Drug metabolism and homologous recombination repair in radiosensitization with gemcitabine.
Drug metabolism and homologous recombination repair in radiosensitization with gemcitabine.
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DOI：
10.1667/rr13807.1
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发表时间：
2015-01
影响因子：
3.4
通讯作者：
Shewach DS
中科院分区：
文献类型：
--
作者：
Im MM;Flanagan SA;Ackroyd JJ;Shewach DS
吉西他滨（二氟脱氧胞苷; dFdCyd）是一种有效的放射增敏剂，以其在体外以非细胞毒性浓度和在患者中以亚化疗剂量辐射时增强细胞毒性的能力而闻名。人肿瘤细胞的放射增敏需要dFdCyd介导的S期细胞蓄积，并抑制核糖核苷酸还原酶，导致≥80%的三磷酸脱氧腺苷（dATP）耗竭和DNA复制错误。对特定DNA复制和修复途径在放射增敏机制中的作用知之甚少。在此，基于HR蛋白XRCC 3的表达，使用一对匹配的CHO细胞系研究了同源重组（HR）与dFdCyd的代谢和细胞周期效应的关系。结果表明，啮齿动物AA 8细胞的放射增敏特性与人肿瘤细胞的放射增敏特性有显著差异。在AA 8细胞中，仅在短时间（≤4 h）细胞毒性孵育下实现放射增敏，放射增敏似乎不需要S期蓄积。相反，人肿瘤细胞系使用非细胞毒性浓度的dFdCyd进行放射增敏，并且需要早期S期蓄积。dFdCyd代谢效应的研究表明，在AA 8和irs 1 SF细胞中，低浓度dFdCyd不会使dATP消耗≥80%。然而，在更高浓度的dFdCyd，未能放射增敏的HR缺陷irs 1 SF细胞不能解释缺乏dATP耗尽或缺乏S期积累。因此，这些参数不对应于CHO细胞中的dFdCyd放射增敏。为了直接评估HR在放射增敏中的作用，用慢病毒递送的shRNA在AA 8细胞中抑制XRCC 3表达。与未转导的（RER = 2.7 ± 0.27; P = 0.012）相比，部分XRCC 3抑制显著降低放射增敏性[放射增强比（RER）= 1.6 ± 0.15]，并且与非特异性shRNA转导的（RER =2.5 ± 0.42; P =0.056）AA 8细胞相比显著降低。尽管结果支持HR在CHO细胞中与dFdCyd的放射增敏中的作用，但基础代谢和细胞周期特征的差异表明，非肿瘤来源的CHO细胞中的dFdCyd放射增敏机制与人肿瘤细胞中的不同。
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.