P16INK4A is implicated in both the immediate and adaptative response of human keratinocytes to UVB irradiation

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Abstract
The p16(INK4A-ARF) locus plays a crucial role in the control of cellular proliferation via both the Rb and P53 pathways. We previously demonstrated that this locus is altered in human skin carcinomas. In the present study we have studied the expression of the p16(INK4A-ARF) locus following UVB irradiation of normal human keratinocytes both at the mRNA (RT-PCR) and at the protein (Western blotting) levels. Our data confirmed that P16(INK4A) protein is induced by UVB at low (30 mJ cm(2)) and high (100 mJ cm(2)) doses and is observed after a single or repeated exposure implying that this response is involved in both the immediate and adaptative response to UVB. The apparent absence of induction p16(INK4A) mRNA suggested that P16(INK4A) protein is upregulated at the post-transcriptional level. Analysis by flow cytometry and BrdU staining indicated that the highest protein level of P16(INK4A) in the cells was associated with a G(2) cell cycle arrest. Comparative analysis of P16(INK4A) and P53 showed that they were differentially modulated in keratinocytes according to the UVB dose and regimen. Low, acute or repeated UVB exposures led to accumulation of both P16(INK4A) and p53, whereas at high UVB doses, P53 and P53-dependent genes were not induced or even downregulated and only a slight but reproducible stabilization of P16(INK4A) protein was observed. In our conditions, P14(ARF) did not seem to participate in the UV response in these cells as P14(ARF) protein did not vary. These results infer that P16(INK4A) plays a role in cell cycle regulation of keratinocytes submitted to UVB irradiation. They also reinforce our previous demonstration of the importance of inactivation of this gene in UV-induced skin carcinogenesis.