Maeda Tomoko, Espino Robin A, Chomey Eugene G, Luong Le, Bano Ather, Meakins Diana, Tron Victor A
Department of Laboratory Medicine and Pathology, University of Alberta, Faculty of Medicine, 4B1 Walter C Mackenzie Health Science Centre, 8440-112th Street, Edmonton, Alberta, Canada T6G 2R7.
Carcinogenesis. 2005 Oct;26(10):1804-10. doi: 10.1093/carcin/bgi140. Epub 2005 May 25.
Ultraviolet light (UV)-induced DNA damage is repaired primarily by the nucleotide excision repair (NER) pathway. Gadd45 is a multifunctional protein that regulates NER. Gadd45-deficient keratinocytes fail to repair UV-induced DNA damage, but the mechanism by which Gadd45 stimulates repair of UV-induced DNA damage is unknown. p21WAF1/Cip1 (p21) is a well-characterized downstream target of p53 that binds to Gadd45 and proliferating cell nuclear antigen (PCNA). The role of p21 in NER is somewhat controversial, however, recent studies appear to suggest that it inhibits DNA repair by inhibiting PCNA activity. Since a physical interplay exists between p21, Gadd45 and PCNA, we hypothesized that Gadd45 promoted DNA repair via p21. Initially, we examined p21 protein expression in Gadd45-deficient and proficient mice and found a higher base level of p21 protein in Gadd45-deficient keratinocytes and in most other tissues. With these results, we next speculated on the role played by p21 in Gadd45 regulated NER, by exposing keratinocytes from wild-type, single and double knockout (Gadd45 and p21) mice to UV, and measuring the responses. We confirmed that Gadd45-deficient keratinocytes were defective in UV-induced NER, but interestingly Gadd45/p21-null keratinocytes had normal NER in response to UV. Furthermore, Gadd45/p21-null keratinocytes were more resistant to UV-induced cell death than Gadd45-deficient keratinocytes. These results support the hypothesis that Gadd45 enhances NER by negatively regulating basal p21 expression in keratinocytes.
紫外线（UV）诱导的DNA损伤主要通过核苷酸切除修复（NER）途径进行修复。Gadd45是一种调节NER的多功能蛋白。Gadd45缺陷的角质形成细胞无法修复UV诱导的DNA损伤，但Gadd45刺激UV诱导的DNA损伤修复的机制尚不清楚。p21WAF1/Cip1（p21）是p53一个特征明确的下游靶点，它与Gadd45和增殖细胞核抗原（PCNA）结合。然而，p21在NER中的作用存在一定争议，最近的研究似乎表明它通过抑制PCNA活性来抑制DNA修复。由于p21、Gadd45和PCNA之间存在物理相互作用，我们推测Gadd45通过p21促进DNA修复。最初，我们检测了Gadd45缺陷和正常小鼠中p21蛋白的表达，发现Gadd45缺陷的角质形成细胞和大多数其他组织中p21蛋白的基础水平较高。基于这些结果，我们接下来通过将野生型、单基因敲除和双基因敲除（Gadd45和p21）小鼠的角质形成细胞暴露于UV并测量其反应，推测p21在Gadd45调节的NER中所起的作用。我们证实Gadd45缺陷的角质形成细胞在UV诱导的NER中存在缺陷，但有趣的是，Gadd45/p21双基因敲除的角质形成细胞对UV的NER反应正常。此外，Gadd45/p21双基因敲除的角质形成细胞比Gadd45缺陷的角质形成细胞对UV诱导的细胞死亡更具抗性。这些结果支持了Gadd45通过负向调节角质形成细胞中基础p21表达来增强NER的假说。
