Novel revertants of H-ras oncogene-transformed R6-PKC3 cells.

Abstract

Rat 6 fibroblasts that overproduce protein kinase C 11 (R6-PKC3 cells) are hypersensitive to complete transformation by the T24 H-ras oncogene; yet T24 H-ras-transformed R6-PKC3 cells are killed when exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA) Mol. Cell. Biol. 9:2641-2647, 1989. Treatment of an R6-PKC3 subclone that harbors a T24 H-ras gene under the control of an inducible mouse metallothionein I promoter with ZnSO4 and TPA is extremely cytocidal. This procedure was used to isolate rare revertants that are resistant to this toxicity. Two revertant lines, R-la and ER-1-2, continue to express very high levels of protein kinase C enzyme activity but, unlike the parental cells, do not grow in soft agar. Furthermore, these revertants are resistant to the induction of anchorage-independent growth by the v-src, v-H-ras, v-raf, and, in the case of the R-la line, v-fos oncogenes. Both revertant lines, however, retain the ability to undergo morphological alterations when either treated with TPA or infected with a v-H-ras virus, thus dissociating anchorage independence from morphological transformation. The revertant phenotype of both R-la and ER-1-2 cells is dominant over the transformed phenotype in somatic cell hybridizations. Interestingly, the revertant lines no longer induce the metallothionein I-T24 H-ras construct or the endogenous metallothionein I and II genes in response to three distinct agents: ZnSO4, TPA, and dexamethasone. The reduction in activity of metallothionein promoters seen in these revertants may reflect defects in signal transduction pathways that control the expression of genes mediating specific effects of protein kinase C and certain oncogenes in cell transformation.(Protein kinase C (PKC) plays a central role in signal transduction, mediating, at least in part, the effects of several oncogene products, growth factors, and cytokines and serving as the cellular receptor for the phorbol ester class of tumor promoters (5,53,54). Activation of PKC has pleiotropic effects, and it has been difficult to determine which effects are most intimately linked to growth control and tumor promotion. Furthermore, the events occurring between activation of PKC and induction of PKC-regulated genes are largely unknown. Dissection of the mechanisms by which PKC transduces signals and influences biological phenomena is complicated by the fact that PKC is encoded by a multigene family comprising at least eight members (4,5,53,54,57). One approach to this problem is the overproduction of single isoforms of PKC in various cell lines of interest (6,7,15,29,41,58,69

Ras Signals to the Cell Cycle Machinery via Multiple Pathways To Induce Anchorage-Independent Growth

“…For example, introduction of Ras oncoprotein into quiescent Swiss 3T3 cells led to both morphological transformation and DNA synthesis, but only the induction of DNA synthesis required activation of protein kinase C (27). Furthermore, a Rat 6 fibroblast-derived mutant cell line, ER-1-2, responded to stable expression of the v-H-ras oncogene with alterations in morphology and gene expression that were nearly indistinguishable from those observed with a matched control cell line yet failed to form colonies in soft agar in response to ras (11,22). Similarly, expression of a dominant negative form of Rac1 in Ras-transformed Rat 1 cells inhibited anchorage-independent growth of these cells but had only marginal effects on their transformed morphology (34).…”

“…Rat 6-and NIH 3T3-derived cell lines were cultured in Dulbecco modified Eagle medium (Gibco) plus 10% bovine calf serum as previously described (16,22). NIH 3T3 cells were obtained from T. Hei (Columbia University).…”

The desmoplastic small round cell tumor t(11;22) translocation produces EWS/WT1 isoforms with differing oncogenic properties

“…Transfectants were selected in medium containing 500 mg ml 71 G418 (neomycin). The virus was collected and used to infect NIH3T3 cells in 100 mm dishes, as described (Krauss et al, 1992). After dilution of the polyclonal cell lines, monoclonal cell lines were isolated by picking individual G418-resistant colonies.…”

The Neuropeptide Processing Enzyme EC 3.4.24.15 Is Modulated by Protein Kinase A Phosphorylation

“…For PKC-PKC was purified from Rat 6 fibroblast cell lines overexpressing PKC ⑀ (calcium independent) or PKC ␤ (calcium dependent) (gift of Dr. Robert Krauss, Mount Sinai School of Medicine) and assays were performed as described elsewhere (24,25), using 3 g of EP24.15; each condition was assayed in triplicate. EGF receptor peptide (RKRTLRRL) served as positive control.…”