A v-H-ras-dependent hemopoietic tumor model involving progression from a clonal stage of transformation competence to autocrine interleukin 3 production.

Autocrine interleukin 3 (IL-3)-secreting tumors were generated from an IL-3-dependent mouse mast cell line (PB-3c) after introduction of the v-H-ras oncogene. Tumor progression was characterized by four distinct phenotypes. The first corresponded to immortalized mast cells unresponsive to the oncogenic effect of v-H-ras. The second was expressed in a clonable subpopulation of PB-3c cells and was marked by the competence to form v-H-ras-dependent tumors (immortalized transformation competence). The third was a direct effect of v-H-ras expression on all PB-3c cells and was characterized in vitro by a reduced IL-3 requirement. Upon injection of v-H-ras-expressing, transformation-competent cells into mice, the final, fully malignant phenotype developed with a long latency period and was marked in vitro by independence of exogenous IL-3 and by autocrine IL-3 stimulation. Northern (RNA) blot analysis and an RNase A-T1 protection assay showed that IL-3 production was strictly associated with the tumor phenotype. Two of six tumors showed an alteration at the 5' region of the IL-3 gene. We conclude that v-H-ras required complementation by IL-3 gene rearrangement or an alternate event to generate autocrine mastocytomas.     

Two dominant inhibitory mutants of p21ras interfere with insulin-induced gene expression.

Insulin induces a rapid activation of p21ras in NIH 3T3 and Chinese hamster ovary cells that overexpress the insulin receptor. Previously, we suggested that p21ras may mediate insulin-induced gene expression. To test such a function of p21ras more directly, we studied the effect of different dominant inhibitory mutants of p21ras on the induction of gene expression in response to insulin. We transfected a collagenase promoter-chloramphenicol acetyltransferase (CAT) gene or a fos promoter-luciferase gene into NIH 3T3 cells that overexpressed the insulin receptor. The activities of both promoters were strongly induced after treatment with insulin. This induction could be suppressed by cotransfection of two inhibitory mutant ras genes, H-ras(Asn-17) or H-ras(Leu-61,Ser-186). In particular, insulin-induced activation of the fos promoter was inhibited completely by H-ras(Asn-17). These results show that p21ras functions as an intermediate in the insulin signal transduction route leading to the induction of gene expression.     

Glucocorticoid dexamethasone reversibly complements EJ-RAS oncogene to transform mouse embryo BALB-3T3 cells

EJ-A is a Balb-3T3 transfectant cell line that bears a small number of EJ-ras oncogene copies/cell, has low EJ-ras expression, and resembles the parental cell line in displaying a non-transformed phenotype. The glucocorticoid hormone dexamethasone reversibly induces transformation traits in EJ-A cells, namely: 1) morphological transformation; 2) increased growth rate and saturation density; 3) reduced G1 length; and 4) independence of the FGF requirement to initiate DNA synthesis. Western blot analysis revealed that dexamethasone does not increase the p21ras protein intracellular level. beta-IFN, added to the culture medium, does not suppress the dexamethasone-induced growth stimulation and morphological transformation. Therefore, glucocorticoid hormones can complement low EJ-ras expression to transform Balb-3T3 cells, by a mechanism that is likely to be independent of p21ras increase and beta-IFN decrease.     

High-level expression of c-H-ras1 fails to fully transform rat-1 cells.

Rat-1 cells were transfected with plasmids encoding normal (Gly-12), nonactivated (Pro-12), and activated (Val-12 and Ile-12) p21H-ras in the presence of an amplifiable dihydrofolate reductase marker. The introduced DNA was amplified by selection in methotrexate to establish the relationship between p21H-ras expression and various hallmarks of cellular transformation. The maximum level of p21H-ras (Gly-12) consistent with cell viability was approximately 0.13% of total cell protein (approximately 60,000 molecules per cell); this is 44-fold greater than the level of the endogenous protein. The maximum tolerated level of a second nontransforming form of p21H-ras (pro-12) was about half of this. Amplification in Rat-1 cells of H-ras genes encoding the highly oncogenic Val-12 and Ile-12 forms of p21H-ras could not be achieved by methotrexate selection, providing strong evidence that synthesis of activated p21H-ras above a certain threshold (about 0.02% of total protein) in Rat-1 cells is incompatible with cell viability. Individual cell lines were isolated and their morphology, anchorage-independent growth, tumorigenicity, and response to and production of growth factors were studied. We report that cell lines expressing near-maximum tolerated levels of either the normal or pro-12 form of p21H-ras were not as transformed as cells expressing much more modest levels of the highly oncogenic (Val-12) form, suggesting that the complete elaboration of the transformed phenotype by ras depends, at least in part, on mutations that distinguish the cellular and viral proteins. We found that cells expressing elevated levels of the normal p21(H-ras) could be fully transformed by the activated (Val-12) form and that such cells continued to overexpress p21(H-ras) (Gly-12), arguing against a role for normal ras genes in suppression of the oncogenic potential of their mutationally activated counterparts.     

Effect of a dominant inhibitory Ha-ras mutation on mitogenic signal transduction in NIH 3T3 cells.

We used a dominant inhibitory mutation of c-Ha-ras which changes Ser-17 to Asn-17 in the gene product p21 [p21(Asn-17)Ha-ras] to investigate ras function in mitogenic signal transduction. An NIH 3T3 cell line [NIH(M17)] was isolated that displayed inducible expression of the mutant Ha-ras gene (Ha-ras Asn-17) via the mouse mammary tumor virus long terminal repeat and was growth inhibited by dexamethasone. The effect of dexamethasone induction on response of quiescent NIH(M17) cells to mitogens was then analyzed. Stimulation of DNA synthesis by epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA) was completely blocked by p21(Asn-17) expression, and stimulation by serum, fibroblast growth factor, and platelet-derived growth factor was partially inhibited. However, the induction of fos, jun, and myc by EGF and TPA was not significantly inhibited in this cell line. An effect of p21(Asn-17) on fos induction was, however, demonstrated in transient expression assays in which quiescent NIH 3T3 cells were cotransfected with a fos-cat receptor plasmid plus a Ha-ras Asn-17 expression vector. In this assay, p21(Asn-17) inhibited chloramphenicol acetyltransferase expression induced by EGF and other growth factors. In contrast to its effect on DNA synthesis, however, Ha-ras Asn-17 expression did not inhibit fos-cat expression induced by TPA. Conversely, downregulation of protein kinase C did not inhibit fos-cat induction by activated ras or other oncogenes. These results suggest that ras proteins are involved in at least two parallel mitogenic signal transduction pathways, one of which is independent of protein kinase C. Although either pathway alone appears to be sufficient to induce fos, both appear to be necessary to induce the full mitogenic response.     

Alpha B-crystallin expression in mouse NIH 3T3 fibroblasts: glucocorticoid responsiveness and involvement in thermal protection.

alpha B-crystallin, a major soluble protein of vertebrate eye lenses, is a small heat shock protein which transiently accumulates in response to heat shock and other kinds of stress in mouse NIH 3T3 fibroblasts. Ectopic expression of an alpha B-crystallin cDNA clone renders NIH 3T3 cells thermoresistant. alpha B-crystallin accumulates in response to the synthetic glucocorticoid hormone dexamethasone. Dexamethasone-treated NIH 3T3 cells become thermoresistant to the same extent as they accumulate alpha B-crystallin. A cell clone in which alpha B-crystallin is superinduced upon heat shock acquires augmented thermotolerance. Expression of the ras oncogene causes a rapid but transient accumulation of alpha B-crystallin within 1 day. Later, sustained ras oncogene expression suppresses the dexamethasone-mediated alpha B-crystallin accumulation. Thus, oncogenic transformation triggered by the ras oncogene interferes with hormone-mediated accumulation of alpha B-crystallin and concomitant acquisition of thermoresistance. Other known heat shock proteins do not accumulate in response to ectopic alpha B-crystallin expression or to dexamethasone treatment. These results indicate that alpha B-crystallin can protect NIH 3T3 fibroblasts from thermal shock.     

Induction of transforming growth factor alpha expression in mouse mammary epithelial cells after transformation with a point-mutated c-Ha-ras protooncogene

NOG-8 ras cells are a normal mouse mammary epithelial cell line transfected with a plasmid containing a glucocorticoid-inducible mouse mammary tumor virus long terminal repeat linked to the activated c-Ha-ras protooncogene. After addition of dexamethasone, there is a rapid induction (within 1-3 h) of p21ras protein that is concomitant with a parallel induction of the c-Ha-ras specific mRNA. After 4-6 days of dexamethasone treatment, NOG-8 ras cells are able to grow as colonies in semisolid medium. Between 9 and 12 days of dexamethasone treatment, there is a 5- to 6-fold increase of transforming growth factor alpha (TGF alpha) activity in the conditioned medium from NOG-8 ras cells. A 60-65% reduction in epidermal growth factor cell surface receptors on NOG-8 ras cells also occurs during this time interval. A 3- to 4-fold increase of the expression of a specific TGF alpha mRNA can be detected within 2 days of dexamethasone treatment, preceding the increase in TGF alpha protein found in the conditioned medium. Exogenous TGF alpha is able to stimulate in a dose-dependent fashion the anchorage-dependent and anchorage-independent growth of NOG-8 ras cells to a level comparable to that observed in dexamethasone treated ras-transformed NOG-8 ras cells. These results suggest that the enhanced expression of TGF alpha after induction of an activated ras protooncogene may be necessary for the anchorage-independent growth and subsequent morphological changes and the enhanced growth rate observed in ras-transformed mammary epithelial cells.     

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.     

Increased tyrosine phosphorylation in ras transformed fibroblasts occurs prior to manifestation of the transformed phenotype

Murine fibroblasts transformed by ras oncogenes exhibited an increased amount of tyrosine phosphorylated proteins compared to normal cells. The pattern of phosphorylation was similar to that observed in cells chronically stimulated with EGF or PDGF, and is probably due to autocrine stimulation of receptor tyrosine kinases. NIH 3T3 cells transfected with H-ras under the control of a glucocorticoid inducible promoter were used to determine the temporal relationship among expression of p21H-ras oncoprotein, increase in tyrosine phosphorylation and appearance of the transformed morphology. Enhanced tyrosine phosphorylation was observed more than 24 hours before evidence of morphological changes. These results suggest that full transformation by ras oncogenes requires cooperation with tyrosine protein kinases.     

Altered ganglioside expression in ras-oncogene-transformed cells

Alteration of ganglioside composition in mouse BALB/3T3 cells transformed either by DNA transfection with viral K-, H-, or cellular H-ras oncogene, or by infection with the K-ras oncogene-carrying murine sarcoma virus (Ki-KSV) was studied using a highly sensitive thin-layer chromatography/enzyme immunostaining method. Marked common decreases in the content of GD3 ganglioside and the increase of its metabolic precursor GM3 were bound in BALB/3T3 cell lines transformed by either K- or H-ras oncogenes. Moreover, a common decrease or loss in the contents of "A" series ganglio-tetraose gangliosides such as GM1a and GD1a was also found in all transformed cell lines, indicating that the alteration of cellular glycosphingolipids by ras oncogenes apparently does not depend on the type of ras-concogenes (K- and H-ras).     

Ras p21 proteins with high or low GTPase activity can efficiently transform NIH/3T3 cells

We sought to determine whether decreased in vitro GTPase activity is uniformly associated with ras p21 mutants possessing efficient transforming properties. Normal H-ras p21-[Gly12-Ala59] as well as an H-ras p21-[Gly12-Thr59] mutant exhibited in vitro GTPase activities at least fivefold higher than either H-ras p21-[Lys12-Ala59] or H-ras p21-[Arg12-Thr59] mutants. Microinjection of as much as 6 X 10(6) molecules/cell of bacterially expressed normal H-ras p21 induced no detectable alterations of NIH/3T3 cells. In contrast, inoculation of 4-5 X 10(5) molecules/cell of each p21 mutant induced morphologic alterations and stimulated DNA synthesis. Moreover, the transforming activity of each mutant expressed in a eukaryotic vector was similar and at least 100-fold greater than that of the normal H-ras gene. These findings establish that activation of efficient transforming properties by ras p21 proteins can occur by mechanisms not involving reduced in vitro GTPase activity.     

Recombinant adenovirus encoding H-ras ribozyme induces apoptosis in laryngeal cancer cells through caspase- and mitochondria-dependent pathways

Previously, we designed a ribozyme that targets the H-ras oncogene at the 12th codon mutation site (Chang et al., 1997). Ribozymes have antisense molecule and site-specific ribonuclease potential. In this study, an adenoviral vector was used to transduce the H-ras ribozyme into laryngeal cancer cells (HEp-2). This served to downregulate the H-ras gene expression in which this ribozyme performed antisense activity due to HEp-2 cells containing wild-type alleles in the 12th H-ras codon. Together, our data demonstrated that the recombinant adenovirus encoding H-ras ribozyme can be broadly regarded as a cytotoxic gene therapy in laryngeal cancer cells regardless of containing wild-type or mutant ras gene. In addition, the mechanism through which the H-ras ribozyme inhibited tumor growth was apoptosis and involved both caspase- and mitochondria-mediated pathways. The activators caspase-8 and -9 as well as the effector caspase-3 in the induction phase of apoptosis and the substrate PARP of caspase-3 in the execution phase were activated 48h following the H-ras ribozyme treatment. Mitochondrial events characterized by the production of superoxide anion and the release of cytochrome c started at 24h. Mitochondrial transmembrane potential loss occurred 48h after the ribozyme treatment. However, Bcl-2 delayed cytochrome c release to the cytosol, but it could not protect the apoptosis effect, suggesting that cytochrome c release from mitochondria may not play a role in H-ras ribozyme-induced apoptosis.     

Ras activation by insulin and epidermal growth factor through enhanced exchange of guanine nucleotides on p21ras.

A number of growth factors, including insulin and epidermal growth factor (EGF), induce accumulation of the GTP-bound form of p21ras. This accumulation could be caused either by an increase in guanine nucleotide exchange on p21ras or by a decrease in the GTPase activity of p21ras. To investigate whether insulin and EGF affect nucleotide exchange on p21ras, we measured binding of [alpha-32P]GTP to p21ras in cells permeabilized with streptolysin O. For this purpose, we used a cell line which expressed elevated levels of p21 H-ras and which was highly responsive to insulin and EGF. Stimulation with insulin or EGF resulted in an increase in the rate of nucleotide binding to p21ras. To determine whether this increased binding rate is due to the activation of a guanine nucleotide exchange factor, we made use of the inhibitory properties of a dominant negative mutant of p21ras, p21ras (Asn-17). Activation of p21ras by insulin and EGF in intact cells was abolished in cells infected with a recombinant vaccinia virus expressing p21ras (Asn-17). In addition, the enhanced nucleotide binding to p21ras in response to insulin and EGF in permeabilized cells was blocked upon expression of p21ras (Asn-17). From these data, we conclude that the activation of a guanine nucleotide exchange factor is involved in insulin- and EGF-induced activation of p21ras.     

Bradykinin-induced oscillations of cell membrane potential in cells expressing the Ha-ras oncogene

Products of ras genes are putative elements of growth factor signal transduction. However, the mechanism of action of these proteins in normal and malignant growth is as yet obscure. To test for functional consequences of ras oncogene expression, electrophysiological experiments were performed on NIH-3T3 fibroblasts transfected with a transforming Ha-ras MMTV-LTR construct expressing the oncogene on treatment with dexamethasone (+ras). Transfected cells in the absence of dexamethasone (-ras) and nontransfected cells in the presence of dexamethasone (oras) served as controls. In -ras and oras, bradykinin induces a single, transient hyperpolarization. In +ras, bradykinin elicits oscillations of cell membrane potential throughout the presence of the hormone by activation of calcium-sensitive K+ channels. The oscillations of cell membrane potential are abolished in the absence of extracellular calcium. As evident from fura 2 fluorescence, bradykinin leads to a transient increase of intracellular calcium both in the presence and absence of extracellular calcium. Oscillations of intracellular calcium could be observed in +ras cells, if bradykinin was applied at reduced extracellular sodium concentration possibly to impair calcium extrusion via the sodium/calcium exchange. Bradykinin induces oscillations of cell membrane potential similarly in -ras cells loaded with GTP[S], a nonhydrolyzable analogue of GTP. Thus, the altered response of ras oncogene expressing cells to bradykinin relates to the GTP binding property of the ras protein. It is concluded that in cells expressing ras oncogene but not in other fibroblasts bradykinin mimicks the effect of growth factors on the cell membrane.     

Involvement of functional protein kinase C in the mitogenic response to the H-ras oncogene product.

Microinjection of purified protein kinase C (PKC) into Swiss 3T3 fibroblasts pretreated with the phorbol ester phorbol-12,13-dibutyrate restores the mitogenic response of the cells to phorbol-12,13-dibutyrate (G. Pasti, J.C. Lacal, B.S. Warren, S.A. Aaronson, and P.M. Blumberg, Nature [London] 324:375-377, 1986). Our present studies demonstrate that the mitogenic activity of the H-ras oncogene in H-ras p21-microinjected quiescent cells is markedly reduced under conditions in which PKC is downregulated by chronic phorbol ester treatment. The ability to reconstitute the mitogenic response upon microinjection of both H-ras p21 and PKC implies involvement of functional PKC in the mitogenic activity of the H-ras oncogene product.