Im Michael M, Flanagan Sheryl A, Ackroyd Jeffrey J, Shewach Donna S
Department of Pharmacology, University of Michigan Medical Center, Ann Arbor, Michigan 48109.
Radiat Res. 2015 Jan;183(1):114-23. doi: 10.1667/RR13807.1. Epub 2015 Jan 7.
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.
吉西他滨（二氟脱氧胞苷；dFdCyd）是一种强效放射增敏剂，以其在体外非细胞毒性浓度以及患者亚化疗剂量下增强辐射细胞毒性的能力而闻名。人肿瘤细胞中的放射增敏作用需要dFdCyd介导细胞在S期积累并抑制核糖核苷酸还原酶，导致三磷酸脱氧腺苷（dATP）消耗≥80%以及DNA复制错误。关于特定DNA复制和修复途径在放射增敏机制中的作用，人们了解较少。在此，使用一对匹配的中国仓鼠卵巢（CHO）细胞系研究同源重组（HR）与dFdCyd的代谢和细胞周期效应之间的关系，这对细胞系基于HR蛋白XRCC3的表达，一个在HR方面功能正常（AA8细胞），另一个存在缺陷（irs1SF细胞）。结果表明，啮齿动物AA8细胞的放射增敏特征与人肿瘤细胞的显著不同。在AA8细胞中，仅在短时间（≤4小时）细胞毒性孵育条件下才能实现放射增敏，且放射增敏似乎不需要S期积累。相比之下，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期积累。对dFdCyd代谢效应的研究表明，低浓度dFdCyd在AA8和irs1SF细胞中不会使dATP消耗≥80%。然而，在较高浓度的dFdCyd下，HR缺陷的irs1SF细胞未能实现放射增敏，这无法用缺乏dATP消耗或缺乏S期积累来解释。因此，这些参数与CHO细胞中的dFdCyd放射增敏不相关。为了直接评估HR在放射增敏中的作用，用慢病毒递送的短发夹RNA（shRNA）抑制AA8细胞中的XRCC3表达。与未转导的细胞（放射增强比[RER]=2.7±0.27；P=0.012）相比，部分XRCC3抑制显著降低了放射增敏作用（RER=1.6±0.15），与非特异性shRNA转导的AA8细胞（RER=2.5±0.42；P=0.056）相比也有大幅下降。尽管结果支持HR在CHO细胞中dFdCyd放射增敏中发挥作用，但潜在的代谢和细胞周期特征差异表明，非肿瘤来源的CHO细胞中的dFdCyd放射增敏在机制上与人肿瘤细胞中的不同。
