Hermann Oberhuber

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.

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 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.

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

SummaryNitrogen 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 μM, 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.

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.

Enhancement of the Antiproliferative Effect of cis-Diamminedichloroplatinum (II) and Other Antitumor Agents by Inhibitors of Enzymes Involved in Growth Factor Signal-Transduction

Progress in our understanding of the biochemical basis of malignant growth has led to the identification of new targets in tumor chemotherapy. In many cases the uncontrolled, autonomous growth of transformed cells has been shown to be caused by one of the following mechanisms: (1) autrocrine production of growth factors; (2) expression of constitutively active growth factor receptors; (3) constitutive activation of elements of growth factor signal transduction1–3. In view of these findings, novel strategies for tumor chemotherapy have been developed including growth factor antagonists4 growth factor receptor blockers5 and inhibitors of enzymes or ion channels involved in intracellular mitogenic signal transfer. Approaches to interfere with growth factor signal transduction include studies on inhibitors of phospholipase C, protein tyrosine kinases, protein kinase C, Na+/H+-antiporter and Ca2+ antagonists6,7. A variety of compounds which were developed along these lines of research exhibit remarkable antitumor activity, some of them are less toxic than established, currently employed antitumor agents. With regard to these low toxicity compounds it seemed intriguing to investigate whether their growth inhibitory activity could be combined with the growth inhibition by established antitumor agents. In such a mixture, the combination of the growth inhibitory effects of the components may exhibit an additive or synergistic behaviour. This could be exploited for a less toxic and/or more effective treatment protocol.

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.

Interference of Ha-ras with inositol trisphosphate-mediated Ca2+-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 Ca2+‐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 Ca2+‐release by IP3 in permeabilized cells. The IP3 concentration required for half‐maximal Ca2+‐release is doubled in Ha‐ras expressing cells. Maximal Ca2+‐release which is obtained with 2,μM IP3 in control cells requires 10μM IP3 in cells expressing Ha‐ras. The desensitization of the IP3 receptors coincides with the desensitization of the Ca2+‐mobilizing system to bombesin. The results indicate that the Ha‐ras mediated desensitization of the Ca2+‐releasing system to bombesin is — at least in part — caused by a decrease in the affinity of the IP3 receptor to inositol trisphosphate.

Role of Na+/H+-Antiporter in Growth Stimulation by Ha-ras

Expression of the transforming Ha-ras oncogene in MMTV-LTR-Ha-ras transfected 3T3 fibroblasts causes a growth factor independent intracellular alkalinization by a dimethylamiloride sensitive mechanism. The phenomenon is accompanied by a desensitization of the intracellular calcium mobilizing system to serum growth factors and an increase in the generation of inositol phosphates. Data are presented which support the assumption that the mobilization of intracellular Ca2+ occurs by a Na+-dependent mechanism. The results are discussed as indicating a constitutive activation of growth factor signal transduction by transforming Ha-ras. The exact sequences of events initiated by the oncogene, however, remains to be elucidated.

Effect of Ha-ras on mitogen-induced Ca2+- 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.