[PubMed] [Google Scholar]Winter C

[PubMed] [Google Scholar]Winter C., Albers P. B suggested that combined NaAsO2, hyperthermia, and cisplatin induced mitotic arrest. However, we observed < 3% mitotic index and phosphorylation of histone Monastrol H3 on serine 10 was undetectable. These results did not confirm mitotic arrest. BUBR1 (BUB1B) also was not phosphorylated, suggesting disrupted mitotic checkpoint. Postmitotic cells accumulated in pseudo-G1 as exhibited by cyclin E stabilization, CDKN1A induction, and hypophosphorylation of retinoblastoma protein. These cells also were positive for Annexin V binding indicating they were apoptotic. In summary, cisplatin plus NaAsO2 and hyperthermia induced pseudo-G1 associated apoptosis in ovarian cancer cells. < 0.05, = 3. RESULTS Flow Cytometry Determination of Cell Cycle Arrest Cisplatin is usually a DNA damaging agent. Cellular response to DNA damage involves cell cycle arrest to allow time to repair damaged DNA (Basu and Krishnamurthy, 2010). Cisplatin is known to cause G2 arrest (Cepeda < 0.05, * = compared with G2/M Monastrol partners. Sodium Arsenite and Hyperthermia Cause Mitotic Arrest in Cisplatin Treated Ovarian Cancer Cells Flow cytometry determination of DNA content using propidium iodide does not distinguish between G2 and M cells because these cells both have double the normal (2C) DNA content. In order to determine if cells are in the G2 or M phase of the cell cycle at 36 h after treatment, the expression of cyclins A and B and cyclin-dependent kinase CDK1 were decided. Furthermore, we decided if sodium arsenite and hyperthermia cotreatment altered the expression of cyclins A and B and CDK1 in response to cisplatin treatment. G2 to M progression requires degradation of cyclin A and accumulation of cyclin B (Malumbres and Barbacid, 2009). Data in Physique ?Physique22 indicate that cisplatin treatment at 37C stabilized CDK1, cyclin A Monastrol and cyclin B (Fig.?2, panel a), suggesting G2 arrest. Adding hyperthermia to cisplatin decreased the levels of both cyclin A and cyclin B in A2780 cells suggesting G1 arrest. In contrast, cyclins A and B were stabilized suggesting G2 arrest in A2780/CP70 cells (Fig.?2, panel b). Cotreatment with cisplatin and sodium arsenite decreased both cyclin A and cyclin B in A2780 cells suggesting G1 arrest. In contrast, cyclin A was undetectable and cyclin B was stabilized, suggesting mitotic arrest in A2780/CP70 cells (Fig.?2, panel c). Combined cisplatin, sodium arsenite, and hyperthermia stabilized cyclin B and CDK1 but attenuated the expression of cyclin A in both cell lines at 36 h after treatment (Fig.?2, panel d), suggesting mitotic arrest. These data suggest that sodium arsenite hyperthermia induced mitotic arrest in cisplatin treated cells. Open in a separate windows FIG. 2. Western blot analyses of G2/M cell cycle regulatory proteins. Representative Western blots of cyclin A and B and CDK1. Cells were treated with their respective IC50 cisplatin (CP) (A2780, 4M; CP70, 40M) or CP plus 20M sodium Igf2r arsenite (CPA) at 37 or 39C (hyperthermia) for 1 h, then washed with PBS and refed with fresh media and incubated at 37C. Cell lysates were prepared at 0, 24, and 36 h. ?-actin is the loading control. Blots shown are representative of three impartial experiments. Sodium Arsenite and Hyperthermia Do Not Enhance Mitotic Index in Cisplatin Treated Cells and Also Fail to Induce Histone H3Ser10 Phosphorylation Data in Physique ?Physique22 suggest that sodium arsenite plus hyperthermia Monastrol is causing cisplatin treated cells to arrest in mitosis. In order to confirm if indeed these cells are in mitosis, we decided mitotic index as described in the Materials and.

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