Positive autoregulation of ras genes expression in fibroblasts.

We have studied the effect of ectopic overexpression of a ras gene on the expression of the other two members of the ras gene family. We obtained NIH3T3 cell lines stably transfected with inducible H-ras and N-ras oncogenes. The expression of these genes is driven by a glucocorticoid-responsive promoter and the addition of dexamethasone resulted in a dramatic induction (10-20-fold) of H- or N-ras mRNA, peaking 4 h after hormone addition. The induction of the expression of ras oncogenes resulted in a transformed phenotype. In quiescent NIH3T3 cells transfected with inducible H-ras oncogenes, the induction of H-Ras was followed 12 h later by a 3-fold increase in the mRNA expression of endogenous K-ras and N-ras. Similarly, in NIH3T3 transfected with inducible N-ras oncogene, the induction of N-ras was followed by an increase in the expression of endogenous K- and H-ras genes. Interestingly, the effect was not limited to the mutated N-ras, as a similar result was obtained in cells transfected with N-ras proto-oncogene. The induction of ras genes expression was not linked to cell cycle progression as it was reproduced in cells arrested in S-phase by pretreatment with hydroxyurea. These results suggest the presence of a positive cross-regulation in the expression among the members of the Ras family. This effect could play a role in Ras-mediated carcinogenesis.

Serum growth factors up-regulate H-ras, K-ras, and N-ras proto-oncogenes in fibroblasts.

We have analyzed the expression of the ras gene family during the transit through quiescence-proliferation. H-, K- and N-ras steady-state mRNA levels and total Ras protein levels did not change significantly when NIH3T3 cells were made quiescent by density arrest in the presence of 10% calf serum. By contrast, levels of ras mRNAs in cells that had been made quiescent by serum deprivation were lower than those in growing cells. An induction of H-, K- and N-ras mRNA levels (3- to 5-fold) was detected in these cells after serum addition. This induction was maximal around 8 h after serum addition for the three ras genes. Like the early-response genes, ras induction was not dependent on protein synthesis; but in contrast to these genes, ras mRNAs showed long half-lives (5-7 h) in NIH3T3 cells. Up-regulation of ras genes by serum was also observed in human primary fibroblasts, indicating that this may be a general effect in mammal cells. We obtained stable transfectants in NIH3T3 cells with the oncogenic N-ras. In these cells, expression of the transforming gene is also induced by serum, and the expression of the transfected N-ras gene did not modify the response to serum of endogenous H-, K-, and N-ras genes. The regions of murine N-ras gene responsible for serum inducibility seem to be intragenic because N-ras up-regulation occurred in cells transfected with a gene construct lacking the sequences upstream from the first exon.(ABSTRACT TRUNCATED AT 250 WORDS)

Down-regulation of c-myc gene is not obligatory for growth inhibition and differentiation of human myeloid leukemia cells.

Suppression of c-myc expression is observed during induced differentiation of several myeloid cell lines and it has been attributed to the cell growth arrest that accompanies terminal differentiation. To dissect the role of c-Myc in the proliferation-differentiation switch we have studied c-myc expression in K562 cells exposed to several chemical agents. This model system allowed us to discriminate between the growth arrest and differentiation phenomena as well as the induction of differentiation along two different lineages (erythroid and myelomonocytic). Our results showed that c-myc expression did not significantly decrease when growth inhibition is reversible, either by treatment with a differentiating agent such as hydroxyurea (which induced erythroid differentiation) or by a non-differentiating agent such as interferon-alpha. In contrast, c-myc expression decreased when cells underwent terminal differentiation, either along the myelomonocytic (by 12-O-tetradecanoylphorbol-13-acetate) or erythroid (by 1-beta-D-arabinofuranosylcytosine) lineages. These results indicated that c-myc down-regulation is not obligatory for growth arrest and non-terminal differentiation of human myeloid cells. In contrast, c-myc down-regulation occurred in terminal differentiation, but induction of myelomonocytic differentiation resulted in a greater loss of c-myc mRNA than induction of erythroid differentiation.

Differential expression of ras protooncogenes during in vitro differentiation of human erythroleukemia cells.

We have compared the expression of the ras protooncogene family (H-, K-, and N-ras) in leukemia cell differentiation utilizing as a model K562 and HEL erythroleukemia cells treated either with 1-beta-arabinofuranosylcytosine or 12-O-tetradecanoylphorbol-13-acetate (TPA). 1-beta-D-Arabinofuranosylcytosine induced terminal erythroid differentiation of K562 cells, while TPA induced myeloid differentiation of K562 and HEL cells, resulting in myelomonocytic-like cells expressing macrophagic and megakaryocytic markers. H-ras mRNA levels showed a dramatic decrease in K562 cells subjected to erythroid and myelomonocytic differentiation. The same result was found at the protein level for p21H-ras. Expression of K-ras and N-ras in K562 cells also decreased with differentiation, although significant mRNA levels remained despite cessation of cell proliferation. The decrease in K-ras expression was greater for TPA-treated cells than for 1-beta-arabinofuranosylcytosine-treated cells. TPA-induced myelomonocytic differentiation in HEL cells also resulted in a dramatic down-regulation of H-ras mRNA levels. Thus, by using a leukemia cell line able to differentiate along two different lineages, our results reveal a lineage-specific modulation of ras gene family expression.