Abstract 5373: Sensitizing pancreatic cancer cells to gemcitabine by inhibition of Chk1 and neutralization of anti-apoptotic Bcl-2 family members
Authors: Beeharry Neil , Smith Mitchell , Yen Timothy 
Published: June 2012

In response to various DNA damaging insults, cells initiate a complex cascade of signaling pathways that arrest the cell cycle in order to facilitate repair. A critical protein in this process is Checkpoint kinase 1 (Chk1). Many chemotherapeutic agents target DNA, and thereby activate Chk1, in order to elicit cell death. Chk1 activation may contribute to drug resistance by providing an opportunity for cancer cells to remain arrested until the drug has been metabolized. Thus, inducing DNA damage and then inhibiting Chk1 is expected to force cells to resume the cell cycle inappropriately. Numerous studies have shown that combinatorial drug-treatment strategies using conventional chemotherapy and Chk1 inhibitors lead to increased cytotoxicity as compared to drug treatment alone. However, the interplay between cell cycle arrest, checkpoint inhibition and induction of cell death remains poorly understood. Here we characterize the response of various pancreatic cancer cell lines to gemcitabine, the standard chemotherapeutic option, along with Chk1 inhibitors. Using live-cell video microscopy, we identified two distinct responses: in one subset of cell lines, addition of various Chk1 inhibitors to gemcitabine arrested cells caused cells to prematurely enter mitosis where they either completed mitosis or died in mitosis. In contrast, a separate set of cell lines treated with gemcitabine and Chk1 inhibitors did not prematurely enter mitosis but instead died in interphase. These findings suggest a link between induction of DNA damage, checkpoint override and induction of cell death.

To establish a link between checkpoint abrogation using Chk1 inhibitors and induction of cell death, we utilized small molecule inhibitors that neutralize the effect anti-apoptotic Bcl-2 family members. Reports have demonstrated a correlation between anti-apoptotic protein levels and resistance to chemotherapeutic drugs. We hypothesized that attenuating the function of these critical cell survival proteins may potentiate cell death. Pancreatic cell lines treated with gemcitabine caused a cell cycle arrest but did not die during over 72h of observation. We tested whether gemcitabine-treated cells would be sensitized to the BH3 mimetic ABT-737. Using viability and apoptosis assays, we observed that gemcitabine, UCN-01 or ABT-737 alone had negligible effect on cell viability. However, co-treatment of BXPC3 cells with gemcitabine and ABT-737 led to an increase in cell death. Cell death was further potentiated by combining gemcitabine, UCN-01 and ABT-737.

Our data demonstrates that Chk1 plays a key role in eliciting checkpoint activation following DNA damage but also suggests a role for maintaining cell survival in the presence of DNA damage. Inhibiting Chk1 blocks cell survival and cell death can be enhanced by nullifying the anti-apoptotic Bcl-2 proteins.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5373. doi:10.1158/1538-7445.AM2011-5373