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B Groner

Publications and source records attributed to B Groner.

At least 145 records · Page 8Linked to original sources
PubMed1989

Overexpression of the c-erbB-2 protein in human breast tumor cell lines.

The c-erbB-2 proto-oncogene is amplified in a high percentage of primary human breast tumors, suggesting that the overexpression of this gene may be involved in the development of human breast cancer. We have investigated five human breast tumor cell lines and have detected amplified c-erbB-2 gene copies in two of them. This amplification leads to overexpression of the c-erbB-2 protein. In addition, two other cell lines have elevated protein levels without gene amplification, suggesting that other mechanisms can lead to overexpression of the c-erbB-2 protein. These results are similar to those that we obtained during a study of primary breast tumors (Berger et al.: Cancer Res 48:1238-1243, 1988). These breast tumor cell lines should be useful for an analysis of c-erbB-2 expression and of the mechanisms that in some cases lead to overexpression.

Blotting, Southern↗
PubMed1989

Regulation of glucocorticoid receptor activity.

Four levels of regulation of glucocorticoid receptor (GR) activity have been investigated. (1) Phosphorylation of the GR was studied in NIH 3T3 cells metabolically labeled with [32P]orthophosphate. A highly specific antiserum against the GR was used to immunoprecipitate 32P-labeled GR, and protein blotting was used to determine the GR concentration. Comparison of the relative specific activities of non-activated and activated receptor revealed a 3-4-fold increase in GR phosphorylation within 60 min upon hormone activation. (2) The affinity of the GR for its hormone response element (GRE) was quantitated in in vitro binding and gel shift experiments. The comparison of monomers, dimers and trimers of the GRE showed that GR binding affinity to multimers is much higher than the affinity for a GRE monomer. (3) The concentration of the GR was determined in quantitative protein blot assays as a function of time after hormone treatment of NIH 3T3 cells. A down-regulation of GR was observed. Only 30% of the maximal GR concentration observed in the absence of hormone remained after 24 h of hormone treatment. (4) The effect of the presence of hormone on the subcellular location of the GR was studied. Hormone treatment and withdrawal experiments indicated that the presence of hormone is not only required to initiate the cascade of events resulting in transcriptional trans-activation. GR translocated to the nucleus upon hormone addition returns rapidly to the cytoplasm upon hormone withdrawal. This indicated an active role for the hormone in the tight nuclear binding of GR.

Animals↗
PubMed1989

Prolactin and glucocorticoid hormones synergistically induce expression of transfected rat beta-casein gene promoter constructs in a mammary epithelial cell line.

We have detected hormone response elements in the promoter region of the rat beta-casein gene that confer the synergistic action of prolactin and glucocorticoid hormones upon transcription of chimeric gene constructs. A 2800-base-pair (bp) rat beta-casein gene fragment containing 2300 bp of 5' flanking sequence was placed in front of a chloramphenicol acetyltransferase (CAT) reporter gene and stably transfected into the mouse mammary epithelial cell line HC11. Addition of prolactin or dexamethasone alone was sufficient for a modest induction of the fusion gene. The simultaneous presence of both hormones produced a strongly synergistic effect, which did not require the presence of insulin. Induction of the beta-casein-CAT gene was only observed in stably transfected confluent cell cultures. Analysis of a 5' deletion series of the beta-casein-CAT gene construct revealed a stepwise loss of hormone inducibility; 285 bp of 5' flanking sequence was sufficient to mediate the synergistic action of lactogenic hormones on expression. The response was reduced by half when compared with the construct containing 2300 bp of the 5' flanking region. Synergistic inducibility further decreased in deletion mutants between -285 and -265 and was completely abolished between -180 and -170. Thus, the 5' flanking region between -285 and -170 contains cis-acting sequences, which are required for mediating the effect of prolactin and dexamethasone.

Animals↗
PubMed1989

New mammary epithelial and fibroblastic cell clones in coculture form structures competent to differentiate functionally.

We have established and characterized a spontaneously immortalized, nontumorigenic mouse mammary cell line, designated IM-2. IM-2 cells synthesize large amounts of the milk protein beta-casein upon addition of lactogenic hormones. The induction of beta-casein occurs rapidly and does not require any exogenous extracellular matrix components. The IM-2 cell line is morphologically heterogeneous and could be separated into cell clones with epithelial and fibroblastic characteristics. In monoculture, none of the epithelial clones could be induced to synthesize caseins. Coculture of epithelial and fibroblastic clones, however, rendered the epithelial cells competent to differentiate functionally; the addition of lactogenic hormones to these cocultures resulted in the synthesis of beta-casein in amounts comparable to that seen with the original IM-2 line. Using this unique cell system, we have investigated the interrelationships between different steps in differentiation leading to hormone-induced casein production. Independent of hormones, epithelial-fibroblastic cell contacts led to the formation of characteristic structures showing the deposition of laminin. We found that the epithelial cells located in these structures also exhibited significantly increased levels of cytokeratin intermediate filament polypeptides. Double immunofluorescence revealed that the cells inducible by hormones to synthesize casein, colocalized exactly with the areas of laminin deposition and with the cells showing greatly intensified cytokeratin expression. These results suggest that hormone-independent differentiation events take place in response to intercellular epithelial-mesenchymal contacts. These events in turn bring about a state of competence for functional differentiation after lactogenic hormonal stimulation.

Animals↗
PubMed1988

Correlation of c-erbB-2 gene amplification and protein expression in human breast carcinoma with nodal status and nuclear grading.

Fifty-one primary human breast tumors were analyzed for amplification of the c-erbB-2 protooncogene. Thirteen (25%) of the DNA samples contained multiple gene copies. Paraffin-embedded tumor sections, available from 47 of the cases, were stained with a c-erbB-2 specific antiserum. Eighty-three % (10 of 12) of the tumors containing amplified c-erbB-2 gene copies stained positively with the c-erbB-2 specific antiserum (P = 0.03). Thirteen tumors containing single copy c-erbB-2 sequences also stained positively with the antiserum. This suggests that mechanisms other than gene amplification may lead to elevated levels of c-erbB-2 protein. Finally, there was a statistically significant correlation between c-erbB-2 protein expression and parameters used in breast cancer prognosis. Positive staining was associated with positive nodal status of the patient (P = 0.02) and with tumors showing a poor nuclear grade (P = 0.02). This is the first study showing that a determination of the level of c-erbB-2 protein in paraffin-embedded tumor sections may have prognostic value for the course of human breast cancer.

Breast Neoplasms↗
PubMed1988

Activation of the receptor kinase domain of the trk oncogene by recombination with two different cellular sequences.

A new chimeric oncogene, trk-2h, has been generated by recombination of two segments of MDA-MB231 human breast carcinoma cell line DNA after transfection in NIH/3T3 cells. The rearranged DNA segments form a fused transcriptional unit. Sequences at the 3' end are homologous to the tyrosine kinase receptor moiety found in the trk oncogene which resembles a truncated growth factor receptor lacking part of its extracellular domain (Martin-Zanca et al., 1986). The 5' sequence of the trk-2h oncogene is contributed by a gene which is expressed in all human cells tested, and is not related to any known gene. Transfection of the receptor kinase domain DNA fragment into NIH/3T3 cells generated another oncogene, trk-3mh, which contains a mouse-specific sequence fused 5' to the receptor kinase. All three trk recombinants have the receptor kinase moiety fused to an activating amino terminus at the same nucleotide in their transcriptional product.

Amino Acid Sequence↗
PubMed1988

Targeted c-myc gene expression in mammary glands of transgenic mice induces mammary tumours with constitutive milk protein gene transcription.

We investigated the consequences of augmented c-myc gene expression in the mammary gland of transgenic mice. For this purpose we directed the expression of a mouse c-myc transgene to the differentiating mammary epithelial cells by subjecting the protein coding region to the 5' regulatory sequences of the murine whey acidic protein gene (Wap). Analogous to the expression pattern of the endogenous Wap gene, the Wap-myc transgene is abundantly expressed in the mammary gland during lactation. The tissue-specific and hormone-dependent expression of the Wap-myc transgene results in an 80% incidence of mammary adenocarcinomas. As early as two months after the onset of Wap-myc expression, tumours occur in the mammary glands of the transgenic animals. The tumours express not only the Wap-myc transgene, but also the endogenous Wap and beta casein genes. The expression of the milk protein genes becomes independent of the lactogenic hormonal stimuli and persists even in transplanted nude mouse tumours.

Adenocarcinoma↗
PubMed1988

Prolactin regulation of beta-casein gene expression and of a cytosolic 120-kd protein in a cloned mouse mammary epithelial cell line.

In order to study the hormonal regulation of gene expression in mammary epithelial cells, we isolated a prolactin-responsive cell clone, HC11, from the COMMA-1D mouse mammary epithelial cell line. Clone HC11 was selected as a unique example of a cloned mouse mammary epithelial cell which has no requirement for complex, exogenously added, extracellular matrix or co-cultivation with other cell types for the prolactin-dependent in vitro induction of the endogenous beta-casein gene by lactogenic hormones. Induction of beta-casein mRNA is rapid and was detected 3 h after hormone stimulation. A prolactin-dependent increase in the rate of transcription of the beta-casein gene was shown in an in vitro nuclear transcription assay. beta-Casein protein was detected in an immunoblot assay after 24 h, and further accumulated during 5 days of hormone treatment. To identify low-abundance proteins induced directly after prolactin stimulation, mRNA was accumulated during 5 h of stimulation of HC11 cells with prolactin in the presence of cycloheximide. Following cycloheximide removal, the mRNA was translated into protein during a 60-min [35S]methionine pulse and the proteins were resolved by DEAE ion exchange HPLC and SDS-PAGE. A strong induction of a 120-kd cytosolic protein was detected which was maximally expressed within 6 h of hormone stimulation.

Animals↗
PubMed1988

Oncogenes modulate cellular gene expression and repress glucocorticoid regulated gene transcription.

The v-mos oncogene was subjected to the transcriptional control of the MMTV LTR and introduced by transfection into NIH 3T3 cells. The LTR v-mos gene was induced by the addition of glucocorticoid hormone to the growth medium of cells synchronized by culturing in 1.5% FCS for 36 h. The effects of p37 v-mos expression were monitored. The endogenous c-myc gene is induced as a consequence of p37 v-mos expression in a transient fashion, reaching a maximum of expression after 8 h. Induction of the c-myc gene was observed at the level of its transcriptional rate and at the level of mRNA concentration. Ornithine decarboxylase (ODC) mRNA was induced constitutively and high levels were found 8 and 25 h after v-mos induction. H4 histone mRNA is elevated at 25 h after hormone addition at a time when the mitogenic stimulus of v-mos causes DNA synthesis. The expression of actin mRNA is not affected by the v-mos oncogene. We have previously described a modulation of glucocorticoid dependent gene expression by oncogenes. In an extension of these observations the consequences of expression of the v-mos and the v-ras oncogenes were also studied in retrovirally infected NIH 3T3 cells. MMTV LTR constructs transfected into the infected cells could only be transiently induced by glucocorticoid hormone. The presence of the p37 v-mos and the p21 v-ras oncoproteins causes a repression of glucocorticoid hormone dependent gene transcription.

Actins↗
PubMed1988

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.

Calcium↗
PubMed1988

Ha-ras and c-myc oncogene expression interferes with morphological and functional differentiation of mammary epithelial cells in single and double transgenic mice.

We studied the effects of the tissue-specific and lactogenic hormone-dependent expression of the recombinant whey acidic protein (Wap)-ras and Wap-myc oncogenes on the differentiation of the mammary epithelium in transgenic mice. The histological appearance of mammary glands in pregnant, lactating, and postlactational animals and their ability to express milk protein genes were analyzed. Activated Ha-ras expression caused a decrease of milk protein synthesis during the lactation period. The formation of glandular epithelium and the postlactational regression of epithelial cells were not affected. c-myc expression impaired the development of the glandular epithelium, and milk protein synthesis was decreased strongly. Epithelial cell proliferation continued during lactation and postlactationally. Coexpression of both oncogenes in double transgenic mice synergistically affected differentiation and resulted in a high number of neoplastic foci. Palpable tumors were observed only after a latency of 3-4 months. Tumor cells utilize the Wap promoter hormone independently, express increased levels of Wap-ras and induce adjacent stromal cells to produce tenascin.

Animals↗
PubMed1988

v-myc alters the response of a cloned mouse mammary epithelial cell line to lactogenic hormones.

Several oncogenes have now been implicated in mammary carcinogenesis. We investigated the phenotypic effects of expressing three representative oncogenes in mammary epithelial cells. v-myc (coding for a nuclear protein), v-Ha-ras (a G-protein homologue) and v-fgr (a tyrosine kinase) genes were introduced into the nontumorigenic clone 14 of the mouse mammary epithelial cell line COMMA-1D. Their effects upon growth and differentiation were determined. Anchorage-independent growth was induced by all three oncogenes with low efficiency. v-Ha-ras and v-fgr induced tumorigenicity in nude mice. The effect of oncogenes upon parameters unique to mammary epithelial cells in vitro was assayed. Both v-myc and v-fgr abolished the ability of clone 14 to grow as three-dimensional branching structures in hydrated collagen gel. v-fgr completely and v-myc partially inhibited the expression of the epithelium specific cytokeratins. Clone 14 can be induced to produce the beta-casein milk protein by the combination of the lactogenic hormones, dexamethasone, insulin, and PRL. Introduction of v-myc into clone 14 cells resulted in an estimated 50-fold increased induction of beta-casein protein and at least a 60-fold increase in beta-casein mRNA. The number of cells stained with anti-beta casein antibodies also showed a 10-fold increase after v-myc introduction. This still required the synergistic action of all three lactogenic hormones. Thus v-myc can alter the normal response of mammary epithelial cells to lactogenic hormones.

Animals↗
PubMed1988

The transformation of primary and established mouse mammary epithelial cells by p21-ras is concentration dependent.

We constructed a retroviral vector (pZSR) which is capable of simultaneously expressing the neomycin resistance gene and the viral ras oncogene. Primary mammary gland epithelial cells were prepared from mid-pregnant mice and infected with this virus. Cell lines with epithelial cell characteristics could be established with a low frequency. High expression of p21 v-ras was observed in these cells. They are tumorigenic and form soft agar colonies dependent on the presence of EGF and insulin in the growth medium but progress to hormone independent growth at higher passage numbers. A cloned cell line of non-tumorigenic, established mammary epithelial cells (NOG8) was also infected with the v-ras expressing virus. Individual cell clones expressing increasing amounts of p21 v-ras were selected. The level of p21 v-ras expression directly influences the morphology of the epithelial cells in culture, determines their cloning efficiency in soft agar and their tumorigenicity.

Animals↗
PubMed1987

The human c-Kirsten ras gene is activated by a novel mutation in codon 13 in the breast carcinoma cell line MDA-MB231.

We have detected amplified human Ki-ras sequences in tumorigenic NIH 3T3 cells transfected with genomic DNA from the human breast carcinoma cell line MDA-MB231. Hybridization of synthetic oligonucleotides specific for human Ki-ras sequences showed a mutation at codon 13. The polymerase chain reaction with Ki-ras specific amplimers revealed a guanosine to adenosine transition at the second position of codon 13, resulting in a substitution of glycine by aspartic acid. The codon 13 mutation is also detected in one Ki-ras allele of the MDA-MB231 cell line.

Animals↗
PubMed1987

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.

Animals↗
PubMed1987

Ha-ras oncogene expression directed by a milk protein gene promoter: tissue specificity, hormonal regulation, and tumor induction in transgenic mice.

The activated human Ha-ras oncogene was subjected to the control of the promoter region of the murine whey acidic protein (Wap) gene, which is expressed in mammary epithelial cells in response to lactogenic hormones. The Wap-ras gene was stably introduced into the mouse germ line of five transgenic mice (one male and four females). Wap-ras expression was observed in the mammary glands of lactating females in two lines derived from female founders. The tissue-directed and hormone-dependent Wap expression was conferred on the Ha-ras oncogene. The signals governing Wap expression are located within 2.5 kilobases of 5' flanking sequence. The other two lines derived from female founders did not express the chimeric gene. In the line derived from the male founder, the Wap-ras gene is integrated into the Y chromosome. Expression was found in the salivary gland of male animals only. After a long latency, Wap-ras-expressing mice developed tumors. The tumors arose in tissues expressing Wap-ras--i.e., mammary or salivary glands. Compared to the corresponding nonmalignant tissues, Wap-ras expression was enhanced in the tumors.

Animals↗
PubMed1986

The v-mos and H-ras oncogene expression represses glucocorticoid hormone-dependent transcription from the mouse mammary tumor virus LTR.

We have subjected the viral mos oncogene (v-mos), the activated human H-ras oncogene [H-ras (A)] and the normal human H-ras protooncogene [H-ras (N)] to the transcriptional regulation of glucocorticoid hormones by in vitro recombination with the promoter region of the mouse mammary tumor virus long terminal repeat (MMTV LTR) and transfection into NIH 3T3 cells. Cell clones were selected which exhibit a transformed phenotype strictly dependent on the presence of hormone in the growth medium. The expression of the chimeric genes as a function of time after hormone stimulation was studied at the level of transcriptional rate, mRNA and protein accumulation. Oncogene expression was stimulated rapidly to high levels, after hormone addition, but declined in the continuous presence of hormone. Measurements of the transcriptional rates in nuclei from LTR v-mos and LTR H-ras (A) transfected cells showed a repression of LTR v-mos and LTR H-ras (A) transcription after the initial stimulation by hormone. LTR H-ras (N) transcription was not affected. An independently transfected LTR H-2Ld construct in LTR v-mos or LTR H-ras (A) containing cells is also transcriptionally repressed. These experiments demonstrated a transcriptional repression effect of the oncogene products on the glucocorticoid hormone-dependent MMTV LTR transcription.

Animals↗