Ruggeri B, Caamano J, Goodrow T, DiRado M, Bianchi A, Trono D, Conti C J, Klein-Szanto A J
Department of Pathology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111.
Cancer Res. 1991 Dec 15;51(24):6615-21.
The two-stage murine skin tumorigenesis model is widely used to study the development of squamous cell neoplasias. We have investigated expression of the p53 and retinoblastoma tumor suppressor genes in eight murine skin tumor cell lines of varied histopathology and malignant potential, in seven in vivo-derived clones from these cell lines, and in 39 primary short-term cultures of similarly induced skin tumors at various stages of tumor progression. One squamous cell carcinoma cell line and three more malignant clones derived from it revealed mutations of the p53 protein by immunoprecipitation analyses despite normal-sized p53 transcripts. Sequence analysis identified the nature of the point mutations in these lines, a G----C transversion in codon 132. Mouse retinoblastoma transcripts and protein were unaltered in all the cell lines examined. Among short-term cultures of skin tumors, the p53 gene appeared normal in all papillomas and early well-differentiated carcinomas by Southern and immunoprecipitation analyses. In contrast, four of eight tumors from later stages of promotion (50-60 weeks) possessed alterations in p53, including loss of the p53 product, and loss of immunoreactivity with a murine-specific antibody recognizing only wild-type p53 protein. Loss of heterozygosity at the p53 locus was similarly observed in several more malignant tumors from later stages of promotion. In contrast retinoblastoma expression was normal regardless of the stage of promotion or histological grade of the tumor. Direct sequence analyses of exons 5 through 8 of the p53 gene in eight advanced murine skin tumors revealed a 25% incidence of p53 mutations. These point mutations were located in codons 245 and 263. Collectively, these data indicate that alterations in the p53 gene occur in 25 to 50% of murine skin tumors induced by the two-stage tumorigenesis protocol and are later events in murine skin tumor progression. Moreover, these alterations are associated with tumors possessing a more malignant and/or poorly differentiated phenotype.
两阶段小鼠皮肤肿瘤发生模型被广泛用于研究鳞状细胞肿瘤的发展。我们研究了p53和视网膜母细胞瘤肿瘤抑制基因在8种组织病理学和恶性潜能各异的小鼠皮肤肿瘤细胞系、这些细胞系的7个体内衍生克隆以及39个处于肿瘤进展不同阶段的类似诱导皮肤肿瘤的原代短期培养物中的表达情况。通过免疫沉淀分析，一个鳞状细胞癌细胞系及其衍生的3个恶性程度更高的克隆尽管p53转录本大小正常，但显示出p53蛋白的突变。序列分析确定了这些细胞系中突变点的性质，即密码子132处的G→C颠换。在所检测的所有细胞系中，小鼠视网膜母细胞瘤转录本和蛋白均未改变。在皮肤肿瘤的短期培养物中，通过Southern印迹和免疫沉淀分析，所有乳头状瘤和早期高分化癌中的p53基因似乎都是正常的。相比之下，在促癌后期（50 - 60周）的8个肿瘤中，有4个p53发生了改变，包括p53产物的缺失以及与仅识别野生型p53蛋白的鼠特异性抗体的免疫反应性丧失。在促癌后期的几个恶性程度更高的肿瘤中同样观察到了p53基因座的杂合性缺失。相比之下，无论促癌阶段或肿瘤的组织学分级如何，视网膜母细胞瘤的表达都是正常的。对8个晚期小鼠皮肤肿瘤中p53基因第5至8外显子的直接序列分析显示，p53突变发生率为25%。这些点突变位于密码子245和263处。总体而言，这些数据表明，在两阶段肿瘤发生方案诱导的小鼠皮肤肿瘤中，25%至50%会发生p53基因改变，且这些改变是小鼠皮肤肿瘤进展过程中的后期事件。此外，这些改变与具有更恶性和/或低分化表型的肿瘤相关。
