2’,2’-Difluoro-2’-Deoxycytidine (dFdCyd, Gemcitabine, Gemzar®)

Metabolism of Gemcitabine dFdCTP rNDP dNTP dNDP DNA dFdCyddFdCMP dFdCDP dFdUrddFdUMP ribonucleotid e reductase deoxycytidine kinase DNA polymerase s dFdCyd Transport

Radiosensitization Corresponds to dATP Depletion

X X X XXX + Gemcitabine dATP Mismatched Nucleotides +  -ray Most lesions repaired correctly Mismatches become mutations XX No further damage X X X X Mismatches not repaired DNA Hypothesis for Radiosensitization by Gemcitabine

Ribonucleotide Reductase Composed of 2 homodimeric subunits R1- 86 kDa Regulatory subunit R2- 43 kDa Catalytic subunit Eklund et al, Progress in Biophysics & Molecular Biology 77 (2001) 177–268 R1 R2

siRNA Protocol Exposed to Transfection Mixture for 24 h Split into T25 24 h prior to treatment Grow 24 h Add Drug 24 h + ionizing radiation Western blot Cytotoxicity Radiosensitization Wash, replete media Allowed to grow 0-72 h

Conclusions and Future Directions p53 status on dFdCyd efficacy –Solid tumors expressing wt or mt p53 should be sensitive to radiosensitization with dFdCyd Manipulation of p53R2 to increase cytotoxicity –Differential sensitivities of R1/p53R2 pairing compared to R1/R2 to anticancer agents –p53R2 suppression increased 5-FU cytotoxicity p53R2 silencing and radiosensitization –In some tumors, p53R2 silencing may enhance radiosensitization –Are the two cell lines inherently different, or are the differences due to incomplete silencing?