ras p21 protein promotes survival and fiber outgrowth of cultured embryonic neurons

Although evidence obtained with the PC12 cell line has suggested a role for the ras oncogene proteins in the signal transduction of nerve growth factor-mediated fiber outgrowth, little is known about the signal transduction mechanisms involved in the neuronal response to neurotrophic factors in nontransformed cells. We report here that the oncogene protein T24-ras, when introduced into the cytoplasm of freshly dissociated chick embryonic neurons, promotes the in vitro survival and neurite outgrowth of nerve growth factor-responsive dorsal root ganglion neurons, brain-derived neurotrophic factor-responsive nodose ganglion neurons, and ciliary neuronotrophic factor-responsive ciliary ganglion neurons. The proto-oncogene product c-Ha-ras also promotes neuronal survival, albeit less strongly. No effect could be observed with truncated counterparts of T24-ras and c-Ha-ras lacking the 23 C-terminal amino acids including the membrane-anchoring, palmityl-accepting cysteine. These results suggest a generalized involvement of ras or ras-like proteins in the intracellular signal transduction pathway for neurotrophic factors.     

The effects of hydrocortisone on c-fos, c-myc and c-ras oncogene expression in IMR-90 fibroblasts

The effects of hydrocortisone on oncogene expression in human IMR-90 fibroblasts was analyzed by Northern blotting of total RNA. In synchronized fibroblasts stimulated with serum alone, there were two time periods of increased c-fos expression during the G1 phase of the cell cycle. There was no significant difference between cells treated with serum plus hydrocortisone, and cells treated with serum alone with respect to c-fos expression. Quiescent cells showed no change in c-fos expression during the G1 phase of the cell cycle. Three peaks of c-fos expression occur when cells are treated with hydrocortisone alone, but hydrocortisone in the absence of serum is insufficient to initiate DNA synthesis. Hydrocortisone has no effect on c-myc or c-Ha-ras expression in the presence or absence of serum in synchronized fibroblasts. Therefore, the control of mRNA production of the nuclear oncogenes c-fos and c-myc, and the cytoplasmic oncogene c-ras are independent and hydrocortisone may enhance DNA synthesis by increasing c-fos expression.     

Differential effects of dioctanoylglycerol on fibronectin localization in normal, partially transformed, and malignant human endometrial stromal cells

In this study, we describe the effects of direct activation of PKC by dioctanoylglycerol (DiC8) on cellular morphology and the localization of fibronectin (Fn) in normal, oncogene-transfected, and malignant human endometrial stromal cells. We questioned whether DiC8, an endogenous specific activator of PKC, would function as a second oncogene in partially transformed human endometrial stromal cells (HESC). Cells utilized were (1) normal HESC, (2) HESC transfected with a plasmid containing an origin-defective temperature-sensitive SV40 large T antigen alone or (3) in combination with an EJ ras oncogene, and (4) an endometrial sarcoma cell line (S7). Cell cultures were treated for 1 h with sn-dioctanoylglycerol (DiC8) and stained with a monoclonal fluorescein-labeled anti-Fn antibody. In normal HESC, DiC8 induced cell rounding and caused Fn localization to revert from the perinuclear region to the cell periphery. All experiments in this investigation were performed when cells were maintained at the permissive temperature for SV40 large T antigen function. In HESC expressing the SV40 large T antigen alone, Fn was localized to the perinuclear region and also occurred as parallel strands between cells. When these cells were treated with DiC8, Fn localization changed to intense punctate regions at the cell periphery or to matrix-like patterns between cells. Also, in these cells, DiC8 induced greater detachment of cells from the substrate than from other cells, resulting in an apparent piling up of cells. Control and treated SV40/EJ ras cells and uterine sarcoma cells expressed Fn in a matrix-like pattern between cells. The rounded cellular morphology of treated HESC and treated cells expressing SV40 resembled the morphology of control or treated SV40/EJ ras cells and uterine sarcoma cells. Thus, treated cells expressing the SV40 large T antigen resembled the SV40/EJ ras cells and uterine sarcoma cells with respect to Fn localization and cellular morphology. DiC8 did not appear to further transform HESC expressing SV40 and EJ ras. However, with regard to cell shape and Fn localization, our results suggest that DiC8 may function as a second oncogene in the signal transduction pathway, in cells expressing SV40 alone. It appears that, with regard to Fn localization, DiC8 may alter signal transduction analogously to that caused by the activated Ha-ras oncogene in HESC expressing the SV40 large T antigen.     

3'-End conjugates of minimally phosphorothioate-protected oligonucleotides with 1-O-hexadecylglycerol: synthesis and anti-ras activity in radiation-resistant cells

Activation of the ras oncogene has been implicated in many types of human tumors. It has been shown that downmodulation of ras expression can lead to the reversion of the transformed phenotype of these tumor cells. Antisense oligodeoxyribonucleotides (ODNs) can inhibit gene expression by hybridization to complementary mRNA sequences. To minimize toxicity associated with all-phosphorothioated ODNs and improve cellular uptake, we used partially phosphorothioate (PPS)-modified ODNs having an additional hydrophobic tail at the 3'-end (PPS-C(16)). The PPS ODNs are protected against degradation by PS internucleotide linkages at both the 3'- and 5'-ends and additionally stabilized at internal pyrimidine sites, which are the major sites of endonuclease cleavage. Here we show that anti-ras PPS-C(16) ODN retains the high sequence-specificity of PPS ODNs and provides maximal inhibition of Ras p21 synthesis with minimal toxicity even without the use of a cellular uptake enhancer. Moreover, treatment of T24, a radiation-resistant human tumor cell line that carries a mutant ras gene, with anti-ras PPS-C(16) ODN resulted in a reduction in the radiation resistance of the cells in vitro. We also demonstrate that the growth of RS504 (a human c-Ha-ras transformed NIH/3T3 cell line) mouse tumors was significantly inhibited by the combination of intratumoral injection of anti-ras PPS-C(16) ODN and radiation treatment. These findings indicate the potential of this combination of antisense and conventional radiation therapy as a highly effective cancer treatment modality.     

Structure of a Moloney murine leukemia virus-virus-like 30 recombinant: implications for transduction of the c-Ha-ras proto-oncogene.

Tumor progression locus 2 (Tpl-2) encodes a novel serine-threonine protein kinase which is activated by provirus integration in the late stages of oncogenesis in Moloney leukemia virus (MoMuLV) induced rat T-cell lymphomas. In this report, we present evidence that the provirus integrated in the Tpl-2 locus in 1 of 10 T-cell lymphomas harboring a Tpl-2 rearrangement (2779) is a recombinant between MoMuLV and virus-like 30 (VL30) sequences (Mo-VL30). Recombination between MoMuLV and VL30 may contribute to the transduction of ras, as suggested by the finding that VL30 flanks the ras oncogene in all of the ras transducing viruses isolated from rats to date. The Mo-VL30 recombinant described here represents evidence that recombination between MoMuLV and VL30 can be uncoupled from the transduction of ras, and it may precede the transduction. Sequence comparison between clones of Mo-VL30, Harvey sarcoma virus (Ha-MSV), and genomic c-Ha-ras revealed that all three share a 124-bp region of 87.3% homology. This region was detected at nucleotide positions -1845 to -1720 of c-Ha-ras and 20 bp 5' of the recombination breakpoint between VL30 and ras in Ha-MSV. On the basis of the sequence comparison between VL30, Ha-MSV, and c-Ha-ras, we are proposing a model which explains how VL30 may have facilitated the transduction of c-Ha-ras and perhaps the other ras proto-oncogenes. According to this model, the sequence homology between VL30 and c-Ha-ras targets this gene for transduction by promoting the integration of the provirus in this locus through homologous recombination.     

Dose effects of transfected c-Ha-rasVal 12 oncogene in transformed cell clones

We have examined the expression of the transformed phenotype in a series of clonal lines of NIH/3T3 cells transfected with the human c-Ha-rasVal 12 oncogene and the neomycin phosphotransferase gene. Cells from individual transformed foci were cloned and subjected to detailed analyses of the ras sequences. Three clones were found that expressed approximately one, 2-4, or 4-8 copies of the human c-ras oncogene, respectively. A fourth clone had multiple copies of the transfected sequences, and expressed abundant c-Ha-ras RNA. Analysis of the transformed phenotype of various clones indicated that cells expressing low levels of mutant c-Ha-ras had lost some of their extracellular fibronectin network, and were barely altered in their cytoskeleton. In contrast, cells expressing abundant c-Ha-ras had lost both their actin and fibronectin networks and showed an increase in plasminogen activator activity. Cells with amplified c-Ha-rasVal 12 grew better in low serum, formed large colonies in soft agar and showed enhanced activity of ornithine decarboxylase, the rate-controlling enzyme in polyamine biosynthesis. These results show that the dosage level of the mutant oncogene makes a significant contribution to the transformed phenotype of c-Ha-ras oncogene-transformed cells.     

In Vitro transformation of LW13 Rat liver epithelial Cells

A rat liver epithelial cell line designated LW 13 was established using a sequential sedimentation method.The cell line retained many normal proerties of liver epithelial cells and showed some structural and functional features resembling those of liver parenchymal cells,LW13 cells became malignant after the intrduction of exogenous transforming EJ Ha ras gene,Tumors produced by inoculation of the transformed cells into baby rats contained areas of poorly differentialted hepatocellular carcinoma,In situ hybridization analysis confirmed the random rather than specific integration of exogenous ras gene into host chromosomes.Furthermore,an at least tenfold increase in the expression of the endogenous c mys gene was detected among transformed cell lines,suggesting the involvement of the c myc proto oncogene in the in vitro transformation of rat liver epithelial cells by EJ Ha ras oncogene.     

Inhibition of MAPK activity, cell proliferation, and anchorage-independent growth by N-Ras antisense in an N-ras-transformed human cell line

Mammalian ras genes encode a family of plasma membrane-bound proteins that function as intermediates in signal transduction pathways involved in cell growth and differentiation. Ras oncogene is frequently involved in neoplastic transformation of different cellular histotypes. In this study, we tested the ability of antisense oligodeoxyribonucleotides (AS-ODN) that have mixed phosphodiester/phosphorothioate backbone, targeted against human N-Ras, to inhibit N-ras gene expression and to specifically interfere with the Ras-dependent activity of mitogen-activated protein kinase (MAPK) in two human cell lines carrying an endogenous N-ras mutated allele at codon 61. Three AS-ODN that inhibit basal MAPK activity have been identified. Moreover, AS-ODN treatment resulted in potent antiproliferative effects in cell culture and great inhibition of N-ras mRNA levels in one of two cell lines. These studies suggest that antisense molecules, targeted against N-Ras, could be of considerable value as a tool to study the N-Ras-specific transduction pathway.     

Inhibition ofras oncogene: A novel approach to antineoplastic therapy

The most frequently detected oncogene alterations, both in animal and human cancers, are the mutations in the ras oncogene family. These oncogenes are mutated or overexpressed in many human tumors, with a high incidence in tumors of the pancreas, thyroid, colon, lung and certain types of leukemia. Ras is a small guanine nucleotide binding protein that transduces biological information from the cell surface to cytoplasmic components within cells. The signal is transduced to the cell nucleus through second messengers, and it ultimately induces cell division. Oncogenic forms of p21(ras) lead to unregulated, sustained signaling through downstream effectors. The ras family of oncogenes is involved in the development of both primary tumors and metastases making it a good therapeutic target. Several therapeutic approaches to cancer have been developed pointing to reducing the altered gene product or to eliminating its biological function: (1) gene therapy with ribozymes, which are able to break down specific RNA sequences, or with antisense oligonucleotides, (2) immunotherapy through passive or active immunization protocols, and (3) inhibition of p21(ras) farnesylation either by inhibition of farnesyl transferase or synthesis inhibition of farnesyl moieties.     

Regulation of tetradecanoyl phorbol acetate-induced responses in NIH 3T3 cells by GAP, the GTPase-activating protein associated with p21c-ras.

Proteins of the ras family of oncogenes have been implicated in signal transduction pathways initiated by protein kinase C (PKC) and by tyrosine kinase oncogenes and receptors, but the role that ras plays in these diverse signalling systems is poorly defined. The activity of ras proteins has been shown to be controlled in part by a cellular protein, GAP (GTPase-activating protein), that negatively regulates p21c-ras by enhancing its intrinsic GTPase activity. Thus, overexpression of GAP provides a tool for determining the step(s) in signal transduction dependent on p21c-ras activity. In this paper, we report that overexpression of GAP blocks the phorbol ester (tetradecanoyl phorbol acetate [TPA])-induced activation of p42 mitogen-activated protein kinase (p42mapk), c-fos expression, and DNA synthesis. GAP overexpression did not block responses to serum or fluoroaluminate. Moreover, not all biochemical events elicited by TPA were affected by GAP overexpression, as increased glucose uptake and phosphorylation of MARCKS, a major PKC substrate, occurred normally. Reduction of GAP expression to near normal levels restored the ability of the cells to activate p42mapk in response to TPA. These findings suggest that ras and GAP together play a key role in a PKC-dependent signal transduction pathway which leads to p42mapk activation and cell proliferation.     

Ras (proto)oncogene induces N-linked carbohydrate modification: temporal relationship with induction of invasive potential

The effect of expression of the ras oncogene on protein glycosylation was studied. VSV G-protein and class I histocompatibility antigens were analysed to monitor ras-mediated changes in glycosylation. Transient expression of the c-Ha-ras oncogene, introduced into NIH 3T3 cells by the DEAE-dextran method, altered protein glycosylation within 25 h of transfection. The same result was obtained after dexamethasone-induced expression of p21-ras in stable NIH 3T3 transfectants containing either an activated Ha-ras oncogene or a normal N-ras proto-oncogene under control of the glucocorticoid-inducible MMTV promoter. The alteration of cell surface carbohydrates, induced by the ras (proto)oncogene and the subsequent acquisition of invasive potential, occurred prior to morphological transformation.     

Suppression of albumin enhancer activity by H-ras and AP-1 in hepatocyte cell lines.

We demonstrated, using a transient transfection assay, that the albumin enhancer increased the expression of the albumin promoter in a highly differentiated, simian virus 40 (SV40)-immortalized hepatocyte cell line, CWSV1, but was not functional in two ras-transformed cell lines (NR3 and NR4) derived from CWSV1 by stable transfection with the T24ras oncogene. A transient cotransfection assay showed that T24ras and normal c-Ha-ras were each able to inhibit the activity of the albumin enhancer in an immortal hepatocyte cell line. DNase I footprinting and gel mobility shift assays demonstrated that the DNA binding activities specific to the albumin enhancer were not decreased in the ras-transformed cells. ras also did not diminish the expression of HNF1 alpha, C/EBP alpha, HNF3 alpha, HNF3 beta, or HNF3 gamma but did significantly increase AP-1 binding activity. Three AP-1 binding sites were identified within the albumin enhancer, and DNA binding activities specific to these AP-1 sites were induced in the ras-transformed hepatocytes. Subsequent functional assays showed that overexpression of c-jun and c-fos inhibited the activity of the albumin enhancer. Site-directed mutagenesis of the AP-1 binding sites in the albumin enhancer partially abrogated the suppressing effect of ras and c-jun/c-fos on the enhancer. These functional studies therefore supported the results of the structural studies with AP-1. We conclude that the activity of the albumin enhancer is subject to regulation by ras signaling pathways and that the effect of ras on the albumin enhancer activity may be mediated by AP-1.     

Lineage-specific transformation after differentiation of multipotential murine stem cells containing a human oncogene.

We transfected the human EJ bladder carcinoma oncogene (Ha-rasEJ-1) into multipotential embryonal carcinoma cell line P19. The transgenic P19(ras+) cells expressed high levels of both the mRNA and the p21EJ protein derived from the oncogene. When cultured in the presence of retinoic acid, P19(ras+) cells differentiated and developed into the same spectrum of differentiated cell types as the parental P19 cells (namely, neurons, astrocytes, and fibroblast-like cells). Thus, it seems unlikely that the Ha-ras-1 proto-oncogene product plays a role in initiation of differentiation or in the choice of differentiated cell lineage. Most of the P19(ras+)-derived differentiated cells contained relatively low levels of p21EJ and were nontransformed, whereas certain cells with fibroblast-like morphology continued to express the Ha-rasEJ-1 gene at high levels and were transformed (i.e., immortal and anchorage independent). Fibroblasts derived from P19 cells did not become transformed following transfection of the Ha-rasEJ-1 oncogene, suggesting that transformation of the fibroblast cells only occurred if the oncogene was present and expressed during the early stages of the developmental lineage.     

Short modified antisense oligonucleotides directed against Ha-ras point mutation induce selective cleavage of the mRNA and inhibit T24 cells proliferation.

We have used derivatized antisense oligodeoxynucleotides both in vitro and in vivo specifically to inhibit translation of the activated human oncogene Ha-ras. The oligonucleotides (5'-CCACACCGA-3') were targeted to a region of Ha-ras mRNA including the point mutation G----T at the 12th codon which leads to a Gly----Val substitution in the ras p21 protein. They were linked to an intercalating agent and/or to a hydrophobic tail, both to increase their affinity for their mRNA target and to enhance their uptake by tumor cells. A cell-free translation system was used to demonstrate an RNase H-dependent specific inhibition of activated ras protein synthesis. 50% inhibition was observed at a concentration of 0.5 microM of the most efficient oligonucleotide (5'-substitution with an acridine derivative and 3'-substitution by a dodecanol chain). This inhibitory effect stems from a point mutation-sensitive cleavage of the mRNA and it mirrors the growth inhibition obtained with T24 bladder carcinoma cells, which carry activated Ha-ras. The proliferation of HBL100 cells (non tumorigenic human mammary cell line) which carry two copies of normal Ha-ras was unaffected. This study shows that it is possible to design antisense agents that will inactivate the mutated oncogene but not the protooncogene which is generally essential to cell survival.