In the United States, the current maximum contaminant level for arsenic in drinking water is 10 µg/L, which is equal to ~0.5 µmol/L of arsenic. A 0.5 or 1.0 µmol/L concentration of arsenic was used for long-term treatment of cells. In light of the fact that concentrations of arsenic in certain areas may be much higher, and in addition, arsenic compounds accumulate within the body upon repeated exposure (14
). A higher concentration of arsenic (10 µmol/L) was selected for a short exposure (12 hours or less), which is comparable to a dose achievable through prolonged exposure to arsenic in contaminated drinking water. We chose untransformed human mammary epithelial cell line MCF-10A cells and TERT-immortalized human keratinocytes in this study for assessing the p53 response. MCF-10A cells were treated for 12 hours with 10 µmol/L of sodium arsenite. 5FU, a DNA-damaging agent able to activate p53, was included as a control. As expected, MCF-10A cells responded to 5FU by marked elevation of p53 abundance and induction of Hdm2 (, left, lane 3
). Interestingly, whereas arsenic treatment was associated with an increase in Hdm2, there was no noticeable change in the p53 protein level (, left, lane 2
). Considering the possibility that the p53 protein can, under certain circumstances, be activated by phosphorylation without a significant increase in protein abundance, we examined serine 15 phosphorylation, a well-documented biochemical marker for stress-induced p53 activation (15
). Probing the membrane with an anti-pS15p53 antibody revealed that this residue was heavily phosphorylated in 5FU-treated cells, whereas no such phosphorylation was detectable in arsenic-treated cells (, left, lane 2
). A similar result was also seen in keratinocytes (, right
). Together, it seems that contrary to 5FU, arsenic treatment resulted in the up-regulation of Hdm2 without apparent p53 activation.
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 (more ...)
is a well-documented gene controlled by p53, its expression can also be regulated by mechanisms independent of p53. The apparent discrepancy of Hdm2 up-regulation without accompanying p53 activation in arsenic-treated cells prompted us to test a p53-independent mode of regulation. We assessed the effect of arsenic in p53-null MEFs. Indeed, the results confirmed a p53-independent mechanism, as evidenced by a marked up-regulation of Hdm2 levels in arsenic-treated p53-null cells (, left
). To substantiate these results, we determined the effects of arsenic on the Hdm2 mRNA level. There are two well-defined promoters, P1 and P2, that control the expression of Hdm2 (), of which P1 is independent and P2 can be either dependent or independent of p53 (15
). Primers corresponding to the sequences of each promoter were generated for probing Hdm2 mRNAs. Arsenic treatment induced the mRNA levels driven by the P2 promoter, whereas no detectable effect on P1 promoter was observed (, right
). This result is further confirmed by a luciferase-based assay (). Together, the results indicate that arsenic induces the expression of Hdm2 via the P2 promoter in a p53-independent fashion.
Hdm2 is an ubiquitin E3 ligase targeting p53 for ubiquitination, which can result in either proteasome-mediated degradation or nuclear export (15
). The p53 abundance did not significantly change in arsenic-treated cells ();we thus analyzed p53 distribution in cells. Immunostaining with an anti-p53 antibody indicated that untreated MCF-10A cells exhibited relatively low levels of nuclear p53 (). As expected, 5FU treatment resulted in an increase in p53 staining that is exclusively nuclear localized (). Interestingly, in arsenic-treated cells, this nuclear localization was markedly reduced with an accompanying increase of cytoplasmic p53 staining (). An almost identical observation was also evident in keratinocytes (). Given the fact that the increased cytoplasmic p53 localization could be attributed to either increased nuclear export or reduced nuclear import, we treated cells with leptomycin B (LMB), which inhibits the activity of the exportin protein Crm1 (16
), to differentiate these two possibilities. Examination of p53 distribution revealed that this nuclear export inhibitor completely abrogated the cytoplasmic p53 accumulation in arsenic-treated cells (). Together, our data show that arsenic exposure is associated with increased p53 nuclear export, leading to its accumulation in the cytoplasm.
Figure 2 Arsenic induces p53 accumulation in the cytoplasm. A, MCF-10A cells were treated as in and were stained with an anti-p53 antibody as described in Materials and Methods. Cells were visualized by using a fluorescence microscope, and representative (more ...)
It is well documented that Hdm2 promotes p53 ubiquitination and subsequent nuclear export. However, the phosphorylation of S15 can also regulate p53 subcellular localization (17
). The serine 15 residue locates within the second nuclear export signal (NES), the phosphorylation of which could result in the inactivation of this NES and thereby p53 nuclear accumulation. Because the S15 of p53 was not detectably phosphorylated in arsenic-treated cells (), the increased p53 nuclear export could be due to the active NH2
-terminal NES. To test this, we examined p53 phosphorylation when arsenic-induced p53 nuclear export was blocked by LMB. Interestingly, in the presence of LMB, arsenic-treated cells displayed strong S15 phosphorylation, whereas LMB alone induced little p53 phosphorylation (, left
). These results suggest that p53 nuclear export was a consequence of arsenic-induced Hdm2 expression and as a consequence, the p53 protein became inaccessible to nuclear localized protein kinases. In agreement with this notion, treatment of cells with wortmannin, an inhibitor of ATM, but not the phosphoinositide-3-kinase (PI3K) inhibitor, suppressed LMB-induced p53 phosphorylation in arsenic-treated cells (, right
It has been previously reported that arsenic treatment can cause changes in gene expression via activating various cellular signaling pathways (2
). To elucidate the cellular pathway that mediates arsenic-induced Hdm2 expression, we selectively inhibited mitogen-activated protein kinase (MAPK), p38, nuclear factor κB, or PI3K pathways by using specific inhibitors. Whereas the inhibitors specific to the p38, nuclear factor κB, and PI3K pathways did not have any detectable effect, the MAPK inhibitor almost completely abrogated arsenic-induced stimulation of the Hdm2 P2 promoter (), suggesting a role for the MAPK pathway in mediating the effects of arsenic. Indeed, arsenic treatment resulted in marked activation of the MAPK pathway, as evidenced by ERK phosphorylation (). Our result is consistent with published data showing activation of ERK by arsenic exposure (18
). To substantiate the result derived from using pharmacologic inhibitors, we used a dominant-negative mutant of MEKK, an upstream kinase of ERK, as a complement approach to block the activity of this pathway. Consistent with the results obtained from the use of chemical inhibitors, expression of dominant-negative MEKK, but not dominant-negative AKT, diminished the effect of arsenic on Hdm2 levels (). Our data are in good agreement with the induction of the P2 promoter of MDM2 by the Raf/MEK/MAPK pathway (19
). Together, our results indicate that the MAPK pathway mediates arsenic-induced up-regulation of Hdm2, which then promotes p53 nuclear export.
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 (more ...)
We next investigated the effect of arsenic on p53 for its biological significance. The ability of arsenic to prevent p53 from nuclear distribution would likely interfere with its function. To test this, we examined the effect of arsenic on the p53 response to UV and 5FU treatment. Significantly, both UV- and 5FU-induced p53 activation were severely compromised in the presence of arsenic, as evidenced by the p53 protein levels and S15 phosphorylation (). To examine the cellular effects of arsenic-mediated interference of stress-induced p53 activation, we determined UV- or 5FU-induced cell killing in the presence or absence of arsenic. As expected, exposure of keratinocytes to 5FU or UV irradiation was associated with the induction of apoptosis. Although sodium arsenite at a concentration of 1 µmol/L did not have a detectable effect on cell viability, pretreatment of cells with arsenic for 24 h significantly suppressed both 5FU- and UV-induced apoptosis (). To further substantiate this observation, we examined the effect of arsenic on p53 response in mice. C57BL/6 mice at an age of 12 weeks were fed with water with or without sodium arsenite (1.0 mg/L) for 5 days. 5FU (30 mg/kg) was then administered i.v. Animals were sacrificed 12 hours later and tissues were harvested. Given the fact that the small intestine is one of the most sensitive tissues in response to DNA damage by undergoing p53-dependent apoptosis (20
), we focused on this tissue to assess the effect of arsenic. To facilitate the visualization of cellular morphology, we stained the tissue with phalloidin (actin) and 4′,6-diamidino-2-phenylindole (nucleus). As shown in , the small intestines are highly organized lumen structures comprised of two single layers of epithelial cells (a
). Mice fed with sodium arsenite–containing water did not show any signs of damage in the small intestine (b
). In contrast, 5FU treatment resulted in marked damage in the small intestine, as evidenced by the drastic disruption of the lumen structures and loss of integrity of the outer epithelium layer (c
). Significantly, the effect of 5FU was almost completely mitigated in mice that had been fed with arsenic-containing water. To determine whether the 5FU-induced damage was caused by apoptosis, we performed the terminal nucleotidyl transferase–mediated nick end labeling assay. The results indicate massive apoptotic cell death in the small intestines isolated from 5FU-treated mice, whereas such 5FU-induced cell death was significantly reduced in arsenic-pretreated animals ().
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 (more ...)