Drug Metabolism and Homologous Recombination Repair in Radiosensitization with Gemcitabine

Abstract

Gemcitabine (difluorodeoxycytidine; dFdCyd) is a potent radiosensitizer, noted for its ability to enhance cytotoxicity with radiation at noncytotoxic concentrations in vitro and subchemotherapeutic doses in patients. Radiosensitization in human tumor cells requires dFdCyd-mediated accumulation of cells in S phase with inhibition of ribonucleotide reductase, resulting in ≥80% deoxyadenosine triphosphate (dATP) depletion and errors of replication in DNA. Less is known of the role of specific DNA replication and repair pathways in the radiosensitization mechanism. Here the role of homologous recombination (HR) in relationship to the metabolic and cell cycle effects of dFdCyd was investigated using a matched pair of CHO cell lines that are either proficient (AA8 cells) or deficient (irs1SF cells) in HR based on expression of the HR protein XRCC3. The results demonstrated that the characteristics of radiosensitization in the rodent AA8 cells differed significantly from those in human tumor cells. In the AA8 cells, radiosensitization was achieved only under short (≤4 h) cytotoxic incubations, and S-phase accumulation did not appear to be required for radiosensitization. In contrast, human tumor cell lines were radiosensitized using noncytotoxic concentrations of dFdCyd and required early S-phase accumulation. Studies of the metabolic effects of dFdCyd demonstrated low dFdCyd concentrations did not deplete dATP by ≥80% in AA8 and irs1SF cells. However, at higher concentrations of dFdCyd, failure to radiosensitize the HR-deficient irs1SF cells could not be explained by a lack of dATP depletion or lack of S-phase accumulation. Thus, these parameters did not correspond to dFdCyd radiosensitization in the CHO cells. To evaluate directly the role of HR in radiosensitization, XRCC3 expression was suppressed in the AA8 cells with a lentiviral-delivered shRNA. Partial XRCC3 suppression significantly decreased radiosensitization [radiation enhancement ratio (RER) = 1.6 ± 0.15], compared to nontransduced (RER = 2.7 ± 0.27; P = 0.012), and a substantial decrease compared to nonspecific shRNA-transduced (RER =2.5 ± 0.42; P =0.056) AA8 cells. Although the results support a role for HR in radiosensitization with dFdCyd in CHO cells, the differences in the underlying metabolic and cell cycle characteristics suggest that dFdCyd radiosensitization in the nontumor-derived CHO cells is mechanistically distinct from that in human tumor cells.

Gemcitabine-Based Chemoradiotherapy Enhanced by a PARP Inhibitor in Pancreatic Cancer Cell Lines

“…In their treatment schedules, cells were treated 24 h before irradiation with gemcitabine and at time of irradiation, approximately 45% of cells were in S-phase [11]. Indeed, Im et al presented that radiosensitization with gemcitabine required a depletion in deoxynucleotide for approximately 4 h with accumulation of cells in S-phase [12]. These observations are coherent with our hypothesis that gemcitabine could synchronize cell lines into S-phase, thus leading to enhanced sensitization after irradiation and olaparib treatment.…”

Inactivation of XPF Sensitizes Cancer Cells to Gemcitabine

“…Our results and the reports from others found that BRCA2 (and likely other genes involved in HR) is required for the cytotoxicity of gemcitabine [ 24 , 25 ]. BRCA2-deficient cells are more resistant to gemcitabine treatment compared to the BRCA2-restored cells ( Figure 4 ).…”

“…Thus, a defect in homologous recombination (HR) is expected to sensitize cells to gemcitabine. However, previous reports indicated that homologous recombination (HR) is indeed required for the cytotoxicity of gemcitabine [ 24 , 25 ]. We examined sensitivity to gemcitabine in BRCA2-defective ovarian cancer cell line PE01 and its BRCA2-revertant PE01(C4-2).…”

Radio-Resistance and DNA Repair in Pediatric Diffuse Midline Gliomas

“…Gemcitabine (difluorodeoxycytidine; dFdCyd) inhibits DNA replication and was found to radio-sensitize a wide range of cancer cell models in vitro [ 104 , 105 ]. Although the mechanism of sensitization is not yet clear, recent evidence suggests that gemcitabine lowers the threshold for radiation-induced apoptosis [ 106 ]. An open-label, single-arm phase I/II trial in newly diagnosed DMGs explored the potential and safety of gemcitabine administered concomitantly with RT.…”