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. 2008 Aug 15;231(1):61-7.
doi: 10.1016/j.taap.2008.03.020. Epub 2008 Apr 9.

Mitotic arrest-associated apoptosis induced by sodium arsenite in A375 melanoma cells is BUBR1-dependent

Samuel C McNeely  1 , B Frazier Taylor, J Christopher States
Affiliations

Mitotic arrest-associated apoptosis induced by sodium arsenite in A375 melanoma cells is BUBR1-dependent

Samuel C McNeely et al. Toxicol Appl Pharmacol. .

Abstract

A375 human malignant melanoma cells undergo mitotic arrest-associated apoptosis when treated with pharmacological concentrations of sodium arsenite, a chemotherapeutic for acute promyelocytic leukemia. Our previous studies indicated that decreased arsenite sensitivity correlated with reduced mitotic spindle checkpoint function and reduced expression of the checkpoint protein BUBR1. In the current study, arsenite induced securin and cyclin B stabilization, BUBR1 phosphorylation, and spindle checkpoint activation. Arsenite also increased activating cyclin dependent kinase 1 (CDK1) Thr(161) phosphorylation but decreased inhibitory Tyr15 phosphorylation. Mitotic arrest resulted in apoptosis as indicated by colocalization of mitotic phospho-Histone H3 with active caspase 3. Apoptosis was associated with BCL-2 Ser70 phosphorylation. Inhibition of CDK1 with roscovitine in arsenite-treated mitotic cells inhibited spindle checkpoint maintenance as inferred from reduced BUBR1 phosphorylation, reduced cyclin B expression, and diminution of mitotic index. Roscovitine also reduced BCL-2 Ser70 phosphorylation and protected against apoptosis, suggesting mitotic arrest caused by hyperactivation of CDK1 directly or indirectly leads to BCL-2 phosphorylation and apoptosis. In addition, suppression of BUBR1 with siRNA prevented arsenite-induced mitotic arrest and apoptosis. These findings provide insight into the mechanism of arsenic's chemotherapeutic action and indicate a functional spindle checkpoint may be required for arsenic-sensitivity.

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Figures

Figure 1
Figure 1
Arsenite does not alter spindle formation like either paclitaxel or nocodazole. A375 cells were treated as indicated for 18 h. Detached cells were collected, centrifuged onto glass coverslips and stained for α-tubulin (green), phospho-Histone H3 (red) and DNA (blue). Bar, 10 μm
Figure 2
Figure 2
Analysis of arsenite-induced alterations of mitotic proteins in A375 cells. A375 cells were treated with indicated concentrations of NaAsO2 for 24 h and examined via western blot.
Figure 3
Figure 3
(A) Mitotic marker phosphorylated Histone H3 and apoptotic marker active caspase 3 colocalize in arsenite-treated A375 cells. (A1–4) A375 cells were treated for 24 h with 5 μM NaAsO2. Detached cells were harvested and centrifuged onto glass coverslips. Cells were stained for (A1) active caspase 3 (green), (A2) phosphorylated Histone H3 (red), and (A3) DNA (blue); (A4) merged color images. (B) Loss of Histone H3 phosphorylation precedes apoptotic DNA fragmentation. A375 cells were treated for 6 h with 5 μM NaAsO2. Detached cells were harvested and centrifuged onto glass coverslips and re-incubated in 5 μM NaAsO2 for indicated times. Cells were stained for phosphorylated Histone H3 and DNA. Data are means ± standard deviation of three independent experiments. (C) Caspase inhibition augments arsenite-induced mitotic arrest. Mitotic indices were determined after A375 cells were treated with 0 or 5 μM NaAsO2 for 24 h in the absence or presence of 50 μM Z-VAD-FMK. Significance of difference between cells treated with NaAsO2 or NaAsO2 plus Z-VAD-FMK determined by student t-test denoted by *; p < 0.05. (D) Western blot showing loss of cleaved caspase 3 in cells treated with Z-VAD-FMK as described for (C).
Figure 4
Figure 4
Arsenite activates CDK1 and induces BCL-2 phosphorylation. A375 cells were treated with indicated concentrations of NaAsO2 for 24 h and analyzed by western blot for the indicated proteins.
Figure 5
Figure 5
CDK1 inhibitor roscovitine promotes mitotic exit and inhibits apoptosis. (A) A375 cells were treated with 5 μM NaAsO2 for 6 h. Detached cells were collected and re-incubated in media containing 5 μM NaAsO2 with and without 20 μM roscovitine. Mitotic index was determined at 0–3 hour of re-incubation. Data are means ± standard deviation of three independent experiments. * p < 0.05 (B1–3) Phase contrast microscopy of A375 cells after 18 h re-incubation either untreated (1) or treated as in (A) in NaAsO2 alone (2) or NaAsO2 plus roscovitine (3) Bar, 50 μm. (C) Cells treated as described in (A) were re-incubated for 6 and 18 h. Cells re-incubated with NaAsO2 and Z-VAD-FMK were also included as negative controls for apoptosis. Samples were collected and analyzed by western blot for indicated proteins.
Figure 6
Figure 6
Suppression of BUBR1 prevents arsenite-induced mitotic arrest in A375 cells. (A) A375 cells were transfected with BUBR1 siRNA, non-specific control siRNA (NSC) or mock-transfected with buffer alone and allowed to recover for 24 h. Suppression of BUBR1 was confirmed via western blot of untreated samples at indicated times after recovery period. (B) Cells treated as in (A) were exposed to indicated concentrations of NaAsO2 for 24 h and harvested for determination of mitotic indices. Data are means ± standard deviation of three independent experiments. * p < 0.05, ** p < 0.01 (C) Cells transfected as in (A) were exposed to indicated concentrations of NaAsO2 for 48 h and assayed for viability. Data are means ± standard deviation of three independent experiments. ** pval < 0.01. (D) Cells transfected as in (A) were treated with 0 or 5 μM NaAsO2 for 24 h and harvested for western blot analysis of the indicated proteins.
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