Frazelle J H, Abernethy D J, Boreiko C J
Carcinogenesis. 1983;4(6):709-15. doi: 10.1093/carcin/4.6.709.
Treatment of low density asynchronous cultures of C3H/10T1/2 Cl 8 mouse embryo fibroblasts with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) initiates the process of transformation and produces significant numbers of transformed foci only when treated cultures are subsequently exposed to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Cell culture variables which influence the outcome of this initiation and promotion system were studied. A TPA concentration of 0.25 micrograms/ml was found to be optimal for the promotion of focus production and the presence of TPA was required both during logarithmic growth and throughout confluence. The lot of fetal calf serum used to cultivate the cells also played a determining role in focus production. Of nine serum lots purchased from four different suppliers, only two were suited for initiation and promotion studies with MNNG and TPA. In contrast, seven of these lots were adequate for transformation studies with 3-methylcholanthrene. Factors which adversely influenced focus production included the use of fungizone or the use of high passage stock cultures. These studies demonstrate that cell culture variables which influence promotion in these cells can be controlled and that this system can be successfully used in studies of the cellular mechanism of in vitro promotion and for the detection of genotoxic substances.
用N-甲基-N'-硝基-N-亚硝基胍（MNNG）处理C3H/10T1/2 Cl 8小鼠胚胎成纤维细胞的低密度异步培养物可启动转化过程，并且只有在处理后的培养物随后暴露于肿瘤促进剂12-O-十四烷酰佛波醇-13-乙酸酯（TPA）时才会产生大量转化灶。研究了影响这种启动和促进系统结果的细胞培养变量。发现TPA浓度为0.25微克/毫升最适合促进灶的产生，并且在对数生长期和整个汇合期都需要TPA的存在。用于培养细胞的胎牛血清批次在灶的产生中也起决定性作用。从四个不同供应商购买的九批血清中，只有两批适合用于MNNG和TPA的启动和促进研究。相比之下，其中七批血清足以用于3-甲基胆蒽的转化研究。对灶的产生有不利影响的因素包括使用制霉菌素或使用高传代的原代培养物。这些研究表明，影响这些细胞促进作用的细胞培养变量可以得到控制，并且该系统可以成功用于体外促进的细胞机制研究和遗传毒性物质的检测。
