Desensitization of the Ca2+-mobilizing system to serum growth factors by Ha-ras and v-mos.

An elevation of the intracellular pH and a rise in the cytoplasmic Ca2+ concentration are considered important mitogenic signals which are observed after stimulation by various growth factors. In a preceding report it was demonstrated that the expression of Ha-ras or v-mos in cells transfected with Ha-ras or v-mos, respectively, leads to an activation of the Na+/H+ antiporter and a concomitant rise in intracellular pH (W. Doppler, R. Jaggi, and B. Groner, Gene 54:145-151, 1987). This report describes the effect of the Ha-ras and v-mos oncogenes on intracellular Ca2+ release. The expression of Ha-ras in NIH 3T3 cells carrying a glucocorticoid-inducible transforming Ha-ras gene caused a desensitization of the Ca2+-mobilizing system to serum growth factors. The induction of p21ras in cells carrying the corresponding glucocorticoid-inducible proto-oncogene did not affect the Ca2+ response to growth factors. Conditions leading to the expression of the transforming Ha-ras gene but not those causing the induction of the normal Ha-ras gene yielded an increase in phosphatidylinositol turnover and a concomitant rise in inositol phosphates. Results similar to those obtained with the transforming Ha-ras gene were seen after the expression of v-mos. The data are consistent with a mechanism in which expression of the transforming Ha-ras gene leads to a release of Ca2+ from intracellular stores via elevated levels of inositol trisphosphate.

Hexadecylphosphocholine inhibits inositol phosphate formation and protein kinase C activity.

Hexadecylphosphocholine (HePC) inhibits protein kinase C (PKC) from NIH3T3 cells in cell-free extracts with a 50% inhibitory concentration of about 7 microM. Inhibition is competitive with regard to phosphatidylserine with a Ki of 0.59 microM. In order to determine whether HePC affects PKC in intact cells, the bombesin or tetradecanoylphorbolacetate-induced, PKC-mediated activation of the Na+/H(+)-antiporter was determined. It is demonstrated that HePC causes a drastic inhibition of this enzyme indicating a similar sensitivity of PKC to HePC in intact cells compared to cell-free extracts. In addition to the effects on PKC, treatment of NIH3T3 cells with HePC depresses the bombesin-induced formation of inositol 1,4,5-trisphosphate and the concomitant mobilization of intracellular Ca2+. Dose-response curves for the inhibition of inositol 1,4,5-trisphosphate formation and Ca2+ mobilization reveal 50% inhibitory concentrations of 2 or 5 microM, respectively. Polyphosphorylated phosphoinositides accumulate in HePC-treated cells indicating that the depression of inositol 1,4,5-trisphosphate generation is not caused by an inhibition of phosphoinositide kinases. Addition of bombesin to HePC-treated cells in the presence of LiCl revealed no evidence for an accelerated rate of inositol 1,4,5-trisphosphate turnover by the phospholipid analogue. It is concluded that HePC inhibits phosphoinositidase C in intact cells. The data strongly suggest that the growth-inhibitory effect of HePC is at least in part explained by the interference with mitogenic signal transduction.

Interference of Ha-ras with inositol trisphosphate-mediated Ca(2+)-release.

Expression of a transforming Ha-ras by dexamethasone in NIH3T3 cells transfected with a glucocorticoid-inducible Ha-ras construct results in a rapid desensitization of the intracellular Ca(2+)-mobilizing system to bombesin. This effect precedes the down-modulation of inositol trisphosphate (IP3) formation by several hours and is, therefore, not explained by an uncoupling of phosphoinositidase C. It is demonstrated that expression of Ha-ras attenuates the Ca(2+)-release by IP3 in permeabilized cells. The IP3 concentration required for half-maximal Ca(2+)-release is doubled in Ha-ras expressing cells. Maximal Ca(2+)-release which is obtained with 2 microM IP3 in control cells requires 10 microM IP3 in cells expressing Ha-ras. The desensitization of the IP3 receptors coincides with the desensitization of the Ca(2+)-mobilizing system to bombesin. The results indicate that the Ha-ras mediated desensitization of the Ca(2+)-releasing system to bombesin is--at least in part--caused by a decrease in the affinity of the IP3 receptor to inositol trisphosphate.

Loss of heterozygosity on chromosome 16q, expression of her2/neu and p53 mutations in endometrial cancer.

We analyzed endometrial adenocarcinomas for mutations of the p53 anti-oncogene, expression of the HER2/neu oncogene and for loss of heterozygosity on chromosome 16q. p53 mutations were found in 3 of 25 tumors. Elevated expression of HER2/neu found in 7 of 24 tumors involves a mechanism other than gene amplification. LOH on chromosome 16q22-24 was observed in 4 of 13 informative tumors. Four of 25 endometrial tumors exhibited two or more alterations. Tumors with the highest HER2/neu protein level exhibited a negative progesterone receptor status. Accumulation of these changes may determine the biological behaviour of a subset of endometrial tumors.

Nitrogen mustard interference with potassium transport systems in Ehrlich ascites tumor cells.

Nitrogen mustard (N-mustard) inhibits the ouabain-sensitive and the furosemide-sensitive Rb uptake of Ehrlich ascites tumor cells, whereas the transport, which is resistant to both inhibitors, is not affected by the alkylating agent. At N-mustard concentrations below 10 microM, the reduction in Rb uptake is predominantly due to an interference with the furosemide-sensitive system. The dose response curve for the inhibition by N-mustard of the furosemide-sensitive Rb uptake closely parallels the dose response curve for the anti-tumor activity of the alkylating drug. This is in contrast to the behaviour of the ouabain-sensitive Rb transport. The inhibition of the furosemide-sensitive Rb uptake is expressed much less in cells which are resistant to N-mustard. The recovery of the furosemide-sensitive transport system after a single exposure to N-mustard is relatively slow and characterized by an initial 4 h lag period, whereas the repair of DNA-interstrand cross-links starts immediately after removal of the drug. At mM concentrations furosemide blocks the multiplication of Ehrlich ascites tumor cells. However, lower concentrations of furosemide which cause a 50% reduction in the furosemide-sensitive Rb uptake do not interfere with cell proliferation. This is in contrast to the behaviour of N-mustard which exerts a clear-cut depression of cell growth at concentrations leading to a 50% inhibition of the furosemide-sensitive Rb transport. It is concluded, therefore, that the inhibition of the furosemide-sensitive system alone is not sufficient to explain the anti-tumor activity of the alkylating agent. The effect is discussed as part of a more extended N-mustard-induced membrane alteration which may be important for the growth inhibitory effect of the alkylating agent.

The phospholipid- and calcium-dependent protein kinase as a target in tumor chemotherapy.

Evidence for a constitutive activation of protein kinase C (EC 2.7.1.37) in Ha-ras transformed 3T3 cells is presented. Several compounds which inhibit protein kinase C in vitro have been studied with regard to their antiproliferative activity in cultured tumor cells. The following agents were investigated: 3-hexadecyl-mercapto-2-methoxy-methyl-propyl-1- phosphocholine (BM 41440); 1-octadecyl-2-methyl-sn-glycero-3-phosphocholine (ET-18-OCH3); quercetin, tamoxifen and staurosporine. All compounds decrease protein kinase C activity in vitro as well as in intact cells and inhibit cell multiplication within the same dose range. The results suggest a causal relation between the antiproliferative effects and the inhibition of protein kinase C. All inhibitors of protein kinase C synergistically enhance the antiproliferative activity of cis-diamminedichloroplatinum(II). Available data suggest that the effects of protein kinase C inhibitors should be exploitable for tumor chemotherapy.

Effect of Ha-ras on phosphatidylinositol metabolism, Na+/H+-antiporter and mobilization of intracellular calcium.

NIH3T3 cells were transfected with activated Ha-ras and the corresponding proto-oncogene was subjected to transcriptional control by recombination in vitro with MMTV-LTR. Induction of p21ras expression in quiescent cells by dexamethasone causes an increased turnover of phosphatidylinositol 4,5-bisphosphate with a concomitant rise in inositol phosphates, and an activation of the Na+/H+-antiporter. Addition of serum growth factors to dexamethasone treated cells does not result in an additional stimulation of phosphatidylinositol metabolism or Na+/H+-exchange. There is also a desensitization to exogenous growth factors of the intracellular Ca2+-mobilizing system, leading to a depression of the transitory increase in cytosolic Ca2+ after addition of serum growth factors. None of these effects are seen after expression of the Ha-ras proto-oncogene. Results are discussed as indicating a constitutive growth factor independent activation of growth factor signal transduction by the activated Ha-ras.

Mechanism and biological significance of the Ha-ras-induced activation of the Na+/H(+)-antiporter.

Expression of the transforming Ha-ras oncogene in MMTV-LTR transfected NIH 3T3 cells leads to a growth factor independent activation of the Na+/H(+)-antiporter. The activation of the antiporter is insensitive to the protein kinase inhibitor staurosporine and equally expressed in protein kinase C-depleted cells. It is concluded that the Ha-ras induced activation of the antiporter occurs by a protein kinase C-independent mechanism. An inhibition of the Na+/H(+)-antiporter by dimethylamiloride or a reduction of the extracellular [Na+] concentration results in a depression of the bombesin induced release of Ca2+ from intracellular stores. These results are explained by a steep pH-dependence of the Ca2(+)-mobilizing system which exhibits a maximum at pH 7.1 in the system studied here. Stimulation by growth factors of quiescent cells with a resting pH below 7 results in a shift of the cytosolic pH towards the optimum for the Ca2+ release. In agreement with the proposed interrelationship, pHi and [Ca2+]i rise and peak simultaneously after addition of bombesin to G0 arrested cells.

Plasma membrane as target of alkylating agents.

N mustard resistant Walker cells exhibit the same frequency of DNA interstrand cross-links and the same rate of cross-link removal as the sensitive parental line. Employing cytostatically active concentrations of chlorambucil covalently bound to polyethyleneimine, the extent of DNA cross-linking is reduced to levels observed in the presence of nontoxic concentrations of free chlorambucil. It is concluded, therefore, that DNA cross-links alone are not sufficient to explain the inhibition of cell multiplication by alkylating agents and that additional mechanisms have to be considered. Evidence for an interference of alkylating agents with several enzymes of the plasma membrane is presented. An inhibition by N mustard of the furosemide-sensitive Na+/K+/Cl- -cotransport and the Na+/H+-antiport is described in greater detail. Considering the fact that the enzymes which are affected by alkylating agents are controlled by growth factors it was investigated whether a synergism between inhibitors of early growth-factor-controlled reactions and alkylating agents is to be seen. It is demonstrated that mepacrine, an inhibitor of phospholipase C, and the calmodulin binding drugs, chlorpromazine and flunarizine, amplify the action of N mustard.

Effect of Ha-ras on mitogen-induced Ca(2+)- and K(+)-fluxes.

Transforming Ha-ras enhances the mitogen-induced activation of both the Ca(2+)-influx and the furosemide-sensitive Na+/K+/2Cl-cotransporter. Both systems represent essential early steps of mitogenic signal transduction in NIH 3T3 fibroblasts.

P53 mutations and loss of heterozygosity on chromosomes 8p, 16q, 17p, and 18q are confined to advanced prostate cancer.

We analysed 39 prostatic carcinomas for loss of heterozygosity on chromosomal arms 8p, 10q, 16q, 17p and 18q and for mutations in the p53 anti-oncogene. Loss of heterozygosity (LOH) on 8p was detected in one out of 5 informative tumors, LOH on 16q in 3 out of 21 tumors, LOH on 17p in 2 out of 18 tumors, and LOH on 18q in 2 out of 17 tumors. No deletions were observed on 10q in 14 informative tumors. p53 alterations occurred in 3 out of 38 examined tumors, comprising two point mutations and a small deletion. Chromosomal deletions and p53 mutations were confined to locally invasive prostatic carcinomas, suggesting that they are associated with the progression of some prostate cancers rather than with tumor initiation.

Partial restoration of pre-transformation levels of lysyl oxidase and transin mRNAs in phenotypic ras revertants.

Neoplastic transformation mediated by ras oncogenes is associated with deregulated expression of genes encoding, for example, various proteases, lysyl oxidase, and smooth-muscle alpha-actin. To define the role of these genes in the initiation or maintenance of the ras-transformed state, we compared their steady-state mRNA levels in two different sets of preneoplastic fibroblast lines, ras-transformed clones, and phenotypic revertants derived from them. Compared with the preneoplastic fibroblasts, the ras-transformed derivatives exhibited elevated levels of cathepsin L (major excreted protein), transin (stromelysin I, matrix metalloproteinase-3), and collagenase I (matrix metalloproteinase-1) mRNA but undetectable levels of lysyl oxidase mRNA. Partial restoration of lysyl oxidase transcription was observed in four of five phenotypic revertants derived from rat FE-8 and NIHpEJcl3 cells. The elevated levels of transin mRNA found in NIHpEJcl3 cells were diminished to the pretransformation level in interferon revertants but were not reduced in phenotypic rat FE-8 revertants expressing a high level of the ras oncoprotein. High steady-state levels of collagenase I mRNA were dependent on ras expression but were not closely associated with the transformed phenotype. High levels of cathepsin L mRNA were associated with neither high ras expression nor neoplastic transformation. The downregulation of smooth-muscle alpha-actin, characteristic of transformed cell lines, was not reversible in phenotypic revertants.

Functional interaction of wild-type and mutant p53 transfected into human tumor cell lines carrying activated ras genes.

We have analyzed the antiproliferative activity of the p53 tumor suppressor gene in human tumor cell lines harboring activated ras genes. The levels of p53 protein and incorporation of bromodeoxyuridine in transiently transfected cells were determined simultaneously by flow cytometry. The human HT1080 fibrosarcoma, EJ bladder carcinoma, and SW480 colon carcinoma cell lines were equally sensitive toward wild-type p53-mediated inhibition of DNA synthesis, independent of the state of the endogenous p53 protein. Overexpression of p53 genes mutated at amino acid codon 143 resulted in increased proliferation of SW480 cells, which have two mutated endogenous p53 alleles. To mimic the genetic constitution of an evolving tumor cell that has sustained a mutation in one p53 allele, we coexpressed both wild-type and mutant p53 genes controlled by strong viral promoters in HT1080 cells. Transiently transfected cells showed a reduced bromodeoxyuridine uptake similar to cells into which only wild-type p53 had been introduced. The wild-type p53 gene is a dominant growth suppressor over the mutant in all three different cell lines analyzed. By immunoprecipitation with antibodies PAb 122, PAb 420, and PAb 1620, we demonstrate the presence of both the mutant and wild-type conformations of the p53 protein in the transfected cells.

Mechanism of desensitization of the Ca2(+)-mobilizing system to bombesin by Ha-ras. Independence from down-modulation of agonist-stimulated inositol phosphate production.

Expression of a transforming Ha-ras gene in NIH 3T3 cells transfected with an inducible Ha-ras construct leads to a rapid desensitization of the intracellular Ca2(+)-mobilizing system to bombesin and serum growth factors. Half-maximal depression of the Ca2+ response is observed 2 h after induction of p21ras. A maximum is obtained after 6 h. Bombesin-induced elevation of inositol 1,4,5-trisphosphate formation is also depressed in cells expressing Ha-ras. This, however, is a relatively late phenomenon and not yet detectable when maximal depression of the Ca2+ signal is observed. We conclude that the rapid densensitization of the Ca2(+)-releasing system to bombesin by Ha-ras is not caused by down-modulation or uncoupling of phospholipase C-coupled bombesin receptors. The inositol 1,4,5-trisphosphate-mediated release of intracellular Ca2+ is reduced in permeabilized cells expressing the Ha-ras oncogene. A depletion of intracellular Ca2+ stores by Ha-ras is unlikely since (i) the Ha-ras-induced growth factor-independent stimulation of inositol phosphate formation occurs several hours after reduction of the Ca2+ response and (ii) the Ca2+ load of intracellular nonmitochondrial Ca2+ stores was found to be unaffected by Ha-ras. We conclude that the desensitization of the Ca2(+)-mobilizing system is caused either by partial inhibition of inositol 1,4,5-trisphosphate-regulated Ca2+ channels or by interference of Ha-ras with Ca2+ translocation between intracellular Ca2+ compartments.

Ha-ras activates the Na+/H+ antiporter by a protein kinase C-independent mechanism.

In quiescent Ha-ras-transfected NIH 3T3 cells, addition of serum growth factors, bombesin or 12-O-tetradecanoylphorbol-13-acetate (TPA) leads to a dimethylamiloride-sensitive intracellular alkalinization which can be inhibited by staurosporine, a potent inhibitor of protein kinase C. Expression of the transforming Ha-ras gene causes a growth factor-independent increase in cytoplasmic pH. This Ha-ras-induced alkalinization is sensitive to dimethylamiloride but is not affected by staurosporine concentrations which prevent the pH response after addition of growth factors or TPA. Protein kinase C depletion by long term exposure to TPA eliminates the pH response to bombesin and phorbol ester but does not effect the Ha-ras-induced intracellular alkalinization. It is concluded that expression of Ha-ras causes an activation of the Na+/H+ antiporter by an as yet unknown protein kinase C-independent mechanism.