Abstract 2553: Gemcitabine fails to radiosensitize normal intestinal epithelial CCD841 cells at concentrations that promote excellent radiosensitization in colorectal tumor cell lines
Authors: Flanagan Sheryl A. , Ackroyd Jeffrey , Shewach Donna S. 
Published: August 2015

Gemcitabine (2′, 2′-difluroro-2′-deoxycytidine;dFdCyd) is a potent radiosensitizer in tumor cells in vitro and commonly used in chemoradiotherapy regimens to enhance radiation induced cell killing (radiosensitization), but its use is limited by normal tissue toxicity. We have studied the mechanism by which dFdCyd promotes radiosensitization in an effort to optimize its use in such regimens. dFdCyd elicits cytotoxicity primarily via incorporation of its triphosphate, dFdCTP, into DNA, whereas inhibition of ribonucleotide reductase (RR) by dFdCDP produces a profound depletion of dATP, which strongly correlates with radiosensitization. DNA replication in the presence of imbalanced dNTPs produces mismatches in DNA, and their persistence underlies the mechanism of radiosensitization by dFdCyd. Importantly, the presence of dFdCyd induced DNA mismatches is necessary for radiosensitization to occur but not for cytotoxicity, and maximal radiosensitization can be elicited at non-toxic concentrations of drug. However, when dFdCyd is currently used as part of chemoradiotherapy regimens, it is administered at the standard and very toxic dose used in monotherapy regimens. The distinction between radiosensitizing and cytotoxic mechanisms of dFdCyd revealed by our studies provides a rationale for optimizing chemoradiotherapy while decreasing normal tissue toxicity. To achieve this, we have evaluated cytotoxicity of dFdCyd + ionizing radiation (IR) in a normal intestinal epithelial cell line, CCD841, as a first approach to identify potential differences in the way tumor versus non-tumor cells respond to dFdCyd ± IR. CCD841 cells were exposed to concentrations of dFdCyd that produce excellent radiosensitization in a variety of colorectal tumor cell lines (10 nM, 30 nM, 100 nM) and produced a range of survival in CCD841 cells (78 ± 17.1%, 46.3 ± 5.5%, 26 ± 3.61%, respectively). Analysis of dNTPs during dFdCyd incubation revealed a profound depletion in dATP. However, all dFdCyd concentrations failed to radiosensitize CCD841 cells (Radiation Enhancement Ratios: 10 nM, 0.92 ± 0.03; 30 nM, 0.82 ± 0.04; 100 nM, 0.67 ± 0.10). Marked differences in cell cycle effects were also noted in CCD841 and tumor cells treated with dFdCyd. While tumor cells accumulate in early S-phase but slowly replicate DNA in the presence of dFdCyd, CCD841 cells arrested right where they were in the cycle at the time of dFdCyd exposure and displayed a striking decrease in DNA synthesis. We propose that this prevents CCD841 cells from undergoing the necessary DNA replication that permits cells to produce the essential DNA mismatches for radiosensitization to occur. Our studies support the use of mechanistically-derived doses and administration schedules for dFdCyd when given with IR to ameliorate normal tissue toxicity while preserving the effectiveness of the chemoradiotherapy regimen.

Citation Format: Sheryl A. Flanagan, Jeffrey Ackroyd, Donna S. Shewach. Gemcitabine fails to radiosensitize normal intestinal epithelial CCD841 cells at concentrations that promote excellent radiosensitization in colorectal tumor cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2553. doi:10.1158/1538-7445.AM2015-2553