Bohm S, Berghard A, Pereswetoff-Morath C, Toftgård R
Karolinska Institute, Center for BioTechnology, NOVUM, Huddinge, Sweden.
Cancer Res. 1990 Mar 1;50(5):1626-33.
Complementary DNA clones representing genes in SENCAR mouse epidermis, the expression of which is induced 4 h after one topical application of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) were isolated. Of 56 isolated complementary DNA clones, 32 were identified to be identical to either metallothioneins (MT-I and MT-II) or endogenous retroviral like (VL30) sequences. In situ hybridization and analysis of mRNA levels in cell fractions separated by density gradient centrifugation revealed that MT induction was restricted to keratinocytes in the basal cell layer. Immunohistochemistry and time-kinetic studies on mRNA levels in mouse epidermis showed that the increase in MT and VL30 RNAs coincide in time with a TPA-induced transient block in basal cell proliferation (3-12 h after TPA treatment). MT immunoreactivity and transcript levels had returned to control values at a time point (24 h after treatment) when epidermis is known to hyperproliferate. Treatment with other types of tumor promoters showed that MT-I and MT-II mRNAs were coordinately induced and indicated that sn-1,2-dioctanoylglycerol, 12-O-retinoylphorbol-13-acetate, and mezerein induced MT to a lesser degree than TPA. The calcium ionophore A23187 induced mRNA levels for MTs as well as VL30. VL30 and MT mRNA levels were not found to be elevated in epidermal tumors whereas the mRNA level corresponding to glyceraldehyde-3-phosphate dehydrogenase was elevated in tumors and induced by TPA with time-kinetics that correlate with a TPA-induced hyperproliferation. These complementary DNA clones provide useful tools in the study of the gene-regulating effects of TPA in a target tissue relevant for tumor promotion.
分离出了代表SENCAR小鼠表皮中基因的互补DNA克隆，这些基因在局部应用肿瘤启动子12 - O - 十四酰佛波醇 - 13 - 乙酸酯（TPA）4小时后表达被诱导。在分离出的56个互补DNA克隆中，32个被鉴定与金属硫蛋白（MT - I和MT - II）或内源性逆转录病毒样（VL30）序列相同。原位杂交以及对通过密度梯度离心分离的细胞组分中mRNA水平的分析表明，MT的诱导仅限于基底细胞层中的角质形成细胞。对小鼠表皮中mRNA水平的免疫组织化学和时间动力学研究表明，MT和VL30 RNA的增加在时间上与TPA诱导的基底细胞增殖的短暂阻滞（TPA处理后3 - 12小时）一致。在已知表皮过度增殖的时间点（处理后24小时），MT免疫反应性和转录水平已恢复到对照值。用其他类型的肿瘤启动子处理表明，MT - I和MT - II mRNA被协同诱导，并且表明sn - 1,2 - 二辛酰甘油、12 - O - 视黄酰佛波醇 - 13 - 乙酸酯和大戟二萜醇对MT的诱导程度低于TPA。钙离子载体A23187诱导了MT以及VL30的mRNA水平。在表皮肿瘤中未发现VL30和MT mRNA水平升高，而与甘油醛 - 3 - 磷酸脱氢酶相对应的mRNA水平在肿瘤中升高，并由TPA诱导，其时间动力学与TPA诱导的过度增殖相关。这些互补DNA克隆为研究TPA在与肿瘤促进相关的靶组织中的基因调节作用提供了有用的工具。
