Trotta R J, Thomson T M, Lacal J C, Pellicer A, Burstein D E
Department of Pathology, New York University School of Medicine, New York 10016.
J Cell Physiol. 1990 Apr;143(1):68-78. doi: 10.1002/jcp.1041430109.
A recombinant N-ras oncogene, under the transcriptional control of a corticosteroid-inducible mouse mammary tumor virus (MMTV) promoter, has been stably transfected into a PC12 rat pheochromocytoma subline. This cell line, designated UR61, undergoes N-ras-induced neurite outgrowth and cessation of division when treated with dexamethasone (Guerrero et al.: Biochemical and Biophysical Research Communications 150:1185-1192, 1988). We have employed the UR61 cell line as a model for ras oncogene-induced neuronal differentiation. In UR61 cells, dexamethasone-induced expression of the recombinant N-ras gene resulted in time-dependent expression of ornithine decarboxylase enzyme (ODC) activity. Prompted by recent reports of possible functional (Lacal et al.: Molecular and Cellular Biology 7:4146-4149, 1987; Wolfman and Macara: Nature 325: 359-361, 1987) and direct (Jeng et al.: Biochemical and Biophysical Research Communications 145:782-788, 1987) interactions between oncogene ras-coded p21 and protein kinase C (PK-C; Ca++/phospholipid-dependent protein kinase), we employed the protein kinase inhibitor H-8 (N-[2-(methylamino)ethyl]-5-isoquinoline sulfonamide dihydrochloride) and phorbol 12,13-dibutyrate (PDBu) to investigate this putative interaction in the UR61 cells, where ODC activity and neurite outgrowth were used as indicators of oncogenic N-ras action. Treatment of UR61 cells with PDBu depleted cells of PK-C and failed to promote neurite outgrowth but enhanced N-ras-induced neurite outgrowth and ODC activity. H-8, which suppressed ODC induction by forskolin and phorbol myristate acetate, enhanced both N-ras-induced ODC activity and neurite outgrowth. Inhibition of ODC activity by difluoromethylornithine (DFMO) did not suppress oncogenic ras-induced neurite outgrowth, suggesting that these two ras-triggered events are mechanistically independent. These findings suggest that certain actions of N-ras can occur in cells depleted of PK-C, and thus, the role of PK-C in ras-induced differentiation differs from its role in ras-induced mitogenesis and transformation.
一种重组的N-ras癌基因，在皮质类固醇诱导型小鼠乳腺肿瘤病毒（MMTV）启动子的转录控制下，已被稳定转染到PC12大鼠嗜铬细胞瘤亚系中。该细胞系命名为UR61，用 dexamethasone处理时会经历N-ras诱导的神经突生长和分裂停止（Guerrero等人：《生物化学与生物物理研究通讯》150:1185 - 1192，1988）。我们将UR61细胞系用作ras癌基因诱导神经元分化的模型。在UR61细胞中，dexamethasone诱导的重组N-ras基因表达导致鸟氨酸脱羧酶（ODC）活性呈时间依赖性表达。受近期关于癌基因ras编码的p21与蛋白激酶C（PK-C；钙/磷脂依赖性蛋白激酶）之间可能存在功能（Lacal等人：《分子与细胞生物学》7:4146 - 4149，1987；Wolfman和Macara：《自然》325: 359 - 361，1987）和直接（Jeng等人：《生物化学与生物物理研究通讯》145:782 - 788，1987）相互作用的报道的启发，我们使用蛋白激酶抑制剂H-8（N-[2-(甲氨基)乙基]-5-异喹啉磺酰胺二盐酸盐）和佛波醇12,13-二丁酸酯（PDBu）来研究UR61细胞中的这种假定相互作用，其中ODC活性和神经突生长用作致癌性N-ras作用的指标。用PDBu处理UR61细胞会耗尽细胞中的PK-C，且未能促进神经突生长，但增强了N-ras诱导的神经突生长和ODC活性。H-8抑制了forskolin和佛波醇肉豆蔻酸酯乙酸盐诱导的ODC，但增强了N-ras诱导的ODC活性和神经突生长。二氟甲基鸟氨酸（DFMO）对ODC活性的抑制并未抑制致癌性ras诱导的神经突生长，这表明这两个ras触发的事件在机制上是独立的。这些发现表明，N-ras的某些作用可以在PK-C耗尽的细胞中发生，因此，PK-C在ras诱导的分化中的作用与其在ras诱导的有丝分裂和转化中的作用不同。
