Beta-casein gene promoter activity is regulated by the hormone-mediated relief of transcriptional repression and a mammary-gland-specific nuclear factor.

Transcription from the beta-casein milk protein gene promoter is induced by the synergistic action of glucocorticoid and prolactin hormones in the murine mammary epithelial cell line, HC11. We analyzed the binding of nuclear proteins to the promoter and determined their binding sites. Site-directed mutagenesis was used to determine the function of nuclear factor binding. During lactogenic hormone induction of HC11 cells, the binding of two nuclear factors increased. The binding of two other nuclear factors, present in uninduced cells, decreased. The basal activity of the promoter could be increased to and above the level of the induced wild-type promoter when the recognition sequences of the negatively regulated factors were mutated. This suggests that the beta-casein promoter is regulated by the relief of the repression of transcription. An essential tissue-specific factor was also found in nuclear extracts from the mammary glands of mice. Mutation of its recognition sequence in the beta-casein promoter led to the abolition of the induction of transcription by lactogenic hormones. The DNA sequences recognized by all five of these nuclear factors are conserved in the promoters of different casein genes from several species, confirming their importance in the regulation of milk protein gene transcription.

A mammary cell-specific enhancer in mouse mammary tumor virus DNA is composed of multiple regulatory elements including binding sites for CTF/NFI and a novel transcription factor, mammary cell-activating factor.

Mouse mammary tumor virus (MMTV) is a milk-transmitted retrovirus involved in the neoplastic transformation of mouse mammary gland cells. The expression of this virus is regulated by mammary cell type-specific factors, steroid hormones, and polypeptide growth factors. Sequences for mammary cell-specific expression are located in an enhancer element in the extreme 5' end of the long terminal repeat region of this virus. This enhancer, when cloned in front of the herpes simplex thymidine kinase promoter, endows the promoter with mammary cell-specific response. Using functional and DNA-protein-binding studies with constructs mutated in the MMTV long terminal repeat enhancer, we have identified two main regulatory elements necessary for the mammary cell-specific response. These elements consist of binding sites for a transcription factor in the family of CTF/NFI proteins and the transcription factor mammary cell-activating factor (MAF) that recognizes the sequence G Pu Pu G C/G A A G G/T. Combinations of CTF/NFI- and MAF-binding sites or multiple copies of either one of these binding sites but not solitary binding sites mediate mammary cell-specific expression. The functional activities of these two regulatory elements are enhanced by another factor that binds to the core sequence ACAAAG. Interdigitated binding sites for CTF/NFI, MAF, and/or the ACAAAG factor are also found in the 5' upstream regions of genes encoding whey milk proteins from different species. These findings suggest that mammary cell-specific regulation is achieved by a concerted action of factors binding to multiple regulatory sites.

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.

Expression level-dependent contribution of glucocorticoid receptor domains for functional interaction with STAT5.

The action of the glucocorticoid receptor (GR) on beta-casein gene transcription serves as a well-studied example of a case where the action of the GR is dependent on the activity of another transcription factor, STAT5. We have investigated the domain-requirement of the GR for this synergistic response in transfection experiments employing GR mutants and CV-1 or COS-7 cells. The results were influenced by the expression levels of the GR constructs. At low expression, STAT5-dependent transactivation by mutants of the GR DNA binding domain or N-terminal transactivation domain was impaired and the antiglucocorticoid RU486 exhibited a weak agonistic activity. When the N-terminal region of the GR was exchanged with the respective domain of the progesterone receptor, STAT5-dependent transactivation was reduced at low and high expression levels. Only at high expression levels did the GR exhibit the properties of a coactivator and enhanced STAT5 activity in the absence of a functional DNA binding domain and of GR binding sites in the proximal region of the beta-casein gene promoter. Furthermore, at high GR expression levels RU486 was nearly as efficient as dexamethasone in activating transcription via the STAT5 dependent beta-casein gene promoter. The results reconcile the controversial issue regarding the DNA binding-independent action of the GR together with STAT5 and provide evidence that the mode of action of the GR depends not only on the type of the particular promoter at which it acts but also on the concentration of the GR. GR DNA binding function appears to be mandatory for beta-casein gene expression in mammary epithelial cells, since the promoter function is completely dependent on the integrity of GR binding sites in the promoter.

STAT-1 expression in human glioblastoma and peritumoral tissue.

BACKGROUND: Glioblastoma is a very aggressive brain tumour with poor prognosis despite radical surgery or radiotherapy. Signal transducers and activators of transcription (STAT) proteins are important elements in intracellular signalling and part of the JAK-STAT pathway. They are activated by growth factors and cytokines and translocate into the nucleus upon activation to exert their function as transcription factors. STAT-1 can be induced by interferons and has also been found to be important in sensitizing tumours to chemotherapeutic drugs. MATERIALS AND METHODS: Forty-six glioblastoma samples have been analysed for the expression of STAT-1 by immunohistochemistry. RESULTS: In our study performed by immunohistochemistry, 22 out of 46 glioblastomas (48%) were strongly positive for staining with a STAT-1 antibody, 9 (20%) showed an intermediate reactivity, 8 (17%) low immunoreactivity, and 7 (15%) were completely negative. In the tumour tissue, STAT-1 expression was mostly localized in the cytoplasm. This location of STAT-1 suggests the predominant presence of an inactive form of STAT-1. Tumour giant cells were frequently strongly stained. Part of the peritumoral brain tissue showed strongly positively reactive glial cells. Interestingly, within the infiltration area strong STAT-1 expression was found in reactive astrocytes, glia, and particularly in microglial components. CONCLUSION: The expression of STAT-1 in the majority of glioblastomas, together with its documented role in apoptosis and in the action of chemotherapeutic drugs on tumour cell lines point to a possible function of this protein in the response of glioblastomas to chemotherapy.

Protection of Ehrlich ascites tumor cells against the antiproliferative effect of mechlorethamine (nitrogen mustard) by 5-N,N-dimethylamiloride.

5-N,N-Dimethylamiloride protects Ehrlich ascites tumor cells against the antiproliferative effect of nitrogen mustard. The drug reduces the frequency of DNA interstrand cross-links introduced by nitrogen mustard. Cells with a defective choline carrier are not protected against nitrogen mustard by dimethylamiloride. As nitrogen mustard is taken up by the choline carrier, it is concluded that the recently reported inhibition of the choline transport system by amiloride and its dimethyl derivatives (W. Doppler et al., Biochem. Pharmacol., 36: 1645-1649, 1987) is responsible for the protection against the alkylating agent.

Inhibition of cell proliferation, protein kinase C, and phorbol ester-induced fos expression by the dihydropyridine derivative B859-35.

The dihydropyridine derivative B859-35 inhibits phospholipid- and calcium-dependent protein kinase C (PKC) in cell-free extracts from NIH3T3 cells. Inhibition is competitive with regard to phosphatidylserine. At 1 microM phosphatidylserine, half-maximal inhibition (IC50) is obtained at approximately 2.5 microM B859-35. 12-O-Tetradecanoylphorbol-13-acetate (TPA)-dependent activation of the Na+/H+ antiporter was used to determine whether the enzyme is also affected in intact cells. The activity of the antiporter was monitored by following the dimethylamiloride-sensitive cytosolic alkalinization. It is demonstrated that B859-35 depresses the TPA-induced alkalinization with an IC50 of 5 microM, indicating that PKC in intact cells and the enzyme in cell-free extracts are equally sensitive to the drug. TPA-induced expression of the c-fos gene was used as an additional marker for intracellular PKC activity. Activation of c-fos expression was determined by measuring chloramphenicol acetyltransferase (CAT) activity in cells transfected with a c-fosCAT construct in which the CAT gene is expressed under the control of the endogenous human c-fos promoter. The studies revealed that 2.5 microM B859-35, a concentration equivalent to the IC50 in cell-free extracts, significantly depresses TPA-induced c-fosCAT expression. B859-35 inhibited cellular proliferation of NIH3T3 cells with an IC50 of approximately 5 microM. This is close to the IC50 for the anti-PKC activity of B859-35. It is suggested that the inhibition of PKC contributes to the growth inhibition following exposure to B859-35.

Gamma-interferon reduces expression of the protooncogene c-erbB-2 in human ovarian carcinoma cells.

The overexpression of the protooncogene c-erbB-2 (HER-2/neu) in ovarian and mammary carcinoma is an important indicator for a bad prognosis. In this study we demonstrate that, in three of four ovarian carcinoma cell lines, there is a gamma-interferon-mediated reduction in c-erbB-2 specific protein, and this effect was found to correlate with the antiproliferative action. It is interesting to note that there is no relation between the absolute amount of c-erbB-2 protein expressed and the sensitivity of the ovarian carcinoma cells for an antiproliferative activity of gamma-interferon. Other chemotherapeutic agents did not affect c-erbB-2 expression, although they inhibited the proliferation. The oncogene expression was lowered only in the ovarian carcinoma cell lines and not in three gamma-interferon-sensitive human breast cancer cell lines. Expression of the oncogene c-erbB-2 is the leading prognostic factor in ovarian cancer. Its modulation might represent a mechanism by which gamma-interferon inhibits cell proliferation.

The BH3-only protein BIM contributes to late-stage involution in the mouse mammary gland.

After cessation of lactation, involution of the mouse mammary gland proceeds in two distinct phases, a reversible and an irreversible one, which leads to the death and removal of alveolar cells. Cell death is preceded by the loss of STAT5 activity, which abrogates cell differentiation and gain of STAT3 activity. Despite early observations implicating BCL2 (B cell lymphoma 2) family proteins in this process, recent evidence suggests that STAT3-controlled cathepsin activity is most critical for cell death at the early stage of involution. Somewhat surprisingly, this cell death associates with but does not depend on the activation of pro-apoptotic effector caspases. However, transgenic overexpression of BCL2, that blocks caspase activation, delays involution while conditional deletion of BclX accelerates this process, suggesting that BCL2 family proteins are needed for the effective execution of involution. Here, we report on the transcriptional induction of multiple pro-apoptotic BCL2 family proteins of the 'BH3-only' subgroup during involution and the rate-limiting role of BIM in this process. Loss of Bim delayed epithelial cell clearance during involution after forced weaning in mice, whereas the absence of related Bmf had minor and loss of Bad or Noxa no impact on this process. Consistent with a contribution of BCL2 family proteins to the second wave of cell death during involution, loss of Bim reduced the number of apoptotic cells in this irreversible phase. Notably, the expression changes observed within the BCL2 family did not depend on STAT3 signalling, in line with its initiating role early in the process, but rather appear to result from relief of repression by STAT5. Our findings support the existence of a signalling circuitry regulating the irreversible phase of involution in mice by engaging BH3-only protein-driven mitochondrial apoptosis.

Activation of c-fos expression by transforming Ha-ras in HC11 mouse mammary epithelial cells is PKC-dependent and mediated by the serum response element.

The mechanism by which transforming Ha-ras induces c-fos expression in HC11 mouse mammary epithelial cells was investigated with regard to controversial data concerning the role of protein kinase C (PKC) and the required promoter elements of the fos gene. HC11 cells carrying a glucocorticoid-inducible Ha-ras (val12) construct were transfected with a chloramphenicol acetyltransferase (CAT) reporter gene under the control of a human fos promoter which includes the serum response element (SRE), the adjacent c-fos AP-1 site (FAP) and the cAMP response element (CRE). Induction of the Ha-ras gene by dexamethasone lead to a transactivation of expression of the transfected fos promoter construct which was inhibited by the PKC inhibitor BM41440 and abrogated in PKC-'depleted' cells. A similar transactivation was observed when the fos promoter construct was co-transfected with a constitutively active ras expression vector. Again, this effect was depressed by the PKC inhibitor and abolished in PKC-'depleted' cells. 'PKC-depletion' was achieved by long-term exposure to 12-O-tetradecanoylphorbol-13-acetate. This procedure was shown to deplete cells of PKC alpha and to reduce significantly PKC epsilon. Long-term exposure to bryostatin 1 selectively depletes PKC alpha. Depletion of PKC alpha by bryostatin 1 does not reduce the transcriptional activation of the SRE-FAP-TK-CAT (TK: thymidine kinase) construct by Ha-ras. In order to delineate the promoter elements mediating the transcriptional activation, constructs which lack the FAP and the CRE sites but contain an intact SRE were co-transfected with the ras construct. Elimination of the FAP and CRE sequences did not affect the transcriptional activation by Ha-ras (val12). It is concluded that in HC11 cells, transforming Ha-ras activates c-fos expression in a PKC-dependent manner, presumably implying PKC epsilon, and that the SRE is sufficient to mediate transcriptional activation.

Prolactin-dependent activation of a tyrosine phosphorylated DNA binding factor in mouse mammary epithelial cells.

The mammary gland factor MGF has been described as a developmentally and environmentally regulated nuclear factor required for transcription of the milk protein gene beta-casein. In the current study the individual role of lactogenic hormones in the activation of MGF DNA binding and the functional relation of MGF to known transcription factors was investigated by electrophoretic mobility shift assays. DNA binding of MGF was rapidly induced by PRL in mammary epithelial cells. The activation was not inhibited by the protein synthesis inhibitor cycloheximide. The effect of PRL on MGF did not require costimulation of cells with the other lactogenic hormones, insulin, and glucocorticoids. Thus, MGF is the first example of a nuclear factor directly regulated by PRL. The MGF complexes formed upon initiation of lactation in the mammary gland and upon stimulation of mammary epithelial cells with PRL migrated at the same position in electrophoretic mobility shift assay, whereas the MGF complex found in mammary gland extracts of pregnant mice exhibited a faster mobility. In cell cultures, PRL-induced activation of MGF as well as up-regulation of beta-casein gene transcription was confined to confluent cultures of mammary epithelial cells and inhibited by long term incubation of cells with epidermal growth factor. MGF was found to be related to the nuclear factors that are activated by tyrosine phosphorylation when cells are stimulated with interferons or cytokines. This notion is supported by experimental evidence for phosphorylation of MGF on tyrosine and by the similar DNA recognition motifs of MGF and cytokine-activated factors.

Prolactin and glucocorticoid hormones control transcription of the beta-casein gene by kinetically distinct mechanisms.

Transcription of the beta-casein milk protein gene in the HC11 mouse mammary epithelial cell line is induced synergistically by the hormones glucocorticoid and PRL. Sequential treatment of HC11 cells with glucocorticoid and PRL demonstrated that the two hormones had different modes of action on beta-casein transcription. Pretreatment with dexamethasone enhanced the response to subsequent induction by PRL, but not vice versa. Dexamethasone increased the sensitivity of the cells to respond to PRL. The increase in sensitivity was slow, extended for 16 days, and could be rapidly reversed by withdrawal of dexamethasone. The dexamethasone-induced sensitivity for the rapid transcriptional regulation by PRL could be observed with transfected rat beta-casein promoter-chloramphenicol acetyltransferase constructs retaining only 175 basepairs upstream from the transcription initiation site. Expression of the endogenous mouse beta-casein gene was regulated identically to that of the promoter constructs with respect to the synergy of the hormones and their different kinetics of action. In contrast to the slow induction of sensitivity toward PRL, dexamethasone rapidly induced the transcription of a mouse mammary tumor virus long terminal repeat controlled gene in HC11. This demonstrated a normal transcriptional activation of the glucocorticoid receptor in this cell line. Thus, glucocorticoid may regulate beta-casein gene transcription indirectly, inducing or repressing other glucocorticoid-regulated genes, whereas the interaction of PRL with its receptor causes a rapid induction of the beta-casein gene promoter.

Lactogenic hormone and cell type-specific control of the whey acidic protein gene promoter in transfected mouse cells.

The whey acidic protein (WAP) is a major milk protein. It is abundantly expressed in mammary epithelial cells, and its gene is controlled by lactogenic hormones. The identification of regulatory cis-acting sequences of the mouse WAP gene was so far dependent on the analysis of transgenic animals. We report here the possibility of analyzing regulatory sequences by gene transfer experiments using the lactogenic hormone-dependent mammary epithelial cell line HC11. A WAP-chloramphenicol acetyltransferase construct containing 2.5 kilobases of the 5'-flanking sequence of the WAP gene was stably transfected into HC11 cells. High chloramphenicol acetyltransferase activity was induced in pools of transfected cells cultured in the presence of the lactogenic hormones glucocorticoid, PRL, and insulin. A lower induction was observed by glucocorticoid hormone alone. PRL by itself was not able to induce the WAP gene promoter above the level observed in the absence of lactogenic hormones. A time course of hormone induction showed a weak initial response with a steady increase over at least 4 days of hormone treatment. Induction was not observable in the mammary epithelial cell line NOG-8 and NIH3T3 fibroblasts, despite the presence of functional glucocorticoid receptor in these cells. This indicates the requirement for a cell type-specific transcription factor present in the mammary epithelial cell line HC11, but not in NOG-8 epithelial cells or NIH3T3 fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of v-mos and activated Ha-ras oncogene expression in quiescent NIH 3T3 cells causes intracellular alkalinisation and cell-cycle progression.

We have tested the effect of the expression of the v-mos oncogene, and the activated human Ha-ras oncogene and its proto-oncogene form on the intracellular pH and on cell cycle progression. The Ha-ras proto-oncogene and the oncogenes under the transcriptional control of the MMTV-LTR promoter were introduced into NIH 3T3 cells. The cells were synchronized at G0/G1 in low-serum medium and the transfected genes were induced by glucocorticoid hormone addition to the growth medium. Expression of the v-mos and the activated form of Ha-ras oncogenes mimics the effect of growth factors and activates the Na+/H+ antiporter. The oncogenes increase the internal pH and provoke initiation of S-phase. The proto-oncogene form of Ha-ras does not induce intracellular alkalinisation and has only a weak mitogenic effect. Our results suggest the oncogenic proteins p21ras and p37mos influence the mitogenic signal transduction pathways at a point prior to the activation of the Na+/H+ antiporter.

A distal enhancer region in the human beta-casein gene mediates the response to prolactin and glucocorticoid hormones.

The 5' flanking region of the human beta-casein gene was investigated for the presence of regulatory sequences mediating the action of the lactogenic hormones prolactin and dexamethasone. DNA encompassing 9389 base pairs of the flanking region was isolated and a sequence comparison performed with regulatory regions previously identified in the beta-casein gene of rodents and ruminants. The analysis revealed the presence of a distal region between -4700 and -4550 with a high percentage of identity to the bovine beta-casein enhancer region, and a proximal region between -1 and -200 similar to the proximal promoter regions found in rodents and ruminants. Reporter gene constructs under the control of the distal or the proximal region of the human beta-casein gene were tested for their responsiveness to prolactin and dexamethasone. In transfection experiments, the distal region functioned as a lactogenic hormone inducible enhancer, whereas the proximal region exhibited low activity. In electromobility shift assays, multiple binding sites for Stat5, CCAAT/enhancer-binding proteins, and Ets domain proteins were identified in the distal human enhancer. These transcription factors have already been demonstrated as important regulators of the transcription of milk protein genes in rodents. Thus, a common set of transcription factors appears to be required for the expression of the human beta-casein gene and of milk protein genes in other species.

Comparative study on the phosphotyrosine motifs of different cytokine receptors involved in STAT5 activation.

Several cytokines and growth factors activate transcription of their target genes via the JAK/STAT signalling pathway. It has been shown that the interaction between SH2 domains of STAT factors and receptor phosphotyrosine residues plays an essential role in the specific recruitment of STATs. For STAT5, however, the importance of receptor tyrosines is still controversial. Using a chimeric receptor system in COS-7 cells, we studied the activation of STAT5 through the interleukin-6 signal transducer gp130. In contrast to previous reports, we did not detect gp130-mediated STAT5 activation. However, STAT5 activation was achieved when tyrosine motifs of other cytokine receptors were fused to the membrane-proximal part of gp130. The comparison of the relative potency of different tyrosine motifs revealed that hydrophobic amino acids, preferentially leucine, in positions +1 and +3, and an aspartate residue in position -1 or -2 with respect to the tyrosine are likely to be required for efficient STAT5 recruitment. In summary, we show here for the first time that phosphotyrosine motifs can confer the ability to activate STAT5 to a heterologous receptor.

Cholesteryl ester transfer protein gene expression is not specifically regulated by CCAAT/enhancer-binding protein in HepG2-cells.

Cholesteryl ester transfer protein (CETP) mediates the exchange of neutral lipids among plasma lipoproteins and is expressed predominantly in liver and intestine. In band shift assays employing nuclear extracts of HepG2 cells we identified C/EBPbeta as the predominant C/EBP isoform involved in binding to the C/EBP consensus sequence within the 5' upstream region of the CETP gene. This was demonstrated by supershift experiments using antibodies specific for C/EBPalpha, C/EBPbeta and C/EBPdelta and an oligonucleotide containing a single point mutation (CAAT-->CTAT) in this site. Expression of a CETP promoter-fragment/luciferase construct in transiently transfected HepG2 and CaCo-2 cells and enhancement of promoter activity by co-transfection with human C/EBPalpha in HepG2 cells could be influenced neither by the mutation in the consensus sequence nor by elimination of this site together with a second potential binding site for C/EBP. Furthermore, transfection of HepG2 with human C/EBPalpha did not influence the synthesis of CETP by these cells. Our results indicate that the expression of C/EBP in HepG2 cells is not able (1) to influence specifically the expression of a transfected CETP promoter dependent reporter through binding to C/EBP sites in the promoter region and (2) to significantly enhance expression of the endogenous CETP gene.

Amiloride and 5-N,N-dimethylamiloride inhibit the carrier mediated uptake of choline in Ehrlich ascites tumor cells.

Amiloride and 5-N,N-dimethylamiloride (DMA) inhibit the choline uptake of Ehrlich ascites tumor cells. The inhibition by DMA is competitive with a KI value of 20 microM. The apparent KM value for choline was determined as 15 microM. Amiloride is approximately three times less potent. Amiloride uptake is not antagonized by choline or impaired in cells characterized by a deficient choline carrier. This indicates that amiloride is not transported into the cell by the choline carrier. The inhibition of the choline uptake by DMA cannot be attributed to a depression of choline kinase (EC 2.7.1.32) and is therefore considered to be due to a direct interaction between DMA and the choline carrier. DMA does not compete with sodium ions for its effect on the choline carrier. It is suggested that the choline carrier of Ehrlich ascites tumor cells exhibits a binding site for DMA similar to the one on the Na+/H+ antiporter.

Mechanism of interaction between the glucocorticoid receptor and Stat5: role of DNA-binding.

The functional interaction between the glucocorticoid receptor (GR) and the signal transducer and activator of transcription-5 (Stat5) was investigated by studying the synergistic activation of beta-cascin gene transcription by prolactin and glucocorticoids. The synergism was shown to be mediated by a complex hormone response region with multiple binding sites for Stat5, the glucocorticoid receptor, and CCAAT/enhancer binding proteins (C/EBP). HC11 mammary epithelial cells, which contain physiological levels of GR and Stat5, and COS-7 cells overexpressing GR and Stat5 were employed. In both cell types intact binding sites for Stat5 and the GR were a prerequisite for the synergism, whereas C/EBP sites were only required in HC11 cells. Interestingly, the GR sites employed for the synergism were nonclassical, half palindromic sites, which did not function in the absence of activated Stat5 to mediate the action of the GR on transcription. The interaction of GR and Stat5 triggered by the unusual configuration of binding sites appears to represent a novel mechanism by which these two distinct types of transcription factors cooperate. The mode of interaction provides an efficient means to restrict gene expression to conditions where both Stat5 and the GR are activated.

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.