Exit from arsenite-induced mitotic arrest is p53 dependent

Abstract

Background: Arsenic is both a human carcinogen and a chemotherapeutic agent, but the mechanism of neither arsenic-induced carcinogenesis nor tumor selective cytotoxicity is clear. Using a model cell line in which p53 expression is regulated exogenously in a tetracycline-off system (TR9-7 cells) , our laboratory has shown that arsenite disrupts mitosis and that p53-deficient cells [p53(-)], in contrast to p53-expressing cells [p53(+)], display greater sensitivity to arsenite-induced mitotic arrest and apoptosis.

Objective: Our goal was to examine the role p53 plays in protecting cells from arsenite-induced mitotic arrest.

Methods: p53(+) and p53(-) cells were synchronized in G2 phase using Hoechst 33342 and released from synchrony in the presence or absence of 5 microM sodium arsenite.

Results: Mitotic index analysis demonstrated that arsenite treatment delayed exit from G2 in p53(+) and p53(-) cells. Arsenite-treated p53(+) cells exited mitosis normally, whereas p53(-) cells exited mitosis with delayed kinetics. Microarray analysis performed on mRNAs of cells exposed to arsenite for 0 and 3 hr after release from G2 phase synchrony showed that arsenite induced inhibitor of DNA binding-1 (ID1) differentially in p53(+) and p53(-) cells. Immunoblotting confirmed that ID1 induction was more extensive and sustained in p53(+) cells.

Conclusions: p53 promotes mitotic exit and leads to more extensive ID1 induction by arsenite. ID1 is a dominant negative inhibitor of transcription that represses cell cycle regulatory genes and is elevated in many tumors. ID1 may play a role in the survival of arsenite-treated p53(+) cells and contribute to arsenic carcinogenicity.

Figure 1

Synchronization of TR9-7 cells expressing or not expressing p53 in G₂ phase. Abbreviations: FL2-A, propidium iodide fluorescence on channel two; Tet, tetracycline. (A) Experimental design. (B) Cell cycle distribution of synchronized cells. Samples were harvested after treatments indicated and analyzed by flow cytometry. Positions of cells with G₁ and G₂/M phase DNA content are indicated.

Figure 2

p53 and ID1 expression in cells after G₂ phase synchronization. Cells were synchronized, treated with 5 μM NaAsO₂, and samples were taken at the indicated times. Levels of p53 and ID1 proteins were assessed by Western blot analysis with β-actin as a loading control. 0 hr = release from Hoechst 33342. A representative example is shown.

Figure 3

Arsenite treatment delays mitotic entry, and p53 deficiency delays mitotic exit in arsenite-treated cells. Abbreviations: As⁽⁺⁾, + 5 μg sodium arsenite; As⁽⁻⁾, 0 μg sodium arsenite. (A) Mitotic indices of TR9-7 cells released from G₂ phase synchronization. Mean ± SD from four independent experiments performed with triplicate cultures are plotted. Not all time points assayed in every experiment. (B) Arsenite-treated TR9-7 cells in late anaphase/early telophase undergoing apoptosis. Cells were released from G₂ phase by feeding with fresh media containing 5 μM NaAsO₂ and monitored by phase microscopy. Photographs in panels 1–8 were taken in order over a 30-min period. Bar, 5 μm. *Significant difference between arsenite-exposed p53(+) and p53(⁻) cells.

Figure 4

Model for exit from mitotic arrest induced by arsenite and subsequent cell cycle progression dependent on p21^CIP1/WAF1 and ID1 induction in p53-expressing cells.