Affiliations of authors: Department of Dermatology, Boston University School of Medicine, Boston, MA (PSA, IV, TMR); Lineberger Cancer Center, University of North Carolina, Chapel Hill (NES); Department of Adult Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA (RADP)
Correspondence to: Thomas M. Rünger, Boston University School of Medicine, Department of Dermatology, 609 Albany St., Boston, MA 02118 (e-mail: truenger{at}bu.edu)
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
In sporadic and familial melanoma, the INK4a/ARF locus at 9p21 often sustains deletions or point mutations (58). This locus encodes two tumor suppressor proteins that regulate p53 and Rb (9). p16INK4a inhibits cyclin-dependent kinase activity, leading to Rb hypophosphorylation and therefore impeding S phase entry and subsequent cell division (10). The other product of the INK4a/ARF locus, p14ARF (p19ARF in mice), inhibits MDM2-mediated degradation of p53, and therefore, in addition to other p53-mediated effects, also impedes subsequent cell division (1114).
Until now, loss of the INK4a/ARF locus has not been directly linked to reduced DNA repair. However such a link is possible, given the association between reduced DNA repair and melanoma. To investigate the possible role of p16INK4a and p19ARF in DNA repair, we measured repair of DNA photoproducts in mouse embryonal fibroblasts lacking p16INK4a and/or p19ARF and in INK4a/ARF-intact littermate control cells (1517). Because the lack of cell cycle arrest of p16INK4a-null and p19ARF-null cells after UV irradiation could alter the processing of DNA photoproducts, we used an assay to assess DNA repair without irradiating the cells with UV. This host cell reactivation assay with the pYZ289 plasmid was performed as reported previously (18,19) but modified slightly by using a liposome-mediated transfection procedure (Lipofectamine Plus; Invitrogen, Frederick, MD) and a different type of DNA damage. The plasmid was irradiated with UVB outside the cells, transfected into the mouse host cells for repair, recovered from the cells after 24 hours, and then assayed for survival by transforming Escherichia coli strain MLB100. Plasmid survival is contingent upon removal of the UVB-induced DNA photoproducts and plasmid replication and therefore assesses DNA repair. The mutagenesis marker gene supF in the plasmid was used to screen for mutations (i.e., the result of error-prone repair) by a color reaction in transformed bacteria.
As expected, plasmid survival decreased with increasing plasmid DNA damage (by increasing UVB dose) in all host cell lines (Fig. 1). However, INK4a/ARF/ cells (lacking both p16INK4a and p19ARF) were found to have a 3.7- to 10.5-fold (P<.001, two-sample t test for difference in means) lower survival of UV-damaged plasmid compared with littermate INK4a/ARF+/+ (wild-type) cells (Fig. 1), indicating a reduced DNA repair capacity. Furthermore, both p16INK4a/ and p19ARF/ cells also showed a reduced (1.6- to 2.2-fold, P<.001 and 2.1- to 3-fold, P<.001, respectively; two-sample t test for difference in means) plasmid survival compared with littermate wild-type cells. Plasmid survival was 1.7- to 6-fold (p16INK4a/) and 1.2- to 5-fold (p19ARF/) higher than survival in the INK4a/ARF/ cells, indicating that both gene products affect DNA damage processing and that a loss of both functions has an additive effect. Transfection with unirradiated or irradiated plasmid did not change the cell cycle profile, as seen in flow cytometric analysis of propidium iodidestained cells of all four cell lines (data not shown). Thus, the difference in DNA repair efficiency cannot be explained by a different reaction of the INK4a/ARF/ cells to the transfection procedure or to DNA damage on the plasmid.
|
|
Stabilization and activation of p53 have been shown to induce global genome nucleotide excision repair (24,25). Therefore, it is perhaps not surprising that loss of p19ARF, an upstream regulator of p53, results in reduced DNA repair efficiency. More surprisingly, our data also link p16INK4a to the repair of DNA photoproducts. Cells expressing human papillomavirus E7 protein with subsequent low levels of hypophosphorylated Rb demonstrate reduced global genome nucleotide excision repair of DNA photoproducts (26). Loss of p16 can also lead to reduced levels of hypophosphorylated Rb by increasing levels of hyperphosphorylated Rb (9), and it is tempting to speculate that the increased levels of hyperphosphorylated Rb might explain deficient DNA repair in p16INK4a/ cells.
Consistent with our results, mice lacking p19ARF have been reported to be highly susceptible to UV-induced melanoma, suggesting that this alteration of DNA repair contributes to tumorigenesis in vivo (27). The model in the study by Kannan et al. was characterized by a single, low-dose UV exposure in RAS-expressing neonatal mice. Although mice lacking p16INK4a only were not tumor-prone, analysis of tumors from these mice demonstrated that Rb pathway inactivation was a required and rate-limiting feature of the model. Therefore, loss of p16INK4a was epistatic to UV treatment in that model. In normal humans, however, many more genetic lesions are no doubt required for melanoma formation, and therefore the decrease in DNA repair induced by p16INK4a deficiency is still likely to be important in the human disease.
Although it is generally thought that p16INK4a and p19ARF suppress malignant proliferation in melanocytes through their acknowledged tumor suppressor roles, our work suggests an additional possibility that reduced repair of UV-induced DNA damage and increased mutation formation with loss of the INK4a/ARF locus might also contribute to melanoma formation. Because the DNA repair defect in xeroderma pigmentosum cells has been shown to affect fibroblasts and melanocytes equally (28), we believe that it is reasonable to assume that the repair deficiency and ultraviolet hypermutability in fibroblasts of INK4a/ARF/ mice is also present in other cell types of these micemelanocytes in particular. It remains to be determined whether INK4a/ARF heterozygous cells are already DNA repairdeficient or whether INK4a/ARF heterozygous individuals are more prone to melanoma because their cells are more likely to become DNA repairdeficient because only one hit instead of two hits is necessary to inactivate INK4a/ARF. Cell-specific differences, e.g., in the apoptotic response to UV exposure (29), might explain why loss of the INK4a/ARF locus does not predispose to other types of UV-induced skin cancer.
![]() |
NOTES |
---|
![]() ![]() ![]() ![]() |
---|
We thank T. Yagi (University of Kyoto, Japan) for kindly providing the shuttle vector plasmid pYZ289.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|
1 Wei Q, Lee JE, Gershenwald JE, Ross MI, Mansfield PF, Strom SS, et al. Repair of UV light-induced DNA damage and risk of cutaneous malignant melanoma. J Natl Cancer Inst 2003;95:30815.
2 Kraemer KH, Lee MM, Scotto J. Xeroderma pigmentosum. Cutaneous, ocular, and neurologic abnormalities in 830 published cases. Arch Dermatol 1987;123:24150.
3 Kraemer KH, Lee MM, Andrews AD, Lambert WC. The role of sunlight and DNA repair in melanoma and nonmelanoma skin cancer. The xeroderma pigmentosum paradigm. Arch Dermatol 1994;130:101821.
4 Baccarelli A, Calista D, Minghetti P, Marinelli B, Albetti B, Tseng T, et al. XPD gene polymorphism and host characteristics in the association with cutaneous malignant melanoma risk. Br J Cancer 2004;90:497502.[CrossRef][ISI][Medline]
5 Chin L, Merlino G, DePinho RA. Malignant melanoma: modern black plague and genetic black box. Genes Dev 1998;12:346781.
6 Hussussian CJ, Struewing JP, Goldstein AM, Higgins PA, Ally DS, Sheahan MD, et al. Germline p16 mutations in familial melanoma. Nat Genet 1994;8:1521.[ISI][Medline]
7 Fitzgerald MG, Harkin DP, Silva-Arrieta S, MacDonald DJ, Lucchina LC, Unsal H, et al. Prevalence of germ-line mutations in p16, p19ARF, and CDK4 in familial melanoma: analysis of a clinic-based population. Proc Natl Acad Sci U S A 1996;93:85415.
8 Haluska FG, Hodi FS. Molecular genetics of familial cutaneous melanoma. J Clin Oncol 1998;16:67082.[Abstract]
9 Sharpless E, Chin L. The INK4a/ARF locus and melanoma. Oncogene 2003;22:30928.[CrossRef][ISI][Medline]
10 Serrano M, Hannon GJ, Beach D. A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4. Nature 1993;366:7047.[CrossRef][ISI][Medline]
11 Zhang Y, Xiong Y, Yarbrough WG. ARF promotes MDM2 degradation and stabilizes p53: ARF-INK4a locus deletion impairs both the Rb and p53 tumor suppression pathways. Cell 1998;92:72534.[ISI][Medline]
12 Kamijo T, Weber JD, Zambetti G, Zindy F, Roussel MF, Sherr CJ, et al. Functional and physical interactions of the ARF tumor suppressor with p53 and Mdm2. Proc Natl Acad Sci U S A 1998;95:82927.
13 Stott FJ, Bates S, James MC, et al. The alternative product from the human CDKN2A locus, p14(ARF), participates in a regulatory feedback loop with p53 and MDM2. EMBO J 1998;17:500114.
14 Pomerantz J, Schreiber-Agus N, Liegeois NJ, Silverman A, Alland L, Chin L, et al. The Ink4a tumor suppressor gene product, p19Arf, interacts with MDM2 and neutralizes MDM2's inhibition of p53. Cell 1998;92:71323.[ISI][Medline]
15 Serrano M, Lee H, Chin L, Cordon-Cardo C, Beach D, DePinho RA. Role of the INK4a locus in tumor suppression and cell mortality. Cell 1996;85:2737.[ISI][Medline]
16 Sharpless NE, Bardeesy N, Lee KH, Carrasco D, Castrillon DH, Aguirre AJ, et al. Loss of p16INK4a with retention of p19ARF predisposes mice to tumorigenesis. Nature 2001;413:8691.[CrossRef][ISI][Medline]
17 Sharpless NE. The preparation and immortalization of primary murine cells. In: Celis J, editor. Cell biology: a laboratory handbook. London (UK): Elsevier Science. In press 2004.
18 Tzung TY, Runger TM. Reduced joining of DNA double strand breaks with an abnormal mutation spectrum in rodent mutants of DNA-PKcs and Ku80. Int J Radiat Biol 1998;73:46974.[CrossRef][ISI][Medline]
19 Moriwaki S, Yagi T, Nishigori C, Imamura S, Takebe H. Analysis of N-methyl-N-nitrosourea-induced mutations in a shuttle vector plasmid propagated in mouse O6-methylguanine-DNA methyltransferase-deficient cells in comparison with proficient cells. Cancer Res 1991;51(23 Pt 1):621923.[Abstract]
20 Eskandarpour M, Hashemi J, Kanter L, Ringborg U, Platz A, Hansson J. Frequency of UV-inducible NRAS mutations in melanomas of patients with germline CDKN2A mutations. J Natl Cancer Inst 2003;95:7908.
21 Seetharam S, Waters HL, Seidman MM, Kraemer KH. Ultraviolet mutagenesis in a plasmid vector replicated in lymphoid cells from patient with the melanoma-prone disorder dysplastic nevus syndrome. Cancer Res 1989;49:591821.[Abstract]
22 Moriwaki S, Tarone RE, Kraemer KH. A potential laboratory test for dysplastic nevus syndrome: ultraviolet hypermutability of a shuttle vector plasmid. J Invest Dermatol 1994;103:712.[Abstract]
23 Moriwaki SI, Tarone RE, Tucker MA, Goldstein AM, Kraemer KH. Hypermutability of UV-treated plasmids in dysplastic nevus/familial melanoma cell lines. Cancer Res 1997;57:463741.[Abstract]
24 Smith ML, Seo YR. p53 regulation of DNA excision repair pathways. Mutagenesis 2002;17:14956.
25 Adimoolam S, Ford JM. p53 and DNA damage-inducible expression of the xeroderma pigmentosum group C gene. Proc Natl Acad Sci U S A 2002;99:1298590.
26 Therrien JP, Drouin R, Baril C, Drobetsky EA. Human cells compromised for p53 function exhibit defective global and transcription-coupled nucleotide excision repair, whereas cells compromised for pRb function are defective only in global repair. Proc Natl Acad Sci U S A 1999;96:1503843.
27 Kannan K, Sharpless NE, Xu J, O'Hagan RC, Bosenberg M, Chin L. Components of the Rb pathway are critical targets of UV mutagenesis in a murine melanoma model. Proc Natl Acad Sci U S A 2003;100:12215.
28 Kraemer KH, Herlyn M, Yuspa S, Clark WH, Townsend GH, Neises G, Hearing V. Reduced DNA repair in cultured melanocytes and nevus cells from a patient with xeroderma pigmentosum. Arch Dermatol 1989;125:2638.[Abstract]
29 Gilchrest BA, Eller MS, Geller AC, Yaar M. The pathogenesis of melanoma induced by ultraviolet radiation. N Engl J Med 1999;340:13418.
30 Werninghaus K, Handjani RM, Gilchrest BA. Protective effect of alpha-tocopherol in carrier liposomes on ultraviolet-mediated human epidermal cell damage in vitro. Photodermatol Photoimmunol Photomed 1991;8:23642.[ISI][Medline]
31 Runger TM, Epe B, Moller K. Processing of directly and indirectly ultraviolet-induced DNA damage in human cells. Recent Results Cancer Res 1995;139:3142.[Medline]
Manuscript received April 8, 2004; revised August 31, 2004; accepted September 10, 2004.
This article has been cited by other articles in HighWire Press-hosted journals:
![]() |
||||
|
Oxford University Press Privacy Policy and Legal Statement |