Miura M, Domon M, Sasaki T, Kondo S, Takasaki Y
Department of Dental Radiology and Radiation Research, Faculty of Dentistry, Tokyo Medical and Dental University, Japan.
Exp Cell Res. 1992 Aug;201(2):541-4. doi: 10.1016/0014-4827(92)90308-u.
We examined the relationship between the formation of proliferating cell nuclear antigen (PCNA) complex with DNA and nucleotide excision repair in human fibroblasts following ultraviolet light (uv) irradiation. PCNA complex formation was detected by the immunofluorescence method after methanol fixation and nucleotide excision repair activity was detected as the unscheduled DNA synthesis (UDS) by autoradiography labeled with [3H]thymidine. Quiescent normal cells showed a strong punctuated pattern of PCNA staining 5 min to 3 h and UDS 3 h after 10 J/m2 of uv irradiation, but they no longer showed PCNA staining and UDS 24 h after irradiation. In contrast, xeroderma pigmentosum group A (XP-A) cells, which lack UDS activity, did not show PCNA staining up to 30 min after irradiation; however, unexpectedly, they were stained 3 h and even 24 h after irradiation with their staining pattern being different from that in normal cells. Namely, the fluorescence spots in XP-A cells were larger in size and much smaller in number than those in normal cells. When XP-A cells were fused with normal cells with polyethylene glycol treatment, nuclei of XP-A cells showed a PCNA staining pattern similar to that of normal cells at 30 min, which was no longer detected 24 h after irradiation. These results suggest that there exist two types of PCNA complex formation, nucleotide excision repair-related and -unrelated, in human fibroblasts following uv irradiation.
我们研究了人成纤维细胞在紫外线（uv）照射后增殖细胞核抗原（PCNA）复合物与DNA的形成以及核苷酸切除修复之间的关系。甲醇固定后通过免疫荧光法检测PCNA复合物的形成，并用[3H]胸腺嘧啶标记通过放射自显影检测核苷酸切除修复活性，即非预定DNA合成（UDS）。静止的正常细胞在10 J/m2紫外线照射后5分钟至3小时显示出强烈的PCNA点状染色模式，照射后3小时显示UDS，但照射后24小时不再显示PCNA染色和UDS。相比之下，缺乏UDS活性的着色性干皮病A组（XP-A）细胞在照射后30分钟内未显示PCNA染色；然而，出乎意料的是，照射后3小时甚至24小时它们被染色，其染色模式与正常细胞不同。即，XP-A细胞中的荧光斑点尺寸更大但数量比正常细胞少得多。当用聚乙二醇处理将XP-A细胞与正常细胞融合时，XP-A细胞的细胞核在30分钟时显示出与正常细胞相似的PCNA染色模式，照射后24小时不再检测到。这些结果表明，在紫外线照射后的人成纤维细胞中存在两种类型的PCNA复合物形成，即与核苷酸切除修复相关和不相关的。
