Guo Z, Heydari A R, Wu W, Yang H, Sabia M R, Richardson A
Department of Physiology, University of Texas Health Science Center, San Antonio, USA.
J Cell Physiol. 1998 Aug;176(2):314-22. doi: 10.1002/(SICI)1097-4652(199808)176:2<314::AID-JCP9>3.0.CO;2-R.
At present, almost all the information on gene-specific DNA repair in mammals comes from studies with transformed cell lines and proliferating primary cells obtained from rodents and humans. In the present study, we measured the repair of specific DNA regions in primary cultures of nondividing rat hepatocytes (parenchymal cells). DNA damage was induced by irradiating the primary cultures of hepatocytes with ultraviolet (UV) light, and the presence of cyclobutane pyrimidine dimers (CPDs) was measured by using T4 endonuclease V in the following: a 21-kb BamHI fragment containing the albumin gene, a 14-kb BamHI fragment containing the H-ras gene, and the genome overall. The frequency of CPDs in the two BamHI fragments and the genome overall were similar and ranged from 0.5 to 1.3 CPDs per 10 kb for UV doses of 5-30 J/m2. However, the removal of CPDs from the DNA fragment containing the albumin gene was significantly higher than from that of the genome overall and the DNA fragment containing the H-ras gene. Within 24 hr, approximately 67% of the CPDs was removed from the DNA fragment containing the albumin gene versus less than 40% for the genome overall and the DNA fragment containing the H-ras gene. The lower repair observed for the 14-kb fragment containing the H-ras gene is probably indicative of repair of the nontranscribed region of this fragment because the H-ras gene makes up only 2.4 kb of the 14-kb fragment. Primary cultures of hepatocytes removed CPDs from the transcribed strand of albumin fragment more efficiently than from the nontranscribed strand; however, no differences were observed in the repair of the two strands of the fragment containing the H-ras gene. These results demonstrate that primary cultures of nondividing rat hepatocytes show differential repair of UV-induced DNA damage that is comparable to what has been reported for transformed, proliferating mammalian cell lines.
目前，几乎所有关于哺乳动物基因特异性DNA修复的信息都来自对从啮齿动物和人类获得的转化细胞系以及增殖原代细胞的研究。在本研究中，我们测量了未分裂大鼠肝细胞（实质细胞）原代培养物中特定DNA区域的修复情况。通过用紫外线（UV）照射肝细胞原代培养物来诱导DNA损伤，并使用T4内切酶V在以下方面测量环丁烷嘧啶二聚体（CPD）的存在：一个包含白蛋白基因的21 kb BamHI片段、一个包含H-ras基因的14 kb BamHI片段以及整个基因组。对于5 - 30 J/m2的紫外线剂量，两个BamHI片段和整个基因组中CPD的频率相似，范围为每10 kb有0.5至1.3个CPD。然而，从包含白蛋白基因的DNA片段中去除CPD的效率明显高于整个基因组和包含H-ras基因的DNA片段。在24小时内，约67%的CPD从包含白蛋白基因的DNA片段中被去除，而整个基因组和包含H-ras基因的DNA片段中这一比例不到40%。观察到的包含H-ras基因的14 kb片段修复率较低，可能表明该片段非转录区域的修复情况，因为H-ras基因仅占14 kb片段的2.4 kb。肝细胞原代培养物从白蛋白片段的转录链中去除CPD的效率比从非转录链中更高；然而，在包含H-ras基因的片段的两条链的修复中未观察到差异。这些结果表明，未分裂大鼠肝细胞的原代培养物显示出紫外线诱导的DNA损伤的差异修复，这与已报道的转化的、增殖的哺乳动物细胞系的情况相当。
