Su Z Z, O'Brian C A, Fisher P B
Department of Pathology, Columbia University, College of Physicians and Surgeons, New York, N.Y. 10032.
Cell Mol Biol. 1992 Feb;38(1):27-39.
The E1A gene of adenovirus type 5 (Ad5) induces morphological transformation and anchorage-independent growth in cloned rat embryo fibroblast (CREF) cells. In contrast, CREF cells transfected with a beta 1 protein kinase C (PKC) gene and expressing low-levels of beta 1 PKC display a CREF-like morphology and do not form colonies when grown in agar. The combination of Ad5 E1A and low-level beta 1 PKC expression in the same CREF cell line results in an enhanced ability to grow when suspended in agar. In Ad5 E1A and Ad5 E1A + low-level beta 1 PKC expressing CREF clones, the tumor promoting agent 12-0-tetradecanoyl-phorbol-13-acetate (TPA) further enhances anchorage-independence. In contrast, TPA does not induce CREF cells or transfected CREF cells expressing low-levels of beta 1 PKC to grow in agar. Low-level beta 1 PKC expression in transfected CREF cells is associated with a modest 1.2 to 1.6-fold increase in binding of [3H]-phorbol-12,13-dibutyrate (PDBu) and only a 2.3-fold increase in PKC enzymatic activity. In contrast, specific beta 1 PKC-retroviral vector transformed CREF clones (CREF-RV-PKC) display higher levels of PKC mRNA, PDBu binding and PKC enzymatic activity. A majority of CREF-RV-PKC clones exhibit a transformed morphology and grow more rapidly in monolayer culture, form macroscopic colonies in agar in the absence of TPA and in many independent clones TPA further enhances anchorage-independent growth. This effect is not directly related to the level of enhanced [3H]-PDBu binding. The present study indicates that the effect of beta 1 PKC on cellular phenotype in immortal rat embryo cells is complex and is affected by its mode of insertion into CREF cells, i.e. transfection versus retroviral insertion. In addition, the combination of a transfected Ad5 E1A and a beta 1 PKC gene in the same CREF clone results in an enhanced expression of the transformed phenotype in both the absence and presence of TPA.
5型腺病毒（Ad5）的E1A基因可诱导克隆的大鼠胚胎成纤维细胞（CREF）发生形态转化并实现不依赖贴壁生长。相比之下，转染了β1蛋白激酶C（PKC）基因且低水平表达β1 PKC的CREF细胞呈现出类似CREF的形态，在琼脂中生长时不形成集落。在同一CREF细胞系中，Ad5 E1A与低水平β1 PKC表达相结合，会增强细胞在琼脂中悬浮生长的能力。在表达Ad5 E1A以及Ad5 E1A + 低水平β1 PKC的CREF克隆中，促肿瘤剂12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯（TPA）进一步增强了不依赖贴壁生长的能力。相比之下，TPA不会诱导CREF细胞或转染后低水平表达β1 PKC的CREF细胞在琼脂中生长。转染的CREF细胞中低水平β1 PKC表达与[³H] - 佛波醇 - 12,13 - 二丁酸酯（PDBu）结合适度增加1.2至1.6倍以及PKC酶活性仅增加2.3倍相关。相比之下，特定的β1 PKC - 逆转录病毒载体转化的CREF克隆（CREF - RV - PKC）表现出更高水平的PKC mRNA、PDBu结合和PKC酶活性。大多数CREF - RV - PKC克隆呈现出转化形态，在单层培养中生长更快，在没有TPA的情况下在琼脂中形成肉眼可见的集落，并且在许多独立克隆中TPA进一步增强了不依赖贴壁生长的能力。这种效应与增强的[³H] - PDBu结合水平没有直接关系。本研究表明，β1 PKC对永生大鼠胚胎细胞中细胞表型的影响是复杂的，并且受其插入CREF细胞的方式影响，即转染与逆转录病毒插入。此外，在同一CREF克隆中转染的Ad5 E1A和β1 PKC基因相结合，在有无TPA的情况下均会增强转化表型的表达。
