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.
J Cell Physiol. 1992 Sep;152(3):639-45. doi: 10.1002/jcp.1041520324.
We examined the role of the factor deficient in xeroderma pigmentosum group A (XP-A) cells in the formation of proliferating cell nuclear antigen (PCNA) complex with DNA in the DNA repair process in human fibroblasts following cis-diamminedichloroplatinum (CDDP)-treatment. Immunofluorescence staining after methanol fixation was used to detect the PCNA complex formation. When quiescent normal cells were PCNA-stained at 3 h after 100 microM CDDP treatment for 1 h, almost all nuclei of the cells showed a punctuated staining pattern. On the other hand, nuclei of XP-A cells were not stained. These results were the same with the findings following 10J/m2 of ultraviolet light (UV)-irradiation. The quantitative analysis of the PCNA immunofluorescence intensity of normal cells revealed that the mean intensity was increased by 4.8 times by the CDDP-treatment and 6.1 times by the UV-irradiation, compared with that of untreated cells. The intensities among nuclei ranged widely in both treatments. In contrast, the mean intensity was not increased in XP-A cells by the same treatments. However, when XP-A cells were fused with normal cells with polyethylene glycol (PEG) treatment, the nuclei of the XP-A cells showed positive PCNA-staining following CDDP-treatment or UV-irradiation in almost all cases. These results suggest that the PCNA complex formation may play a role in the DNA repair process after the step where the factor deficient in XP-A cells is involved following CDDP-treatment as well as following UV-irradiation.
我们研究了人成纤维细胞经顺二氯二氨铂(CDDP)处理后,在DNA修复过程中,着色性干皮病A组(XP - A)细胞中缺乏的因子在增殖细胞核抗原(PCNA)与DNA形成复合物过程中的作用。采用甲醇固定后的免疫荧光染色检测PCNA复合物的形成。当静止的正常细胞在100 microM CDDP处理1小时后3小时进行PCNA染色时,几乎所有细胞的细胞核都呈现点状染色模式。另一方面,XP - A细胞的细胞核未被染色。这些结果与10J/m2紫外线(UV)照射后的结果相同。对正常细胞PCNA免疫荧光强度的定量分析表明,与未处理细胞相比,CDDP处理使平均强度增加了4.8倍,UV照射使平均强度增加了6.1倍。在两种处理中,细胞核间的强度范围都很广。相比之下,相同处理并未使XP - A细胞的平均强度增加。然而,当用聚乙二醇(PEG)处理使XP - A细胞与正常细胞融合时,几乎在所有情况下,CDDP处理或UV照射后,XP - A细胞的细胞核都显示PCNA阳性染色。这些结果表明,在CDDP处理以及UV照射后,PCNA复合物的形成可能在DNA修复过程中,在涉及XP - A细胞中缺乏的因子的步骤之后发挥作用。