We found that when noncytotoxic concentrations of gemcitabine were used, an EPZ5676 incubation period of 24

hours was associated with increased radiosensitization compared to treatment just before LDR. This finding is consistent with prior reports showing that it takes several hours to deplete dNTP pools [13] and [14]. Additionally, an increase in the number of cells in S phase was seen with our dosing schedule consistent with conditions needed for radiosensitization with gemcitabine [13] and [14]. 5-FU’s main mechanism of action is through inhibition of thymidylate synthase [15]. In our study, 5-FU was associated with a pronounced S phase arrest in both HCC cell lines tested. Pretreatment for 24 hours was associated with improved radiosensitivity at noncytotoxic concentrations. Because high levels of enhancement were seen at noncytotoxic concentrations, the mechanism of radiosensitivity is not simply related to killing of radioresistant cells in S phase. More likely, treatment Vincristine price with 5-FU leads to inappropriate S phase progression during LDR [16] and [17]. The findings from our study suggest that

LDR and gemcitabine or 5-FU have complementary effects on cell cycle distribution leading to enhanced radiosensitivity. LDR alone was associated with G2 arrest which persisted for ≥ 24 hours after the 16-hour course of LDR was complete. LDR-induced G2 arrest is well established and is the basis of the inverse dose rate effect [18]. Treatment with gemcitabine plus LDR was associated with a higher percentage of cells in S phase compared to LDR alone in addition to G2 arrest. Additionally, treatment Progesterone with 5-FU

produced S phase arrest in both cell lines. Abnormal progression through S phase in conjunction with LDR-induced G2 arrest would be predicted to lead to increased radiosensitivity. The formation and resolution of γH2AX foci provide insight into the induction of DNA damage and subsequent repair after LDR. As expected, in our study, we saw increased DNA damage and impaired DNA double-strand break repair when 5-FU or gemcitabine was added to LDR. A surprising finding was that DNA double-strand break repair was impaired for a longer period of time after LDR compared to cells treated with SDR at the same dose (4 Gy). Prior reports show that DNA damage is repaired during a course of LDR [19]. Therefore, one might predict less DNA damage after LDR compared to SDR therapy at the same dose. In this study, the percentage of γH2AX-positive cells was relatively similar for each dose rate when cells were examined shortly after radiation therapy; however, at 24 hours, treatment with gemcitabine and LDR was associated with a higher percentage of γH2AX-positive cells compared to treatment with SDR and gemcitabine.