The effects of dFdCyd and ionizing irradiation on p53R2 and R2 expression during the cell cycle

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Gemcitabine (2’, 2’-difluoro-2’-deoxycytidine; dFdCyd) is a nucleoside analog that is clinically effective in the treatment of solid tumors. In addition to its effects as a cytotoxic agent, it can also enhance the ability of ionizing radiation to kill cells, a process known as radiosensitization. dFdCyd is administered as a prodrug and is activated via phosphorylation to two metabolites: dFdCDP, which is a potent inhibitor of ribonucleotide reductase and results primarily in depletion of dATP in solid tumor cells, and dFdCTP, which is incorporated into DNA. Cytotoxicity correlated most strongly with dFdCTP incorporation into DNA, whereas radiosensitization with dFdCyd has correlated with dATP depletion. Ribonucleotide reductase consists of two subunits, R1, which is constitutively expressed, and R2, which is expressed primarily in S-phase. Recent studies resulted in the discovery of p53R2, a p53-inducible homolog of R2, which has been shown to interact with the R1 subunit to produce dNTPs following DNA damage. Because of the importance of RR inhibition to radiosensitization, we were interested in determining whether induction of p53R2 affects this process. Previously, our lab has demonstrated that MCF-7 breast cancer cells, which express wild-type p53, can activate p53R2 in response to DNA damage by dFdCyd or ionizing irradiation. It had been proposed by others that, following DNA damage, p53R2 induction led to R2 downregulation. However, our studies demonstrated that, after treatment with dFdCyd at non-cytotoxic or cytotoxic concentrations, p53R2 was expressed concurrently with R2. Since R2 is a cell cycle regulated protein, we evaluated expression of p53R2 and R2 with time following treatment with dFdCyd or ionizing irradiation along with cell cycle distribution. Treatment with dFdCyd for 24 h produced a dose dependent S-phase accumulation, and cells remained in S-phase for at least 24 h after washout. Both p53R2 and R2 were induced within 4 h of dFdCyd addition, with p53R2 expression remaining elevated for up to 48 h after washout. However, R2 expression decreased 24 h after washout, corresponding to progression of cells into G2/M and G1. After treatment with 5 Gy ionizing irradiation, p53R2 was induced and remained elevated for up to 72 h post-irradiation. In contrast, treatment with 5 Gy ionizing irradiation resulted in a decrease in R2 expression over time, which corresponded to cells progressing out of S-phase and accumulating in G1. These results indicate that R2 expression is not decreased due to p53R2 upregulation, but due to changes in cell cycle distribution, whereas p53R2 can be expressed throughout the cell cycle.