Abstract #460: The relationship between G₂/M cell cycle arrest, and activation of autophagy and apoptosis following MMR processing of 6-thioguanine

DNA mismatch repair (MMR) maintains genome stability by recognizing and repairing mis-incorporated bases that can arise during DNA replication. MMR also mediates the cytotoxicity of certain clinically important chemotherapeutic drugs such as 5-fluorouracil (5-FU), temozolomide and 6-thioguanine (6-TG). In response to these DNA damage agents, MMR has been implicated in activating a G₂/M cell cycle arrest, apoptosis and autophagy as recently reported from our laboratory. In this study, we determined the potential relationship between a G₂/M cell cycle arrest and the activation of autophagy and apoptosis following MMR processing of 6-TG. We treated human colon cancer HCT116 cells stably transfected with MLH1 expression vectors (MLH1⁺, MMR⁺) with 6-TG (3uM) for 24 hours and then monitored the dynamic changes of G₂/M cell cycle arrest and apoptosis using propidium iodide staining and flow cytometry as well as autophagy using GFP-LC3 staining daily over a period of 5 days. We found that a G₂/M cell cycle arrest reached a peak at day 1. In agreement, our immunoblot analysis using anti-phospho-Chk1 (Ser³⁴⁵) showed that Chk1 activation also peaked at day 1, consistent with its role as essential regulator of MMR-mediated G₂/M cell cycle arrest. Conversely, both autophagy and apoptosis exhibited a peak response at day 3 after MMR processing of 6-TG. Since our earlier studies showed that p53 played an essential role in mediating 6-TG-induced autophagy, we measured the kinetics of p53 activation over a period of 5 days using immunoblot analysis with anti-p53. Our results show that p53 activation peaked at day 3. Overall, these results from the time-course studies indicate that Chk1 is activated to cause a G₂/M cell cycle arrest shortly after 6-TG is incorporated into DNA during S-phase and 6-TG-induced DNA mismatches are recognized by MMR. Since 6-TG induced DNA damage is not repairable, DNA strand breaks ensue and autophagy and apoptosis are subsequently activated through induction of p53 or other signaling proteins. To further determine whether or not G₂/M cell cycle arrest affects activation of autophagy and apoptosis following MMR processing of 6-TG, We performed siRNA-mediated silencing of Chk1 expression to inhibit 6-TG-induced G₂/M cell cycle arrest. Interestingly, suppression of Chk1 expression by Chk1 siRNA also dramatically increased 6-TG-induced autophagy and apoptosis. In conclusion, our results show that a Chk1-activated G₂/M cell cycle arrest produced an inhibitory effect on the induction of autophagy and apoptosis following MMR processing of 6-TG. These data suggest that if chemical inhibitors are developed and used to inhibit Chk1 activity, synergistic cytotoxic effects of MMR-processing of chemotherapeutic drugs such as 5-FU and 6-TG combined with Chk1 inhibitors may be obtained. Supported by NIH grants CA050595 and CA112963 to TJK and the Richard A. Green Foundation.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 460.