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. 2008 Nov 15;68(22):9131-6.
doi: 10.1158/0008-5472.CAN-08-3025.

Induction of cytoplasmic accumulation of p53: a mechanism for low levels of arsenic exposure to predispose cells for malignant transformation

Yelin Huang  1 , Jianglin Zhang, Kevin T McHenry, Mihee M Kim, Weiqi Zeng, Vanessa Lopez-Pajares, Christian C Dibble, Joseph P Mizgerd, Zhi-Min Yuan
Affiliations

Induction of cytoplasmic accumulation of p53: a mechanism for low levels of arsenic exposure to predispose cells for malignant transformation

Yelin Huang et al. Cancer Res. .

Abstract

Although epidemiologic studies have linked arsenic exposure to the development of human cancer, the mechanisms underlying the tumorigenic role of arsenic remain largely undefined. We report here that treatment of cells with sodium arsenite at the concentrations close to environmental exposure is associated with the up-regulation of Hdm2 and the accumulation of p53 in the cytoplasm. Through the mitogen-activated protein kinase pathway, arsenite stimulates the P2 promoter-mediated expression of Hdm2, which then promotes p53 nuclear export. As a consequence, the p53 response to genotoxic stress is compromised, as evidenced by the impaired p53 activation and apoptosis in response to UV irradiation or 5FU treatment. The ability of arsenite to impede p53 activation is further demonstrated by a significantly blunted p53-dependent tissue response to 5FU treatment when mice were fed with arsenite-containing water. Together, our data suggests that arsenic compounds predispose cells to malignant transformation by up-regulation of Hdm2 and subsequent p53 inactivation.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Figures

Figure 1
Figure 1
Arsenic induces Hdm2 expression via stimulating the P2 promoter in a p53-independent manner. A, MCF-10A cells (left) or keratinocytes (right) were treated with sodium arsenite (10 µmol/L) or 5FU (375 µg/mL) for 12 h. Cells were lysed in radioimmunoprecipitation assay buffer, analyzed by SDS-PAGE, and Western blot with the indicated antibodies. B, p53-null MEFs were treated as in (A) and divided into two aliquots; one subjected to Western analysis (left) and the other reverse transcription-PCR using primers specific for P1 or P2 promoter of hdm2 (right). C, schematic representation of the hdm2 gene and its promoters. D, plasmid-encoding luciferase driven by the P1 or P2 hdm2 promoter along with Renilla (pRL-TK) as a transfection efficiency control. Sodium arsenite or 5FU was added 24 h after transfection and luciferase expression was measured 36 h posttransfection. Columns, mean luciferase activities from experiments done in triplicate; bars, SD.
Figure 2
Figure 2
Arsenic induces p53 accumulation in the cytoplasm. A, MCF-10A cells were treated as in Fig. 1 and were stained with an anti-p53 antibody as described in Materials and Methods. Cells were visualized by using a fluorescence microscope, and representative fields were photographed. 4′,6-Diamidino-2-phenylindole (DAPI) staining is used to locate the nucleus. B, keratinocytes were analyzed as in A. C, MCF-10A cells were treated with 10 µmol/L of sodium arsenite for 12 h. LMB (50 nmol/L) was added to cultures 6 h before fixing. Cells were analyzed as in A. D, MCF-10A cells were treated with as in C and subjected to Western analysis. 5FU-treated cells were included as a control.
Figure 3
Figure 3
Arsenic induces Hdm2 expression by the ERK pathway. A, p53-null MEFs were transfected with the hdm2 P2 promoter-driven luciferase vector. Cells were treated with 10 µmol/L of sodium arsenite 24 h after transfection, and the indicated inhibitors (Calbiochem) were added 6 h later. The cells were incubated for another 6 h and then harvested for luciferase assay. Columns, mean luciferase activities from experiments done in triplicate; bars, SE. B, MCF-10A cells were serum starved overnight and then treated as indicated for 3 h. Cells were analyzed by Western blotting using the indicated antibodies. C, MCF-10A cells were treated with 10 µmol/L of sodium arsenite for 6 h and the indicated inhibitors were added to cultures for another 6 h. Cells were analyzed by Western blotting using the indicated antibodies. D, MCF-10A cells were transfected with plasmids encoding dominant-negative mutant of MEKK (MEKKDN) or AKT (AKTDN). Cells were treated with arsenic for 24 h, after transfection for 12 h, and then analyzed by Western blot.
Figure 4
Figure 4
Low levels of arsenic exposure impedes DNA damage-induced p53 response. A, keratinocytes were pretreated with or without 1.0 µmol/L of sodium arsenite for 24 h and then treated as indicated for 12 h. Cells were analyzed by Western blotting using the indicated antibodies. B, keratinocytes were treated as in A and harvested 24 h later for FACS analysis of sub-G1 population. Columns, mean percentages of sub-G1 cells from experiments done in triplicate; bars, SD. C, C57BL/6 mice at 10 wk of age were randomly divided into four groups (three mice/group) and fed with water with or without sodium arsenite (1.0 mg/L) for 3 d. 5FU (30 mg/kg) was then administered by tail vein injection. Animals were sacrificed 12 h later and tissues were harvested. Images are representative staining of small intestine. D, terminal nucleotidyl transferase–mediated nick end labeling assay was performed according to the instructions of the manufacturer (Invitrogen). Apoptotic cells are shown in green.
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