Barret J M, Calsou P, Salles B
Laboratoire de Pharmacologie et Toxicologie Fondamentales, CNRS, Toulouse, France.
Carcinogenesis. 1995 Jul;16(7):1611-6. doi: 10.1093/carcin/16.7.1611.
DNA repair activity in human peripheral blood lymphocytes (PBL) has been investigated by various techniques. Here, we report the use of an in vitro assay in order to assess nucleotide excision repair activity (NER). The mechanism of this major repair process relies on two broad steps: first, recognition, incision and excision of the damaged DNA; second, repair synthesis on the gapped DNA. Briefly, damaged plasmids were incubated with whole cell extracts which allows one to quantify DNA repair synthesis. When NER was determined on plasmid DNA damaged with UV-light or cisplatin, PBL extracts showed no repair synthesis for unstimulated lymphocytes. Using a new in vitro assay measuring only the damage-specific DNA incision activity in cell extracts, we found that the incision step in the repair reaction was blocked in unstimulated PBL. By mixing PBL with XP (group A, B, C, D) extracts, no restoration of NER activity was observed. In addition, these lymphocytes also lacked DNA replication activity as determined with pre-incised plasmid substrate. However, a phytohemagglutinin treatment of PBL led to an extent of repair synthesis similar to that observed with extracts from lymphoblastoid cells. When lymphocytes were incubated in 20% serum medium with and without phytohemagglutinin, the repair activity increased dramatically after 24 h. During the activation of lymphocytes, the extent of repair synthesis was proportional to the percentage of cells in S phase of the cell cycle. Our results suggest that the blockage of the cell cycle in G0/G1 in PBL may be responsible for their lack of NER activity.
人们已通过多种技术对人类外周血淋巴细胞（PBL）中的DNA修复活性进行了研究。在此，我们报告了一种体外测定法的应用，以评估核苷酸切除修复活性（NER）。这一主要修复过程的机制主要依赖两个广泛的步骤：首先，识别、切割和切除受损DNA；其次，在有缺口的DNA上进行修复合成。简而言之，将受损质粒与全细胞提取物一起孵育，这样就能对DNA修复合成进行定量。当对经紫外线或顺铂损伤的质粒DNA测定NER时，PBL提取物显示未刺激的淋巴细胞没有修复合成。使用一种仅测量细胞提取物中损伤特异性DNA切割活性的新体外测定法，我们发现未刺激的PBL中修复反应的切割步骤被阻断。通过将PBL与着色性干皮病（A、B、C、D组）提取物混合，未观察到NER活性的恢复。此外，如用预先切割的质粒底物所测定的，这些淋巴细胞也缺乏DNA复制活性。然而，用植物血凝素处理PBL会导致修复合成程度与淋巴母细胞提取物所观察到的相似。当淋巴细胞在含和不含植物血凝素的20%血清培养基中孵育时，24小时后修复活性显著增加。在淋巴细胞激活过程中，修复合成程度与细胞周期S期细胞的百分比成正比。我们的结果表明，PBL中细胞周期在G0/G1期的阻滞可能是其缺乏NER活性的原因。
