The Effects of p53 and p53R2 Expression on Gemcitabine-Mediated Cytotoxicity and Radiosensitization.

Influence of P53 and P53R2 on DFdCyd-mediated Radiosensitization;Radiosensitization;Ribonucleotide Reductase;Nucleoside Analogs;Pharmacy and Pharmacology;Health Sciences;Pharmacology

Barber, JoeLjungman, Mats ;

University of Michigan

关键词: Influence of P53 and P53R2 on DFdCyd-mediated Radiosensitization;    Radiosensitization;    Ribonucleotide Reductase;    Nucleoside Analogs;    Pharmacy and Pharmacology;    Health Sciences;    Pharmacology;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/62229/joebarb_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Gemcitabine (dFdCyd) is a nucleoside analog that produces synergistic killing of tumor cells with ionizing radiation (radiosensitization).Previously, radiosensitization correlated with dATP depletion, mediated by dFdCyd diphosphate inhibition of ribonucleotide reductase, and S-phase accumulation, whereas dFdCyd triphosphate and its incorporation into DNA contributed to cytotoxicity but not radiosensitization.DNA damage caused by dFdCyd can induce p53, and this dissertation examines the roles of wild-type (wt) p53 and a p53-inducible ribonucleotide reductase subunit, p53R2, in the radiosensitizing mechanism. Based on previous findings, it was hypothesized that p53 expression could prevent S-phase accumulation by inducing a G1 block after dFdCyd exposure.Although p53 was induced strongly and rapidly with IC90 dFdCyd, p53 induction occurred as MCF-7 breast cancer cells accumulated in S-phase resulting in radiosensitization.The data demonstrate that p53 induction does not prevent radiosensitization in MCF-7 cells at clinically relevant concentrations of dFdCyd. p53R2 is induced by DNA damage, providing dNTPs for DNA repair.Since dATP depletion is pivotal for radiosensitization, it was important to determine if dFdCyd could induce p53R2 to alter dATP depletion and radiosensitization.p53R2 was induced in response to dFdCyd, even at radiosensitizing but non-cytotoxic concentrations, regardless of cell cycle distribution.Expression of the replication-associated ribonucleotide subunit, R2, increased as cells accumulated in S-phase following dFdCyd addition, and decreased after ionizing radiation as cells accumulated in G1 and G2/M.MCF-7 breast and HCT116 colon carcinoma (wt p53) cells expressed p53R2 after dFdCyd exposure, but dATP depletion and radiosensitization were unaffected compared to p53-deficient counterparts.This suggests that p53R2 induction could not maintain dNTP pools following dFdCyd exposure in these cell lines. The role of p53R2 in radiosensitization was evaluated more directly using siRNA-mediated p53R2 suppression. It was hypothesized that p53R2 silencing would increase dFdCyd-mediated dNTP depletion and radiosensitization.Although p53R2 silencing did not alter cytotoxicity or radiosensitization in the MCF-7 cell line, radiosensitization was increased in the A549 non-small cell lung cancer cells following p53R2 suppression, due to improved dNTP depletion.The data suggest that, in some cell lines, p53R2 is induced to sufficient levels to maintain dNTP pools after dFdCyd exposure, thereby preventing radiosensitization.