Tronov V A, Loginova M Iu, Kramarenko I I
Genetika. 2008 May;44(5):686-92. doi: 10.1134/s1022795408050128.
Total repair capability is a widely used phenotypic marker of predisposition to cancer. Evaluation of this parameter implies using a challenge mutagen in an in vitro system to unmask latent genetic instability and repair insufficiency in the target cells. Traditionally, these investigations involve two tests, evaluation of mutagenic susceptibility (chromosomal aberrations) and genotoxic effect (DNA comet assay). The present study was focused on analysis of the effect of methylnitrosourea (MNU) on resting and PHA-stimulated lymphocytes from healthy donors and patients with gynecological cancer. Cytotoxic effect of MNU (apoptotic lymphocyte death) was estimated using two parameters, interaction of the cells with the annexin V-FITC complex, and morphological changes of the nuclei after their staining with the mixture of two DNA tropic dyes. The genotoxic effect of MNU, namely, secondary double-strand DNA breaks, was scored using the neutral comet assay, modified for the calculation of the comets produced exclusively by BrUdr-labeled proliferating lymphocytes. The proportion of these comets was represented as the proliferative cell index. It was shown that resting lymphocytes were resistant to genotoxic and cytotoxic effects of MNU. The response of proliferating cells to the action of MNU was expressed as the development of secondary DNA breaks (P <0.01), along with the increased frequency of apoptosis (P <0.05). The genotoxic effect of MNU on stimulated lymphocytes of gynecological cancer patients was fourfold lower compared to healthy donor lymphocytes. In response to the MNU action, patient lymphocytes did not change their proliferative index, while in healthy donor lymphocytes proliferative index was two times decreased in response to the MNU action. The data obtained pointed to the association between the cytotoxic response of the lymphocytes to the action of MNU and gynecological cancer. Since only proliferating lymphocytes response to the genotoxic effect of MNU, and the effect is revealed a day after the mutagen action, it is suggested that this phenomenon is associated with postreplicative repair, MMR, the substrate of which is O6-methylguanin. The MMR deficiency in patient lymphocytes determines their tolerance to the action of MNU. Genotoxic effect of lymphocytes to the action of MNU can serve as a marker of MMR, as well as of the MMR deficiency-associated gynecological cancer.
总修复能力是一种广泛用于癌症易感性的表型标志物。对该参数的评估意味着在体外系统中使用诱变剂来揭示靶细胞中潜在的遗传不稳定性和修复不足。传统上，这些研究涉及两项测试，即诱变敏感性评估（染色体畸变）和遗传毒性效应评估（DNA彗星试验）。本研究的重点是分析甲基亚硝基脲（MNU）对健康供体以及妇科癌症患者的静息淋巴细胞和PHA刺激的淋巴细胞的影响。使用两个参数评估MNU的细胞毒性作用（凋亡性淋巴细胞死亡），即细胞与膜联蛋白V-FITC复合物的相互作用，以及用两种DNA嗜染染料混合物染色后细胞核的形态变化。使用中性彗星试验对MNU的遗传毒性作用，即继发性双链DNA断裂进行评分，该试验经过改良以计算仅由BrUdr标记的增殖淋巴细胞产生的彗星。这些彗星的比例表示为增殖细胞指数。结果表明，静息淋巴细胞对MNU的遗传毒性和细胞毒性作用具有抗性。增殖细胞对MNU作用的反应表现为继发性DNA断裂的出现（P<0.01），同时凋亡频率增加（P<0.05）。与健康供体淋巴细胞相比，MNU对妇科癌症患者刺激淋巴细胞的遗传毒性作用低四倍。响应MNU的作用，患者淋巴细胞的增殖指数没有变化，而健康供体淋巴细胞的增殖指数在响应MNU的作用时降低了两倍。所获得的数据表明淋巴细胞对MNU作用的细胞毒性反应与妇科癌症之间存在关联。由于只有增殖淋巴细胞对MNU的遗传毒性作用有反应，并且这种作用在诱变剂作用一天后才显现出来，因此提示这种现象与复制后修复、错配修复（MMR）有关，其底物是O6-甲基鸟嘌呤。患者淋巴细胞中的MMR缺陷决定了它们对MNU作用的耐受性。淋巴细胞对MNU作用的遗传毒性作用可作为MMR以及与MMR缺陷相关的妇科癌症的标志物。
