Abstract #5602: Inhibition of RNA polymerase II activity enhances radiosensitivity of human non_small cell lung carcinoma in vitro
Authors: Zhi-Qiang Wang, Uma Raju, David Molkentine, Luka Milas
Journal: Cancer Research (2009)

Background:  We previously reported that the enhancement in cell radiosensitivity induced by a broad spectrum cdk inhibitor, flavopiridol, was mediated by several mechanisms including inhibition of DNA repair, increase in apoptosis induction and cell cycle redistribution (Raju et al., Radiotherapy & Oncology, 2006, 80:185-191). Because these effects of flavopiridol were associated with inhibition of RNA polymerase II activity, the present study investigated whether specific inhibition of this enzyme by a reversible transcription inhibitor, 5, 6-dichloro-l-\#946;-D-ribofuranosylbenzimidazole (DRB) could also enhance cell radiosensitivity.  Methods:  H460, a human non-small cell lung cancer (NSCLC) line, cells were treated with 10 to 100 \#956;M of DRB for 2 - 24 h and then irradiated with graded single doses of ionizing radiation (2-6 Gy \#947;-rays). In addition, H460 cells were first irradiated and then 7 h later treated with 100 \#956;M DRB for 24 h. The treatment endpoint was clonogenic cell survival determined 10 days after irradiation. Treated cells were also analyzed for molecular changes involved in DNA damage and repair, specifically nuclear \#947;-H2AX foci expression using immunocytochemisty. Cell cycle changes and apoptosis induction were also assessed using flowcytometry. DRB effect on cell transcription was assessed by  3 H-Uridine incorporation.  Results:  DRB significantly enhanced H460 cell radiosensitivity in a dose-dependent manner, with a dose of 100 \#956;M given for 24 h before radiation being the most effective [radioenhancement factor (EF) of 1.56 at the cell survival level of 0.1]. This treatment abolished the \#8220;shoulder\#8221; region of the cell survival curve (EF at the clinically relevant dose of 2 Gy was 2.6), suggesting that DRB strongly inhibited cellular repair. DRB given 7 h after cell irradiation failed to affect cell radiosensitivity which further suggests that the effect of DRB was primarily mediated via inhibition of cellular repair. DRB treatment prolonged the presence of radiation-induced nuclear \#947;-H2AX foci, a measure of DNA double strand breaks. In addition, DRB caused a further increase in the radiation-induced apoptotsis, suggesting that induction of apoptosis as yet another mechanism by which DRB enhanced cell radiosensitivity. Cells exhibiting DRB-enhanced radiosensitization, had a significantly reduced expression of RNA polymerase II phosphorylation and also showed a considerably reduced incorporation of  3 H-Uridine incorporation, confirming that the DRB treatment indeed inhibited cellular transcription.  Conclusions:  Treatment of H460 NSCLC cells with DRB strongly enhanced their radiosensitivity. The effect was achieved when DRB was given before but not after irradiation. The underlying mechanisms include DNA repair and induction of apoptosis. Thus, our findings suggest that DRB has potential to increase the efficacy of radiotherapy in NSCLC.   Citation Information:  In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 5602.