Li G, Ho V C, Mitchell D L, Trotter M J, Tron V A
Department of Medicine, Vancouver Hospital and Health Sciences Centre, British Columbia, Canada.
Am J Pathol. 1997 Apr;150(4):1457-64.
The role of the tumor suppressor p53 in repair of ultraviolet light (UV)-induced DNA damage was evaluated using a host-cell reactivation (HCR) assay. HCR determines a cell's ability to repair UV-damaged DNA through reactivation of a transfected CAT reported plasmid. Most UV damage is removed through nucleotide excision repair (NER). Primary murine keratinocytes isolated from p53-deficient and wild-type p53 mice were used in the HCR assay. The NER was reduced in p53-/- keratinocytes as compared with p53+/+ keratinocytes. The reduced DNA repair in p53-/- mice was confirmed with a radioimmunoassay comparing cyclobutane dimers (CPDs) and (6-4) photoproducts in p53+/+ and p53-/- keratinocytes after the cells were exposed to UV irradiation. Our results demonstrate that wildtype p53 plays a significant role in regulating NER. Furthermore, as there is evidence that p53 protein levels decrease after keratinocytes become differentiated, we sought to determine whether p53 plays a role in NER in differentiated keratinocytes. Differentiation of the keratinocytes by increasing the Ca2+ concentration in the culture media resulted in a marked reduction in NER equally in both p53+/+ and p53-/- groups. This finding suggests that reduced DNA repair after differentiation is p53 independent. A similar reduction in HCR was confirmed in differentiated human keratinocytes. These data, taken together, indicate that p53 or p53-regulated proteins enhance NER in basal undifferentiated keratinocytes but not in differentiated cells. As nonmelanoma skin cancers originate from the basal keratinocytes, our findings suggest that loss of p53 may contribute to the pathogenesis of this common skin cancer.
使用宿主细胞再激活（HCR）试验评估肿瘤抑制因子p53在紫外线（UV）诱导的DNA损伤修复中的作用。HCR通过转染的CAT报告质粒的再激活来确定细胞修复UV损伤DNA的能力。大多数UV损伤通过核苷酸切除修复（NER）去除。从p53缺陷型和野生型p53小鼠中分离出的原代小鼠角质形成细胞用于HCR试验。与p53+/+角质形成细胞相比，p53-/-角质形成细胞中的NER降低。在用放射免疫测定法比较p53+/+和p53-/-角质形成细胞在暴露于UV照射后环丁烷二聚体（CPD）和（6-4）光产物时，证实了p53-/-小鼠中DNA修复减少。我们的结果表明，野生型p53在调节NER中起重要作用。此外，由于有证据表明角质形成细胞分化后p53蛋白水平会降低，我们试图确定p53在分化的角质形成细胞的NER中是否起作用。通过增加培养基中的Ca2+浓度使角质形成细胞分化，导致p53+/+和p53-/-组中的NER均显著降低。这一发现表明分化后DNA修复减少与p53无关。在分化的人角质形成细胞中也证实了HCR有类似的降低。综合这些数据表明，p53或p53调节的蛋白在未分化的基底角质形成细胞中增强NER，但在分化细胞中则不然。由于非黑素瘤皮肤癌起源于基底角质形成细胞，我们的发现表明p53的缺失可能有助于这种常见皮肤癌的发病机制。
