■Lynch综合征患者由于DNA错配修复(MMR)基因缺乏而具有遗传的癌症倾向,如果暴露于电离辐射,可能会导致更高的癌症风险。这项初步研究旨在揭示CT相关低剂量(20mGy)和较高治疗剂量(2Gy)下MMR缺乏与放射敏感性之间的关联。
■使用细胞和细胞遗传学分析,在暴露于辐射之前和之后,分析了具有(dMMR)或没有MMR缺陷(pMMR)的人类结直肠癌细胞系。克隆形成测定以确定细胞生殖死亡;姐妹染色单体交换(SCE)测定以检测姐妹染色单体之间的DNA交换;γH2AX测定以分析DNA损伤修复;和凋亡分析以比较细胞死亡反应。在体外评估了这些测定的优点和局限性,并对其适用性和可行性进行了研究,以了解其用于进一步研究的潜力。
■克隆测定的结果表明,在2GyX辐射后,pMMR细胞系(HT29)比dMMR细胞系(HCT116,SW48和LoVo)显着更具放射抗性。细胞类型和辐射剂量都对SCE/染色体的产量有显著影响。当辐射样品的SCE/染色体的产量(2Gy)相对于对照进行归一化时,细胞系之间没有观察到显著差异。对于γH2AX测定,在暴露后30分钟(min)的时间点检查0、20mGy和2Gy,4和24h(h)。统计分析显示,HT29仅比MLH1缺陷细胞系显着更具放射抗性,但不是缺乏MSH2的细胞系。细胞凋亡分析(4Gy)显示HT29比HCT116明显更具放射抗性,尽管观察到非常少的凋亡细胞。
■总的来说,这项研究表明,在一些测定中,MMR熟练细胞系的放射性抗性,但不是在其他人。本研究中使用的所有方法都已得到验证;然而,由于与癌细胞系相关的限制,下一步将是在临床样本中使用这些检测方法,以了解辐射对Lynch患者的生物学和机制影响以及对健康的影响.
Lynch syndrome patients have an inherited predisposition to cancer due to a deficiency in DNA mismatch repair (MMR) genes which could lead to a higher risk of developing cancer if exposed to ionizing radiation. This pilot study aims to reveal the association between MMR deficiency and radiosensitivity at both a CT relevant low dose (20 mGy) and a therapeutic higher dose (2 Gy).
Human colorectal cancer cell lines with (dMMR) or without MMR deficiency (pMMR) were analyzed before and after exposure to radiation using cellular and cytogenetic analyses i.e., clonogenic assay to determine cell reproductive death; sister chromatid exchange (SCE) assay to detect the exchange of DNA between sister chromatids; γH2AX assay to analyze DNA damage repair; and apoptosis analysis to compare cell death response. The advantages and limitations of these assays were assessed in vitro, and their applicability and feasibility investigated for their potential to be used for further studies using clinical samples.
Results from the clonogenic assay indicated that the pMMR cell line (HT29) was significantly more radio-resistant than the dMMR cell lines (HCT116, SW48, and LoVo) after 2 Gy X-irradiation. Both cell type and radiation dose had a significant effect on the yield of SCEs/chromosome. When the yield of SCEs/chromosome for the irradiated samples (2 Gy) was normalized against the controls, no significant difference was observed between the cell lines. For the γH2AX assay, 0, 20 mGy and 2 Gy were examined at post-exposure time points of 30 min (min), 4 and 24 h (h). Statistical analysis revealed that HT29 was only significantly more radio-resistant than the MLH1-deficient cells lines, but not the MSH2-deficient cell line. Apoptosis analysis (4 Gy) revealed that HT29 was significantly more radio-resistant than HCT116 albeit with very few apoptotic cells observed.
Overall, this study showed radio-resistance of the MMR proficient cell line in some assays, but not in the others. All methods used within this study have been validated; however, due to the limitations associated with cancer cell lines, the next step will be to use these assays in clinical samples in an effort to understand the biological and mechanistic effects of radiation in Lynch patients as well as the health implications.