 2030.

44. Sakata, K. (1996) Alterations of tumor suppressor genes and the H-ras oncogene in oral squamous cell carcinoma. J. Oral Pathol. Med. 25, 302-307.

45. T&minen, V. and G. Pfeifer (1992) Mapping of UV photoproducts within ras proto-oncogenes in UV-irradiated cells: correlation with mutations in human skin cancer. Oncogene 7, 1729-1736.

46. Chapon, D., E. Chen, A. Levinson, P. Seeburg and D. Goeddel (1983) Complete nucleotide sequences of the T24 human bladder carcinoma oncogene and its normal homologue. Nature 302, 33-37.

47. Hsieh, L., H. Chen, J. Hsieh, S. Jee, G. Chen and C. Chen (1994) Arsenic-related Bowens disease and paraquat-related skin cancerous lesions show no detectable ras and p53 alterations. Cancer Lett. 86, 59-65.

48. Pai, E., U. Krengel, G. Petsko, R. Goody, W. Kabsch and A. Wittinghofer (1990) Refined crystal structure of the triphosphate conformation of H-ras p21 at 1.35 A resolution: implications for the mechanism of GTP hydrolysis. EMBO J. 9, 2351-2359.

49. Valencia, A., M. Kjeldgaard, E. Pai and C. Sander (1991) GTPase domains of ras p21 oncogene protein and elongation factor Tu: analysis of three-dimensional structures, sequence families, and functional sites. Proc. Natl. Acad. Sci. USA 88, 5443-5447.

50. Corominas, K., J. Leon and A. Pellicer (1989) Oncogene activation in human benign tumors of the skin (keratoacanthomas):

is HRAS involved in differentiation as well as proliferation. Proc. Natl. Acad. Sci. USA 86, 6372-6376.

51. Pierceall, G., M. Tainsky, T. Mukhopadhyay and H. Ananthaswamy (1991) Ras gene mutation and amplification in human nonmelanoma skin cancers. Mol. Carcinogen. 4, 196-202.

52. Wang, D., D. Kreutzer and J. Essigmann (1998) Mutagenicity and repair of oxidative DNA damage: insights from studies using defined lesions. Mutat. Res. 400, 99-115.

53. Ananthaswamy, H. and W. Pierceall (1990) Yearly review: Molecular mechanisms of ultraviolet radiation carcinogenesis. Photochem. Photobiol. 52, 1119-1136.

54. Czerniak, B., G. Cohen, P. Etkind, D. Deitch, H. Simmons, F. Herz and L. Koss (1992) Concurrent mutations of coding and regulatory sequences of the Ha-ras gene in urinary bladder carcinomas. Hum. Pathol. 23, 1199-1204.

55. Matsui, M., C. Nishigori, S. Toyokuni, J. Takada, M. Akaboshi, M. Ishikawa, S. Imamura and Y. Miyachi (1999) The role of oxidative DNA damage in human arsenic carcinogenesis: detection of 8-hydroxy-2'-deoxyguanosine in arsenic-related Bowen's disease. J. Invest. Dermatol. 113, 26-31.

56. Seidl, H., H. Kreimer-Erlacher, B. Back, H. Soyer, G. HOfler, H. Kerl and P. Wolf (2001) UV exposure as the main initiator of p53 mutations in basal cell carcinomas from psoralen and UVA (PUVA)-treated patients with psoriasis. J. Invest. Dermatol. (In press)

57. Zarbl, H., S. Sukumar, A. Arthur, 0. Martin-Zanca and M. Barbacid (1985) Direct mutagenesis of Ha-ras-1 oncogenes by N-- nitroso-N-methylurea during initiation of mammary carcinogenesis in rats. Nature 315, 382-385.

58. Lewin, B. (1997) Genes VI. Oxford University Press, Oxford. 59. Milburn, M., L. Tong, A. Brunger, Z. Yamaizumi, S. Nishimura and S. Kim (1990) Molecular switch for signal transduction: structural differences between active and inactive forms of protooncogenic ras proteins. Science 247, 939-945.

60. De Vos, A., L. Tong, M. Milburn, P. Matias, J. Jancarik, S. Noguchi, S. Nishimura, K. Miura, E. Ohtsuka and S. Kim (1988) Three-dimensional structure of an oncogenic protein: catalytic domain of human c-H-ras p21. Science 239, 888-893.

61. Pai, E., W. Kabsch, U. Krengel, K. Holmes, J. John and A. Wittinghofer (1989) Structure of the guanine-nucleotide-binding domain of the Ha-ras oncogene product p21 in the triphosphate conformation. Nature 341, 209-214.

62. Serrano, L., M. McCurrach, D. Beach and S. Lowe (1997) Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16 INK4a. Cell 88, 593-602.

63. Harwood, C., P. Spink, T. Surentheran, I. Leigh, J. Hawke, C. Proby, J. Breuer and J. McGregor (1998) Detection of human papillomavirus DNA in PUVA-associated non-melanoma skin cancers. J. Invest. Dermatol. 111, 123-127.

64. Favre, M., G. Orth, S. Majewski, S. Baloul, A. Pura and S. Jablonska (1998) Psoriasis: a possible reservoir for human papillomavirus type 5, the virus associated with skin carcinomas of epidermodysplasia verruciformis. J. Invest. Dermatol. 110, 311317.

65. Weissenborn, S., R. Hopfl, F. Weber, H. Smola, J. Pfister and P. Fuchs (1999) High prevalence of a variety of epidermodysplasia verruciformis-associated human papillomaviruses in psoriatic skin of patients treated or not treated with PUVA. J. Invest. Dermatol. 113, 122-126.

66. Berkhout, R. J., L. M. Tieben, H. L. Smits, J. N. Bavinck, B. J. Vermeer and J. ter Schegget (1995) Nested PCR approach for detection and typing of epidermodysplasia verruciformis-associated human papillomavirus types in cutaneous cancers from renal transplant recipients. J. Clin. Microbiol. 33, 690-695.

67. Boxman, I. L., R. J. Berkhout, L. H. Mulder, M. C. Wolkers, J. N. Bouwes Bavinck, B. J. Vermeer and J. ter Schegget (1997) Detection of human papillomavirus DNA in plucked hairs from renal transplant recipients and healthy volunteers. J. Invest. Dermatol. 108, 712-715.

68. Burns, P., C. Kemp, J. Gannon, D. Lane, R. Bremner and A. Balmain (1991) Loss of heterozygosity and mutational alteration of the p53 gene in skin tumours of interspecific hybrid mice. Oncogene 6, 2363-2369.

69. Kemp, C., L. Donehower, A. Bradley and A. Balmain (1993) Reduction of p53 gene dosage does not increase initiation or promotion but enhances malignant progression of chemically induced skin tumors. Cell 74, 813-822.

70. Hinds, P., C. Finlay, R. Quartin, S. Baker, E. Fearon, B. Vogelstein and A. Levine (1990) Mutant p53 DNA clones from human colon carcinomas cooperate with ras in transforming primary rat cells: a comparison of the "hot spot" mutant phenotypes. Cell Growth Differ. 1, 571-580.

71. Lehman, T., W. Bennett, R. Metcalf, J. Welsh, J. Ecker, R. Modali, S. Ullrich, J. Romano, E. Appella and J. Testa (1991) p53 mutations, ras mutations, and p53-heat shock 70 protein complexes in human lung carcinoma cell lines. Cancer Res. 51, 4090-4096.

72. Mitsudomi, T., S. Steinberg and M. Nau (1992) p53 gene mutations in non-small-cell lung cancer cell lines and their correlation with the presence of ras mutations and clinical features. Oncogene 7, 171-180.

73. Pierceall, W., M. Kripke and H. Ananthaswamy (1992) N-ras mutation in ultraviolet radiation-induced murine skin cancers. Cancer Res. 52, 3946-3951.

74. Evans, M., B. Taffe, C. Harris and V. Bohr (1993) DNA strand bias in the repair of the p53 gene in normal human and xeroderma pigmentosum group C fibroblasts. Cancer Res. 53, 53775381.

Heidemarie Kreimer-Erlacher, Hannes Seidl, Barbara Back, Helmut Kern and Peter Wolf*

Department of Dermatology, Karl-Franzens University, Graz, Austria

Received 4 January 2001; accepted 5 May 2001

(par)Posted on the website on 8 June 2001.

*To whom correspondence should be addressed at: Department of Dermatology, Karl-Franzens University, Auenbrugger Platz 8, A-- 8036 Graz, Austria. Fax: 43-316-385-2466; e-mail: peter.wolf@kfunigraz.ac.at

Copyright American Society of Photobiology Aug 2001
Provided by ProQuest Information and Learning Company. All rights Reserved