Paramio J M, Segrelles C, Laín S, Gómez-Casero E, Lane D P, Lane E B, Jorcano J L
Project on Cell and Molecular Biology and Gene Therapy, Centro de Investigaciones Energéticas Medioambientales y Tecnológicas, Madrid, Spain.
Mol Carcinog. 2000 Dec;29(4):251-62. doi: 10.1002/1098-2744(200012)29:4<251::aid-mc1007>3.0.co;2-o.
The p53 phosphoprotein acts as a tumor-suppressor gene product through the inhibition of cell growth and induction of apoptosis in a transcription-dependent manner. These functions require p53 activation through different biochemical postranslational modifications. Given the relevance of this protein in ultraviolet light-induced carcinogenesis, whose targets are primarily skin keratinocytes, we studied the functions of p53 in epidermal cell differentiation. We selected HaCaT cells, a human keratinocyte cell line bearing point-mutated, transcriptionally inactive, but highly stable p53, which facilitates immunochemical and biochemical analysis. In addition, a reliable in vitro differentiation system has been developed with these cells (Paramio et al. Oncogene 17:949, 1998). We report that during HaCaT differentiation there is a loss of immunoreactivity of p53 against antibodies that specifically recognize epitopes located at the carboxyl terminus of the protein. Because treatment with phosphatase restores this immunoreactivity, we conclude that p53 is phosphorylated at the carboxyl terminus during keratinocyte differentiation. This biochemical modification has been associated with the transcriptional activation of the molecule, and because p53 is involved in differentiation processes in other cell types, we investigated the potential functions of p53 during epidermal differentiation. To this end, we generated HaCaT clones expressing a murine temperature-sensitive p53 (Mp53ts) by transfection because the endogenous p53 is not functional even with phosphorylation. We characterized the expression and effects of the transfected protein in different selected clones. The ultraviolet-light response of these clones was restored, demonstrating the functionality of Mp53ts in these cells. We also observed that, with induction of differentiation, Mp53ts transfected cells differentiate faster than the parental or vector-transfected control cells, demonstrating that p53 promotes epidermal differentiation. The sustained expression of p53 in differentiating cells leads to massive cell death and detachment, a phenomenon that may be similar to epidermal terminal differentiation. In addition, we observed that the expression of p53-dependent genes such as p21waf/cip1 and mdm2 (which are known to participate in epidermal differentiation) increases during HaCaT differentiation, i.e., in a p53-independent manner.
p53磷酸化蛋白作为一种肿瘤抑制基因产物，通过转录依赖性方式抑制细胞生长并诱导细胞凋亡。这些功能需要通过不同的生化翻译后修饰来激活p53。鉴于该蛋白在紫外线诱导的致癌作用中的相关性，其主要靶点是皮肤角质形成细胞，我们研究了p53在表皮细胞分化中的功能。我们选择了HaCaT细胞，这是一种人角质形成细胞系，其p53存在点突变、转录无活性但高度稳定，这便于进行免疫化学和生化分析。此外，已经利用这些细胞开发了一种可靠的体外分化系统（Paramio等人，《癌基因》17:949，1998）。我们报告，在HaCaT细胞分化过程中，p53针对特异性识别位于该蛋白羧基末端表位的抗体的免疫反应性丧失。因为用磷酸酶处理可恢复这种免疫反应性，我们得出结论，在角质形成细胞分化过程中p53在羧基末端被磷酸化。这种生化修饰与该分子的转录激活有关，并且由于p53参与其他细胞类型的分化过程，我们研究了p53在表皮分化过程中的潜在功能。为此，我们通过转染产生了表达鼠温度敏感型p53（Mp53ts）的HaCaT克隆，因为即使发生磷酸化，内源性p53也无功能。我们在不同的选定克隆中表征了转染蛋白的表达和作用。这些克隆的紫外线反应得以恢复，证明了Mp53ts在这些细胞中的功能。我们还观察到，随着分化的诱导，转染了Mp53ts的细胞比亲本细胞或载体转染的对照细胞分化得更快，表明p53促进表皮分化。p53在分化细胞中的持续表达导致大量细胞死亡和脱落，这一现象可能类似于表皮终末分化。此外，我们观察到，在HaCaT细胞分化过程中，即通过一种不依赖p53的方式，p21waf/cip1和mdm2等p53依赖性基因（已知参与表皮分化）的表达增加。
