Tron V A, Trotter M J, Tang L, Krajewska M, Reed J C, Ho V C, Li G
Department of Pathology, Vancouver Hospital and Health Science Centre, University of British Columbia, Canada.
Am J Pathol. 1998 Aug;153(2):579-85. doi: 10.1016/S0002-9440(10)65600-3.
Previous studies from our laboratory, using p53 transgenic mice, have suggested that ultraviolet (UV) light-induced keratinocyte apoptosis in the skin is not affected by overexpression of mutant p53 protein. To further elucidate a possible role for p53 in UV-induced keratinocyte cell death, we now examine apoptosis in skin and isolated keratinocytes from p53 null (-/-) mice and assess the influence of cell differentiation on this process. In vivo, using this knockout model, epidermal keratinocytes in p53-/- mice exhibited only a 5.2-fold increase in apoptosis after 2000 J/m2 UVB irradiation compared with a 26.3-fold increase in normal control animals. If this p53-dependent apoptosis is important in elimination of precancerous, UV-damaged keratinocytes, then it should be active in the undifferentiated cells of the epidermal basal layer. To test this hypothesis, we examined the effect of differentiation on UV-induced apoptosis in primary cultures of murine and human keratinocytes. Apoptosis was p53-independent in undifferentiated murine keratinocytes, which exhibited relative resistance to UVB-induced killing with only a 1.5-fold increase in apoptosis in p53+/+ cells and a 1.4-fold increase in p53-/- cells. Differentiated keratinocytes, in contrast, showed a 9.4-fold UVB induction of apoptosis in p53+/+ cells, almost three times the induction observed in p53-/- cells. This UV-induced difference in apoptosis was observed when keratinocytes were cultured on type IV collagen substrate, but not on plastic alone. Western blotting of UV-irradiated, differentiated keratinocytes did not support a role for either Bax or Bcl-2 in this process. In support of these findings in mice, cell death in human cultured keratinocytes also occurred in a differentiation-associated fashion. We conclude that p53-induced apoptosis eliminates damaged keratinocytes in the differentiated cell compartment, but this mechanism is not active in the basal, undifferentiated cells and is therefore of questionable significance in protection against skin cancer induction.
我们实验室之前利用p53转基因小鼠开展的研究表明，皮肤中紫外线（UV）诱导的角质形成细胞凋亡不受突变型p53蛋白过表达的影响。为了进一步阐明p53在紫外线诱导的角质形成细胞死亡中可能发挥的作用，我们现在检测了p53基因敲除（-/-）小鼠皮肤和分离出的角质形成细胞中的凋亡情况，并评估细胞分化对这一过程的影响。在体内，利用这种基因敲除模型，与正常对照动物26.3倍的凋亡增加相比，p53基因敲除小鼠的表皮角质形成细胞在2000 J/m2紫外线B照射后凋亡仅增加了5.2倍。如果这种依赖p53的凋亡在消除癌前紫外线损伤的角质形成细胞中很重要，那么它应该在表皮基底层的未分化细胞中发挥作用。为了验证这一假设，我们检测了分化对小鼠和人类角质形成细胞原代培养物中紫外线诱导凋亡的影响。在未分化的小鼠角质形成细胞中，凋亡不依赖p53，这些细胞对紫外线B诱导的杀伤表现出相对抗性，p53+/+细胞凋亡仅增加1.5倍，p53-/-细胞凋亡增加1.4倍。相比之下，分化的角质形成细胞中，p53+/+细胞的紫外线B诱导凋亡增加了9.4倍，几乎是p53-/-细胞中观察到的诱导倍数的三倍。当角质形成细胞在IV型胶原基质上培养时，观察到了这种紫外线诱导的凋亡差异，但仅在塑料上培养时未观察到。对紫外线照射的分化角质形成细胞进行蛋白质免疫印迹分析，不支持Bax或Bcl-2在此过程中发挥作用。为支持在小鼠中的这些发现，人类培养角质形成细胞中的细胞死亡也以与分化相关的方式发生。我们得出结论，p53诱导的凋亡消除了分化细胞区室中受损的角质形成细胞，但这种机制在基底未分化细胞中不活跃，因此在预防皮肤癌发生方面的意义值得怀疑。
