Dose Dependent Effects on Cell Cycle Checkpoints and DNA Repair by Bendamustine

Neil Beeharry,Alfonso Bellacosa,Jerome B Rattner,Timothy J Yen,Mitchell R Smith,Toru Ouchi

doi:10.1371/journal.pone.0040342

Neil Beeharry, Alfonso Bellacosa + Show 4 more

Open Access

https://doi.org/10.1371/journal.pone.0040342

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Journal: PloS one	Publication Date: Jun 29, 2012
Citations: 49	License type: CC BY 4.0

Affiliation: Fox Chase Cancer Center, University of Calgary

Abstract

Bendamustine (BDM) is an active chemotherapeutic agent approved in the U. S. for treating chronic lymphocytic leukemia and non-Hodgkin lymphoma. Its chemical structure suggests it may have alkylator and anti-metabolite activities; however the precise mechanism of action is not well understood. Here we report the concentration-dependent effects of BDM on cell cycle, DNA damage, checkpoint response and cell death in HeLa cells. Low concentrations of BDM transiently arrested cells in G2, while a 4-fold higher concentration arrested cells in S phase. DNA damage at 50, but not 200 µM, was efficiently repaired after 48 h treatment, suggesting a difference in DNA repair efficiency at the two concentrations. Indeed, perturbing base-excision repair sensitized cells to lower concentrations of BDM. Timelapse studies of the checkpoint response to BDM showed that inhibiting Chk1 caused both the S- and G2-arrested cells to prematurely enter mitosis. However, whereas the cells arrested in G2 (low dose BDM) entered mitosis, segregated their chromosomes and divided normally, the S-phase arrested cells (high dose BDM) exhibited a highly aberrant mitosis, whereby EM images showed highly fragmented chromosomes. The vast majority of these cells died without ever exiting mitosis. Inhibiting the Chk1-dependent DNA damage checkpoint accelerated the time of killing by BDM. Our studies suggest that BDM may affect different biological processes depending on drug concentration. Sensitizing cells to killing by BDM can be achieved by inhibiting base-excision repair or disrupting the DNA damage checkpoint pathway.

Highlights

Bendamustine (BDM) represents one of the earliest rationally designed anticancer drugs that incorporated three functional groups; a benzimidazole ring, a mechlorethamine group and a butanoic acid residue
The treatment of the nonHodgkin lymphoma cell line SU-DHL-1 with BDM led to a greater increase in the expression of several p53-responsive genes and DNA damage/repair genes when compared to other alkylators
Cell lines derived from pancreatic (PANC1 and BXPC3 cells), breast (MCF7 and MDA-MB-435), and ovarian (OVCAR 5 and 10) cancers responded to HeLa cells (Fig. 1 and Figure S1) in that 50 mM BDM caused a G2 arrest while 200 mM BDM resulted in an S phase arrest

Summary

Introduction

Bendamustine (BDM) represents one of the earliest rationally designed anticancer drugs that incorporated three functional groups; a benzimidazole ring, a mechlorethamine group and a butanoic acid residue. These groups putatively endowed BDM with both alkylator and anti-metabolite activities. A previous report suggested that the type of DNA damage induced by BDM was different to that caused by alkylating agents [3]. The treatment of the nonHodgkin lymphoma cell line SU-DHL-1 with BDM led to a greater increase in the expression of several p53-responsive genes and DNA damage/repair genes when compared to other alkylators. By interrogating CLL and mantle cells from patients, Roue et al found no correlation between p53 status and BDM cytotoxicity [4]

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