Abstract 3664: Bendamustine induces a dose-dependent cell cycle arrest in Hela cells that results in different mitotic outcomes
Authors: Beeharry Neil , Stobbe Corinne , Smith Mitchell R. , Yen Timothy J. 
Published: February 2011

Abstract
Bendamustine (BDM) was originally synthesized in the 1960s but has only recently been approved by the FDA for the treatment of chronic lymphocytic leukemia and B-cell non-Hodgkin lymphoma. BDM consists of a bifunctional alkylating nitrogen mustard group and a benzimidazole ring and has been suggested to have both alkylator and purine analogue activities. Insights into the molecular mechanisms by which efficacy is achieved are scarce. Furthermore, the effects of BDM on solid tumors also remains under-investigated. Here we evaluate the effects of BDM on Hela cells and attempt to discern its effects on the cell cycle. Hela cells were treated with different concentrations of BDM, with the antimetabolite gemcitabine, or with the alkylator MMS. Interestingly, we found dose-dependent effects of BDM on cell cycle progression. Using synchronized cells, we observed that a low dose of BDM blocked cells in G2, similar to MMS, while a 4-fold higher dose of BDM arrested cells in S phase, as seen with gemcitabine.
We next investigated the effect of abrogating the BDM-induced S and G2 cell cycle arrests with the Chk1 inhibitor UCN-01. To track the fates of cells time-lapse microscopy was conducted on cells expressing H2B: GFP. Cells were first arrested in G2 with low BDM or MMS before addition of UCN-01. Cells were observed to enter mitosis, align their chromosomes but then were delayed from entering anaphase. The cells eventually exited mitosis, with a small number of cells displaying lagging chromosomes. In contrast, cells arrested in S phase with high dose BDM or with gemcitabine, followed by UCN-01 showed highly aberrant mitotic figures. Although these cells were also delayed in mitosis, many of them died in mitosis rather than exit. We stained the mitotic cells generated by G2 or S phase inhibitors and found that cells arrested in G2 and forced into mitosis displayed normal looking metaphases, with aligned centromere/kinetochores on the spindle. However, the cells arrested in S phase followed by UCN-01 led to the generation of ‘mitosis with unreplicated genomes’ (MUGs). This phenomenon is characterized by fragmented mitotic chromatin and detached centromeres and kinetochores away from bulk DNA, as confirmed by electron microscopy (see accompanying poster Beeharry et al.). Furthermore, UCN-01 only induces formation of MUGs in cells arrested in with high BDM or gemcitabine. Although cells arrested in G2 by low BDM or MMS can also enter mitosis after UCN-01 treatment, they do not exhibit fragmented centromeres (MUGs).
These observations are consistent with the notion that BDM can act as a nucleoside analogue or as an alkylator, depending on the dose. BDM must therefore inhibit different processes in a dose-dependent manner. Furthermore, tumor cell response may differ depending on the sensitivity of these two cellular processes to BDM. These findings maybe critical for predicting sensitization by inhibitors of Chk1 kinase.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3664.