Hennings H, Robinson V A, Michael D M, Pettit G R, Jung R, Yuspa S H
Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, Bethesda, Maryland 20892.
Cancer Res. 1990 Aug 1;50(15):4794-800.
To facilitate the study of skin tumor promotion, a cell culture model system with characteristics analogous to initiated mouse epidermis was established. Cells of the keratinocyte cell line 308, derived from adult mouse skin initiated with 7,12-dimethylbenz[a]anthracene, display the initiated phenotype, since papillomas are produced when the cells are grafted to the backs of athymic mice. Coculture of a small number of these initiated cells with confluent normal primary keratinocytes resulted in the inhibition of growth of colonies of 308 cells. Addition of fresh keratinocytes weekly was required to sustain the inhibition for 3-4 weeks. Inhibition of 308 cell colonies required culture medium with a calcium concentration of 1.2 mM; normal keratinocytes did not inhibit 308 cells in medium with 0.05 mM calcium. Growth of 308 cells was not inhibited by coculture with confluent fibroblasts or by 1.2 mM calcium medium conditioned by either keratinocytes or fibroblasts. During continuous exposure of the cocultures to tumor promoters, 308 cell colonies became apparent within 2-3 weeks. A limited number of promoters were tested in this model system and 12-O-tetradecanoylphorbol-13-acetate, 12-O-retinoylphorbol-13-acetate, mezerein, and benzoyl peroxide were all active. The number of colonies which developed during promoter exposure in cocultures showed a dose-response curve which differed from the dose-response curve for stimulation of growth of 308 colonies in the absence of normal keratinocytes. Simultaneous treatment with 12-O-tetradecanoylphorbol-13-acetate and known inhibitors of skin tumor promotion, such as retinoic acid, fluocinolone acetonide, and bryostatin 1, blocked colony formation of 308 cells in cocultures but not in cultures with only 308 cells. In this model system, the actions of promoters and inhibitors both appear to be mediated by normal keratinocytes.
为便于研究皮肤肿瘤促进作用，建立了一种细胞培养模型系统，其特征类似于起始的小鼠表皮。角质形成细胞系308的细胞源自经7,12 - 二甲基苯并[a]蒽起始处理的成年小鼠皮肤，表现出起始表型，因为当将这些细胞移植到无胸腺小鼠的背部时会产生乳头状瘤。将少量这些起始细胞与汇合的正常原代角质形成细胞共培养会导致308细胞集落生长受到抑制。每周添加新鲜角质形成细胞以维持抑制作用3 - 4周。抑制308细胞集落需要钙浓度为1.2 mM的培养基；正常角质形成细胞在钙浓度为0.05 mM的培养基中不会抑制308细胞。308细胞的生长不会因与汇合的成纤维细胞共培养或与角质形成细胞或成纤维细胞条件化的1.2 mM钙培养基共培养而受到抑制。在共培养物持续暴露于肿瘤促进剂期间，308细胞集落在2 - 3周内变得明显。在该模型系统中测试了有限数量的促进剂，12 - O - 十四酰佛波醇 - 13 - 乙酸酯、12 - O - 视黄酰佛波醇 - 13 - 乙酸酯、大戟二萜醇酯和过氧化苯甲酰均具有活性。在共培养物中促进剂暴露期间形成的集落数量显示出剂量反应曲线，该曲线与在无正常角质形成细胞的情况下刺激308集落生长的剂量反应曲线不同。同时用12 - O - 十四酰佛波醇 - 13 - 乙酸酯和已知的皮肤肿瘤促进抑制剂（如视黄酸、醋酸氟轻松和苔藓抑素1）处理，可阻断共培养物中308细胞的集落形成，但在仅含308细胞的培养物中则不会。在该模型系统中，促进剂和抑制剂的作用似乎均由正常角质形成细胞介导。
