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. 2010 Mar 15;243(3):399-404.
doi: 10.1016/j.taap.2009.12.014. Epub 2009 Dec 28.

Arsenite induced poly(ADP-ribosyl)ation of tumor suppressor P53 in human skin keratinocytes as a possible mechanism for carcinogenesis associated with arsenic exposure

Elena V Komissarova  1 , Toby G Rossman
Affiliations

Arsenite induced poly(ADP-ribosyl)ation of tumor suppressor P53 in human skin keratinocytes as a possible mechanism for carcinogenesis associated with arsenic exposure

Elena V Komissarova et al. Toxicol Appl Pharmacol. .

Abstract

Arsenite is an environmental pollutant. Exposure to inorganic arsenic in drinking water is associated with elevated cancer risk, especially in skin. Arsenite alone does not cause skin cancer in animals, but arsenite can enhance the carcinogenicity of solar UV. Arsenite is not a significant mutagen at non-toxic concentrations, but it enhances the mutagenicity of other carcinogens. The tumor suppressor protein P53 and nuclear enzyme PARP-1 are both key players in DNA damage response. This laboratory demonstrated earlier that in cells treated with arsenite, the P53-dependent increase in p21(WAF1/CIP1) expression, normally a block to cell cycle progression after DNA damage, is deficient. Here we show that although long-term exposure of human keratinocytes (HaCaT) to a nontoxic concentration (0.1 microM) of arsenite decreases the level of global protein poly(ADP-ribosyl)ation, it increases poly(ADP-ribosyl)ation of P53 protein and PARP-1 protein abundance. We also demonstrate that exposure to 0.1 microM arsenite depresses the constitutive expression of p21 mRNA and P21 protein in HaCaT cells. Poly(ADP-ribosyl)ation of P53 is reported to block its activation, DNA binding and its functioning as a transcription factor. Our results suggest that arsenite's interference with activation of P53 via poly(ADP-ribosyl)ation may play a role in the comutagenic and cocarcinogenic effects of arsenite.

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Figures

Fig. 1
Fig. 1. Toxicity of arsenite to HaCaT cells in clonal survival assay using continuous arsenite exposure
Cells were plated at a density 500 cells/60mm dish with different concentrations of arsenite added 4h later. After 10 days incubation with arsenite-containing medium, colonies were fixed and stained (see Materials and Methods). The results were obtained from three separate experiments with each assay in triplicate and expressed as the mean plus/minus standard error of the mean. The plating efficiency of untreated HaCaT cells was about 50%.
Fig. 2
Fig. 2. Reduction in poly(ADP-ribosyl)ation of proteins in HaCaT cells exposed to arsenite
HaCaT cells were treated with 0.1μM arsenite for various periods prior to lysis. Poly(ADP-ribosyl)ated proteins were detected with PAR antibody.
Fig. 3
Fig. 3. Treatment with arsenite results in up-regulation of PARP-1 protein
HaCaT cells were exposed to 0.1μM arsenite for various periods of time prior to lysis. PARP-1 and ß-actin proteins were detected with specific mouse monoclonal antibodies.
Fig. 4
Fig. 4. Poly(ADP-ribosyl)ation of p53 protein in HaCaT cells exposed to arsenite
HaCaT cells were exposed to 0.1μM arsenite for various periods of time before lysis. P53 protein was precipitated with P53 specific rabbit polyclonal antibody then detected in Western blot with P53 specific mouse monoclonal antibody. Poly(ADP-ribosyl)ated P53 protein was detected with PAR-specific antibody.
Fig. 5
Fig. 5. Effect of arsenite on p21 expression in HaCaT cells
Total RNA was isolated from HaCaT cells exposed to 0.1μM arsenite for various periods of time. p21 transcript was detected by RT-PCR using p21 specific primers. Housekeeping gene ß-actin transcript was detected in the same samples with ß-actin specific primers.
Fig. 6
Fig. 6. Effect of arsenite on P21 protein level in HaCaT cells
Cells were exposed to 0.1μM arsenite for various periods of time before lysis. P21 and ß-actin proteins were detected in Western blot with specific mouse monoclonal antibodies.
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