Morgan D, Welty D, Glick A, Greenhalgh D, Hennings H, Yuspa S H
Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, Bethesda, Maryland 20892.
Cancer Res. 1992 Jun 1;52(11):3145-56.
Initiation and promotion in mouse skin carcinogenesis produce multiple benign tumors, squamous papillomas, but only a few squamous cell carcinomas. The spontaneous conversion from the benign to the malignant phenotype occurs over many months and in stages, but induced malignant conversion can be accomplished more rapidly by exposure of papilloma-bearing mice to mutagens or by transfection of papilloma cell lines with specific oncogenes. The analysis of genetic targets responsible for carcinogen-induced neoplastic progression would be facilitated by the development of in vitro models where the process is rapid, focal, and quantitative. To this end, primary newborn mouse keratinocytes were initiated in vitro by the introduction of the v-rasHa oncogene via a defective retrovirus. Recipient cells produce squamous papillomas and have a high proliferation rate in culture medium with 0.05 mM Ca2+, but fail to grow in medium with 0.5 mM Ca2+ which is permissive for growth of malignant keratinocytes. When v-rasHa-keratinocytes were exposed to mutagens in vitro, proliferative foci emerged after culture in 0.5 mM Ca2+ for 4 weeks. These foci stained intensely red with rhodamine stain, could be easily quantitated, and readily incorporated bromodeoxyuridine. Dose-response studies with several mutagens indicated that the number of foci increased with concentration to the point where excessive cytotoxicity developed. Mutagens varied in potency for producing foci in the following order: cis-diamminedichloroplatinum greater than or equal to benzo(a)pyrene diolexpoxide I greater than N-methyl-N'-nitro-N-nitrosoguanidine greater than or equal to 4-nitroquinoline-N-oxide greater than N-acetoxy-acetyl- aminofluorene. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate was inactive in the assay. A subset of cell lines derived from foci produced malignant tumors in vivo, while others were not tumorigenic. Analysis of DNA from cell lines and tumors revealed that most tumorigenic cell lines maintained the v-rasHa genome, whereas the viral sequences were deleted in nontumorigenic cell lines. Immunohistochemical analysis indicated that proliferative foci and quiescent v-rasHa keratinocytes expressed keratin 8, a marker of v-rasHa expression in cultured keratinocytes. Cells in foci, but not v-rasHa control cells, expressed keratin 13, a marker which is strongly associated with the malignant progression of skin tumors in vivo. This in vitro assay provides a quantitative model to study chemically induced focal neoplastic progression at the cellular level and to identify agents which may be selective for enhancing malignant conversion.
小鼠皮肤癌发生过程中的启动和促进阶段会产生多个良性肿瘤，即鳞状乳头瘤，但只有少数鳞状细胞癌。从良性表型到恶性表型的自发转变会在数月内分阶段发生，但通过将携带乳头瘤的小鼠暴露于诱变剂或用特定癌基因转染乳头瘤细胞系，可更快地实现诱导性恶性转变。体外模型的建立有助于分析致癌剂诱导肿瘤进展的遗传靶点，在这种模型中，该过程快速、局部且可定量。为此，通过缺陷型逆转录病毒导入v-rasHa癌基因，在体外启动新生小鼠原代角质形成细胞。受体细胞在含有0.05 mM Ca2+的培养基中产生鳞状乳头瘤且增殖率高，但在含有0.5 mM Ca2+的培养基中无法生长，而该培养基允许恶性角质形成细胞生长。当v-rasHa-角质形成细胞在体外暴露于诱变剂时，在含有0.5 mM Ca2+的培养基中培养4周后出现增殖灶。这些灶用罗丹明染色呈深红色，易于定量，且易于掺入溴脱氧尿苷。对几种诱变剂的剂量反应研究表明，灶的数量随浓度增加而增加，但到产生过度细胞毒性时达到顶点。诱变剂产生灶的效力顺序如下：顺二氯二氨铂≥苯并(a)芘二环氧物I＞N-甲基-N'-硝基-N-亚硝基胍≥4-硝基喹啉-N-氧化物＞N-乙酰氧基-乙酰氨基芴。肿瘤促进剂12-O-十四酰佛波醇-13-乙酸酯在该试验中无活性。从灶衍生的一部分细胞系在体内产生恶性肿瘤，而其他细胞系则无致瘤性。对细胞系和肿瘤的DNA分析表明，大多数致瘤细胞系保留了v-rasHa基因组，而非致瘤细胞系中病毒序列被删除。免疫组织化学分析表明，增殖灶和静止的v-rasHa角质形成细胞表达角蛋白8，这是培养的角质形成细胞中v-rasHa表达的标志物。灶中的细胞而非v-rasHa对照细胞表达角蛋白13，该标志物与体内皮肤肿瘤的恶性进展密切相关。这种体外试验提供了一个定量模型，用于在细胞水平研究化学诱导的局部肿瘤进展，并识别可能对增强恶性转变具有选择性的药物。
