Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
Involvement of p16CDKN2A in cell cycle delays after low dose UV irradiation
Introduction
Skin cancer is the most common human malignancy and is strongly associated with exposure to ultraviolet (UV) radiation. The UV radiation in sunlight is subdivided into three wavebands, UVC (190–280 nm) which is absorbed by the earth's atmosphere, while UVB (280–320 nm) and UVA (320–400 nm) both penetrate the earth's ozone layer. UVB is the major waveband involved in the development of skin cancer, primarily targeting DNA, producing 6–4 photoproducts and cyclobutane pyrimidine dimers, which are also produced by UVC exposure in vitro (reviewed in Ref. [1]). The depletion of the ozone layer is expected to contribute to increased penetration of UV radiation with a consequently increased rate of UV-induced DNA damage and higher incidence of skin cancer [2].
Exposure to UV radiation induces a variety of cellular responses including transcriptional induction of a number of genes including c-jun, c-fos and TP53 (Ref. [3]and references therein) and cell cycle effects 4, 5, 6, 7. Little is known regarding the mechanisms by which UV light affects the cell cycle machinery. In mammalian cells, cell cycle progression is controlled by the sequential formation and activation of cyclin/cyclin-dependent kinase (cdk) complexes (reviewed in Ref. [8]). The recent identification of low molecular weight inhibitors of cyclin/cdk complexes, the p16 and p21 families of proteins, has introduced a new mechanism for regulating cell cycle progression (reviewed in Ref. [9]). The expression of some of these proteins has been shown to be upregulated in response to cellular stress. For example, the p53 tumour suppressor protein has been shown to participate in DNA damage-activated cell cycle checkpoints 4, 10, in part by up regulating the expression of p21 7, 11, 12, 13.
p16 is the founding member of a rapidly growing family of inhibitors of cyclin/cdk complexes which specifically bind and inhibit the activity of cdk4 and cdk6 14, 15, which are in turn required for the transition from G1 in to S phase in the eukaryotic cell cycle [16]. Deletion or mutational inactivation of CDKN2A, the gene encoding p16, could lead to disruption of the cell cycle and as a consequence, contribute to tumorigenesis.
CDKN2A maps to the chromosomal region 9p21–22 and has been identified as a melanoma susceptibility gene (reviewed in Ref. [17]). Analysis of a number of CDKN2A mutations identified in affected members of melanoma kindreds showing linkage to 9p21–22, has confirmed that they encode functionally compromised p16 proteins [18]. This suggests that p16 is a tumour suppressor and that loss of p16 function may be a predisposing event in familial melanoma. The CDKN2A gene is also mutated or deleted in cell lines and primary tumour cells from a wide variety of cancer types 19, 20, 21, indicating that it may have a more general role in tumour suppression.
Previous work in this laboratory has demonstrated that in response to low doses of UVC radiation, HeLa cells were delayed in the S and G2/M phases of the cell cycle [5]. Similar UV-induced delays have been observed by others 4, 6, 7. In HeLa cells, the G2 delay correlated with elevated p16 levels, and loss of p16 expression resulted in the loss of the UVC-induced cell cycle delay [22]. We were interested to see whether this UV response was abrogated in melanoma cell lines which express mutated p16 or else carry homozygous deletions of CDKN2A. We have therefore investigated the cell cycle and growth effects of both UVC and UVB radiation on a number of different melanoma cell lines. We now report that induction of p16 following UV irradiation results in a G2 cell cycle delay in those cells expressing functional p16, but that melanoma cell lines which have lost functional p16 expression do not delay in G2 following irradiation, and accumulate significantly more DNA damage.
Section snippets
Cell lines and culture conditions
Melanoma cell lines, MM96L, MM384, MM473, SK-MEL-13, SK-MEL-28 and A2058, 18-11-Ti (HPV-transformed human keratinocytes) and HeLa (human cervical carcinoma) were cultured as monolayers in RPMI-1640 medium supplemented with 0.1 mg/ml streptomycin, 100 U/ml penicillin and 10% fetal bovine serum or 5–10% fetal bovine serum. All cell lines used were shown to be free of mycoplasma infection.
Ultraviolet radiation
Irradiation with UVC was performed using a UVS-52 Mineralite lamp (Ultraviolet Products, San Gabriel, CA) with
UVC and UVB irradiation produce similar cell cycle and growth effects
Equitoxic doses of UVC and UVB radiation (10 J m−2 and 100 J m−2, respectively) produced similar cell cycle delays in G2/M at 24 h in both HeLa cells and the melanoma line A2058 (Fig. 1A). Another melanoma line, MM96L, showed little change in its cell cycle distribution, even with higher doses (30 J m−2 UVC; data not shown), although a small increase in the G2/M population was consistently observed at 24 h (Fig. 1B). Both cell lines overcome the G2/M delay by 48 h and return to a normal cell
Discussion
The work described here provides further support for a role for elevated levels of p16 in the initial G2/M delay seen in response to both UVC and the physiologically relevant UVB radiation. This data is summarised in Table 1. In 3/3 cell lines (HeLa, A2058 and 18-11-Ti) expressing wild type p16 which accumulated to greater than 4-fold following UV irradiation, an initial G2/M delay was observed. The delay in HeLa and A2058 cells is due to a block in progression through G2 phase, as the
Acknowledgements
The authors are grateful to Dr. Peter Parsons for the cell lines and Dr. Marina Castellano for helpful discussions. This work was supported by the Queensland Cancer Fund. BG is a QIMR Fuiczek Fellow.
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