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. 2008 Jul 15;230(2):235-46.
doi: 10.1016/j.taap.2008.02.030. Epub 2008 Mar 14.

Arsenite-induced mitotic death involves stress response and is independent of tubulin polymerization

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

Arsenite-induced mitotic death involves stress response and is independent of tubulin polymerization

B Frazier Taylor et al. Toxicol Appl Pharmacol. .

Abstract

Arsenite, a known mitotic disruptor, causes cell cycle arrest and cell death at anaphase. The mechanism causing mitotic arrest is highly disputed. We compared arsenite to the spindle poisons nocodazole and paclitaxel. Immunofluorescence analysis of alpha-tubulin in interphase cells demonstrated that, while nocodazole and paclitaxel disrupt microtubule polymerization through destabilization and hyperpolymerization, respectively, microtubules in arsenite-treated cells remain comparable to untreated cells even at supra-therapeutic concentrations. Immunofluorescence analysis of alpha-tubulin in mitotic cells showed spindle formation in arsenite- and paclitaxel-treated cells but not in nocodazole-treated cells. Spindle formation in arsenite-treated cells appeared irregular and multi-polar. gamma-tubulin staining showed that cells treated with nocodazole and therapeutic concentrations of paclitaxel contained two centrosomes. In contrast, most arsenite-treated mitotic cells contained more than two centrosomes, similar to centrosome abnormalities induced by heat shock. Of the three drugs tested, only arsenite treatment increased expression of the inducible isoform of heat shock protein 70 (HSP70i). HSP70 and HSP90 proteins are intimately involved in centrosome regulation and mitotic spindle formation. HSP90 inhibitor 17-DMAG sensitized cells to arsenite treatment and increased arsenite-induced centrosome abnormalities. Combined treatment of 17-DMAG and arsenite resulted in a supra-additive effect on viability, mitotic arrest, and centrosome abnormalities. Thus, arsenite-induced abnormal centrosome amplification and subsequent mitotic arrest is independent of effects on tubulin polymerization and may be due to specific stresses that are protected against by HSP90 and HSP70.

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Figures

Figure 1
Figure 1. Paclitaxel and nocodazole but not arsenite disrupt adherent cell tubulin architecture after 1 h
A. TR9-7 and B. HeLa cells were grown on poly-D-lysine coated coverslips and treated with either nocodazole, paclitaxel or arsenite at the concentrations shown for 1 h before fixation and subsequent staining for cytoskeletal proteins α-tubulin. Cells were analyzed and photographed via fluorescence microscopy. C. Both cell lines were treated with 0 or 5 μM arsenite for 1 h and total cellular lysate proteins were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and probed for HSP70i and β-actin.
Figure 2
Figure 2. Analysis of spindle in mitotic cells
HeLa cells were grown in drug free media in 15 cm dishes, while cells treated with 90 ng/ml nocodazole, 100 nM paclitaxel, or 5 μM arsenite were grown in 6 cm dishes for 15 h. Detached cells were shaken, pelleted, and adhered to poly-D-lysine slides before fixation and staining with anti-α-tubulin (AlexaFluor 488, green), anti-histone H3-S10-P (a mitotic marker, AlexaFluor 594, red), and DAPI (blue). Cells were analyzed and photographed via fluorescence microscopy. Representative examples of three independent experiments are shown. White bar represents 50 micrometers.
Figure 3
Figure 3. Analysis of centrosome number and location in mitotic cells
A. HeLa cells were grown in drug free media in 15 cm dishes, while cells treated with 90 ng/ml nocodazole (Noc 100 nM paclitaxel (Tax), or 5 μM arsenite (As) were grown in 6 cm dishes for 15 h. Detached cells were collected and adhered to poly-D-lysine slides before fixation and staining with anti-γ-tubulin (AlexaFluor 488, green), anti-histone H3-S10-P (a mitotic marker, AlexaFluor 594, red), and DAPI (blue). Cells were analyzed and photographed via fluorescence microscopy. Representative examples of three independent experiments are shown. White bar represents 50 micrometers. B. A minimum of 200 mitotic cells per experiment were analyzed and each mitotic cell was scored for number of centrosomes. Data from three independent experiments were graphed as a stacked mean percentage ± SD. Statistically significant differences between treatments are labeled (*** = p < 0.01).
Figure 4
Figure 4. Western blot analysis of protein markers of mitotic arrest and apoptosis
A375, HeLa, TR9-7 p53(+), and TR9-7 p53(-) cells were untreated (UT), or treated with 5 μM arsenite (A), 90 ng/ml nocodazole (N), 7 nM paclitaxel (LP), or 100 nM paclitaxel (HP) for 24 h. Total cellular lysate proteins were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and probed for cleaved caspase 3, PARP, Bcl-2, Bcl-2-S70-P, histone H2A.X-S139-P, survivin, HSP70, HSP70i, HSP90. α-tubulin and β-actin as indicated from top to bottom, respectively. Representative results of triplicate experiments are shown.
Figure 5
Figure 5. Immunofluorescence analysis of centrosomes in mitotic cells after heat shock
HeLa cells were subjected to heat shock by incubation for 1 h at 39° C. Mitotic cells were harvested at 0, 1, 3 and 6 h post-heat shock and analyzed by ICC for centrosome quantification. A. Representative examples of abnormal centrosome amplification induced by arsenite exposure or heat shock and allowed to recover for 1 h. White bar represents 50 micrometers. B. Quantification of centrosome number per mitotic cell. A minimum of 200 mitotic cells per experiment were analyzed and each mitotic cell was scored for number of centrosomes. Data from three independent experiments were graphed as a stacked mean percentage ± SD. Statistically significant differences between cells with one hour recovery and either untreated or cell after 6 h recovery (*** = p < 0.01). C. Cells were also collected for western blot analysis and total cellular lysate proteins were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and probed for HSP70i and β-actin.
Figure 6
Figure 6. Cytotoxicity of HSP90 inhibitor 17-DMAG co-treatment with arsenite
A. A375, HeLa, and TR9-7 cells were plated on 96 well dishes in triplicate and exposed to 0-300 nM 17-DMAG in combination with 0, 0.5, 1, 2.5 or 5 μM arsenite for 48 h prior to analysis with AlamarBlue fluorescence assay. Data are graphed as mean ± SD from three independent experiments. Data are presented as percent untreated control either with 17-DMAG concentration on the X-axis (top graphs) or arsenite concentration on the X-axis (bottom graphs).
Figure 7
Figure 7. HSP90 inhibitor 17-DMAG enhances arsenite-induced mitotic arrest and centrosome abnormalities
A. A375 and HeLa cells were plated in 6 well dishes and treated with 0, 3 or 5 μM arsenite, 10 or 30 nM 17-DMAG, or 10 nM 17-DMAG in combination with 3 or 5 μM for 24 h before mitotic index analysis. Interphase nuclei, mitotic spreads and mitotic catastrophe were scored in 1,800 cells per condition. Statistically significant differences between mitotic index (MI) and mitotic catastrophe index (MCI) are labeled (MI: * = p < 0.1, ** = p < 0.05, *** = p < 0.01; MCI: † = p < 0.1, †† = p < 0.05, ††† = p < 0.01). B. HeLa and A375 cells were plated on 15 or 6 cm dishes exposed to 0, 3 or 5 μM arsenite, 10 or 30 nM 17-DMAG, or 10 nM 17-DMAG in combination with 3 or 5 μM for 15 h. Detached cells were collected and adhered to poly-D-lysine slides before fixation and staining with anti-γ-tubulin, anti-histone H3-S10-P, and DAPI. Centrosomes per mitotic cell were quantified. in a minimum of 200 mitotic cells per experiment. Data from three independent experiments are graphed as a stacked mean percentage ± SD. Statistically significant differences between treatments are labeled (* = p < 0.1, ** = p < 0.05, *** = p < 0.01). C. Total cellular lysate proteins of cells treated for 24 h were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and probed for PARP, cleaved caspase 3, HSP90 and β-actin. Treatment conditions are: untreated (UT), 3 μM (A375, 3A) or 5 μM (HeLa, 5A) arsenite, 10 nM 17-DMAG (10D), 3 μM (for A375) or 5 μM (for HeLa) arsenite plus 10 nM 17-DMAG (A/D), and 30 nM 17-DMAG (30D).
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