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C M Bamberger

Publications and source records attributed to C M Bamberger.

34 records · Page 2Linked to original sources
PubMed1997

Transcriptional regulation of the human 'leukemia inhibitory factor' gene: modulation by glucocorticoids and estradiol.

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine implicated in various pathological conditions, such as rheumatoid arthritis and osteoporosis. Despite its possible importance as a therapeutic target, very little is known about the regulation of human LIF. In particular, its regulation at the promoter level has not been studied so far, and was, therefore, the focus of the present work. After showing that Jurkat T lymphoma cells can be induced to express endogenous LIF mRNA, we used this cell line as a model to study the regulation of the human LIF promoter in transient transfection assays. For this purpose, a 666 bp fragment of the human LIF 5'-flanking region, amplified from genomic DNA by nested polymerase chain reaction (PCR), was used for the construction of a luciferase reporter plasmid (hLIF666-Luc). In unstimulated Jurkat cells, the human LIF promoter showed low constitutive activity. The promoter was induced upon stimulation with phorbol ester (TPA). Combined stimulation with TPA and the calcium ionophore ionomycin resulted in strong synergistic induction of luciferase activity from the LIF promoter. Transfection experiments with deletion constructs (hLIF274-Luc and hLIF82-Luc) located the region required for this induction to a 192 bp portion of the promoter, which carries two putative c-ets binding sites. We then investigated the effect of glucocorticoids and estradiol by cotransfecting the respective receptors. Both hormones strongly inhibited the stimulation of the LIF promoter by TPA and ionomycin. Since LIF is implicated in the pathogenesis of inflammatory and degenerative conditions, such as rheumatoid arthritis and osteoporosis, the finding that therapeutic agents employed in the treatment of such conditions, i.e. glucocorticoids and estrogens, can modulate the induction of LIF at the transcriptional level, is of particular interest.

Blotting, Southern↗
PubMed1997

PKC isoenzyme expression and cellular responses to phorbol ester in JEG-3 choriocarcinoma cells.

Protein kinase C (PKc) is a key regulatory enzyme involved in the transduction of extracellular growth signals to the cell nucleus. It occurs in several isoforms, the exact functional roles of which have not been established as yet. The tumor-promoting agent 12-O-tetradecanoyl-phorbol acetate (TPA) is the classic activator of PKC and modulates the activity of the activating protein-1 (AP-1) transcription factor complex via this pathway. AP-1, in turn, induces cell proliferation in many tissues. In the present study, the PKC isoenzyme expression pattern in JEG-3 choriocarcinoma cells was analyzed. The results were compared with those obtained in HEC-1B endometrium adenocarcinoma cells, which had previously been characterized in this respect. To gain insight into the possible functional consequences of different PKC expression patterns, cell proliferation rates and AP-1 activity in response to TPA in both cell lines was studied. Western blot analysis of the PKC isoenzyme expression pattern revealed that JEG-3 cells are deficient in the PKC alpha, delta, and epsilon isoforms. These isoenzymes are strongly expressed in HEC-1B cells, with the alpha and delta being constitutively active. As opposed to HEC-1B cells, JEG-3 cells did not show an enhanced proliferation rate in response to TPA. Furthermore, TPA-treated JEG-3 cells did not exhibit any change in cell shape and refractility as observed in HEC-1B cells. AP-1 activity, as determined by a transfected AP-1-luciferase reporter plasmid, was induced 10-fold by TPA in JEG-3 cells, yet only threefold in HEC-1B cells. It is concluded from these data that differential expression of a subset of PKCs, e.g., the alpha, delta, and epsilon isoforms, may serve as an indicator of the proliferative potential in response to growth factors and mitogens. Furthermore, our data indicate that the inducibility of AP-1 activity does not necessarily reflect the proliferative capacity of a given cell type in response to classical tumor promoters such as phorbol ester.

Blotting, Western↗
PubMed1997

Inhibition of mineralocorticoid activity by the beta-isoform of the human glucocorticoid receptor.

Mineralocorticoids and glucocorticoids are important regulators of electrolyte homeostasis and arterial blood pressure. Their effects are mediated by the mineralocorticoid (MR) and the glucocorticoid receptor (GR), respectively. The present study was designed to determine how the two isoforms of the human GR, the "classic" GR alpha and the non-hormone-binding GR beta, interfere with the transcriptional effects of the hormone-activated human MR. COS-7 monkey kidney cells were transfected with different mineralocorticoid-responsive reporter plasmids and a vector expressing the human MR protein. Different amounts of either control, GR alpha, or GR beta plasmid were co-transfected, and luciferase activity was measured after stimulation with aldosterone and/or dexamethasone. MR-mediated stimulation of transcription was enhanced by co-transfection of the GR alpha expression vector. In contrast, MR-mediated stimulation of transcription was strongly inhibited by co-transfection of equal amounts of the GR beta expression vector. Reverse transcription-polymerase chain reaction (RT-PCR) showed expression of both GR isoforms as well as of MR in the human kidney. These data indicate that the two isoforms of the human GR exert opposite effects on mineralocorticoid activity. We conclude that the ratio between GR alpha and GR beta can define the sensitivity of mineralocorticoid target tissues to aldosterone. Imbalances of this ratio may participate in clinical syndromes of impaired or augmented mineralocorticoid sensitivity, such as certain cases of pseudohypoaldosteronism or, possibly, primary arterial hypertension.

Aldosterone↗
PubMed1997

Regulation of the human leukaemia inhibitory factor (LIF) promoter in HEC-1B endometrial adenocarcinoma cells.

Leukaemia inhibitory factor (LIF) is a pleiotropic cytokine which has been found to be expressed in the human endometrium and to play an important role in human reproduction. In the present study we investigated expression and regulation of the human LIF promoter in HEC-1B endometrial adenocarcinoma cells using a luciferase reporter plasmid bearing a 666 bp promoter fragment (h666LIF-Luc) in transient transfection assays. HEC-1B cells were first shown by reverse transcription-polymerase chain reaction (RT-PCR) to be able to produce endogenous LIF mRNA. The LIF promoter was efficiently transcribed in HEC-1B cells, showing much higher levels of basal activity than in the previously studied Jurkat T-lymphoma cells and SKUT-1B uterine mesodermal tumour cells. The activity of the LIF promoter was stimulated in HEC-1B cells by a combination of phorbol ester (TPA) and ionomycin, which we had previously found to strongly induce its activity in Jurkat T-lymphoma cells. We next studied the effect of progestin (medroxyprogesterone acetate; MPA) on the LIF promoter activity in HEC-1B cells. The LIF promoter was not stimulated by MPA treatment in the presence of transfected progesterone receptor B (PR-B) expression vector in HEC-1B cells, while we had previously described its induction by MPA in SKUT-1B cells. This indicates that progestin-dependent regulation of the LIF promoter in uterine tumour cells is different in cells of epithelial and mesodermal origin.

DNA, Recombinant↗
PubMed1997

Expression of the apoptosis-inducing Fas ligand (FasL) in human first and third trimester placenta and choriocarcinoma cells.

The Fas (Apo-1/CD95) ligand (FasL) belongs to the tumor necrosis factor family and acts through its receptor (FasR/ Apo-1/CD95) to induce apoptosis in target cells. FasL is expressed in several immunologically privileged sites. Induction of apoptosis by FasL in invading lymphocytes acts as a mechanism of immune privilege and is important in preventing graft rejection. Furthermore, FasL is expressed in certain malignancies and it has been implicated as a possible key mechanism in immune privilege of these tumors. Since the invading placental trophoblast is another very important site with a privileged immune status, we investigated whether FasL is expressed in the normal and tumoral human placenta. For this purpose, mRNA was extracted from first and third trimester human placental samples as well as from JEG3 choriocarcinoma cells and reverse transcribed to obtain cDNAs. These were used as templates for PCR analysis of FasL expression, in which specific primers were employed to amplify an 853 bp fragment spanning the whole FasL coding region. A product of the appropriate length was amplified from normal placenta as well as from the choriocarcinoma cells. Expression of FasL protein was confirmed by Western Blot and was localized to trophoblast by immunohistochemistry using a FasL-specific antibody. Expression of FasL in the human placenta indicates that induction of apoptosis in lymphocytes by the invading trophoblast could be an important mechanism implicated in the immune tolerance of the fetal semi-allograft.

Apoptosis↗
PubMed1996

Protein kinase C (PKC) isoenzyme expression pattern as an indicator of proliferative activity in uterine tumor cells.

The protein kinase C (PKC) signal transduction pathway is the prototype of a growth factor-responsive intracellular signaling system, which is activated by various cytokines, growth factors and tumor promoters, such as the phorbol ester 12-O-tetradecanoyl-phorbol acetate (TPA). To date, a large number of different PKC isoforms has been identified, the physiological relevance of which is unknown. Moreover, the expression pattern of PKC isoforms in uterine cells has not been studied as yet. To study the functional role of differential PKC isoform expression in uterine tumor progression, we have compared the proliferative response to TPA, changes in cell morphology induced by TPA, and the PKC isoform expression pattern in two uterine tumor cell lines of different origin. The moderately differentiated endometrial HEC-1-B adenocarcinoma cell line showed a marked increase in proliferative activity and a profound morphological change in response to TPA. In contrast, TPA did not induce cell proliferation and/or morphological changes in the well-differentiated SKUT-1-B mixed mesodermal cell line. Analysis of the PKC isoform expression profile by Western blot revealed that PKC alpha, betaI, delta, epsilon, and zeta were expressed at a much higher level in HEC-1-B as compared to SKUT-1-B cells. PKC beta11 was the only isoenzyme to exhibit a higher expression level in SKUT-1-B cells. This is the first study analyzing the PKC isoform expression profile in uterine tumor cells. Our data demonstrate that the proliferative response to TPA correlates with the expression levels of the majority of PKC isoforms in these cells. Overexpression of PKC isoforms indicates a higher proliferative capacity, and may, thus, represent an important step in the pathogenesis of certain uterine malignancies.

Adenocarcinoma↗
PubMed1996

Modulation of AP-1 activity by the human progesterone receptor in endometrial adenocarcinoma cells.

The composite transcription factor activating protein 1 (AP-1) integrates various mitogenic signals in a large number of cell types, and is therefore a major regulator of cell proliferation. In the normal human endometrium, proliferation and differentiation alternate in a cyclic fashion, with progesterone being largely implicated in the latter process. However, the effects of progesterone and the progesterone receptor (hPR) on AP-1 activity in the human endometrium are not known. To address this issue, HEC-1-B endometrial adenocarcinoma cells, which are devoid of hPR, were transfected with luciferase reporter constructs driven by two different AP-1-dependent promoters. Unexpectedly, cotransfection of hPR caused a marked induction of luciferase activity in the absence of ligand on both promoters. The magnitude of this induction was similar to that observed in response to the phorbol ester TPA. Addition of ligand reversed the stimulating effect of the unliganded hPR on AM activity in these cells. These effects were specific for hPR, and were not observed with either human estrogen receptor or human glucocorticoid receptor. Furthermore, they strictly depended on the presence of AP-1-responsive sequences within target promoters. Finally, the described effects of hPR on AP-1 activity were shown to be cell-type specific, because they could not be demonstrated in SKUT-1-B, JEG-3, and COS-7 cells. To our knowledge this is the first report of an unliganded steroid receptor stimulating AP-1 activity. This effect and its reversal in the presence of ligand suggest a novel mechanism, through which hPR can act as a key regulator of both proliferation and differentiation in the human endometrium.

Adenocarcinoma↗
PubMed1996

Molecular determinants of glucocorticoid receptor function and tissue sensitivity to glucocorticoids.

Glucocorticoids play an essential role in maintaining basal and stress-related homeostasis, and lack of glucocorticoid action is incompatible with life in primates. Most known effects of glucocorticoids are mediated by the intracellular GR. The magnitude of a cell's response to glucocorticoids depends both on the hormone level it is exposed to and on its glucocorticoid sensitivity, i.e. the efficiency of GR-mediated signal transduction. In this review, we have summarized the multiple endogenous and exogenous factors that have been shown to be involved in this signaling cascade and, thus, to alter glucocorticoid sensitivity.

Animals↗
PubMed1995

The glucocorticoid receptor and RU 486 in man.

RU 486 is a prototype glucocorticoid antagonist with strong antiglucocorticoid activity in vitro and in vivo. Studies of its molecular mechanism of action have provided invaluable insights in the complex activation cascade of the GR itself. RU 486 effectively antagonizes glucocorticoids in most glucocorticoid-sensitive systems. Agonist effects, however, have been observed in some in vitro and in vivo systems, but remain the exception. In humans, administration of RU 486 causes generalized glucocorticoid resistance with high levels of cortisol compensating for the peripheral receptor blockade. This state is similar to that of patients with generalized familial glucocorticoid resistance. Because of its HPA axis stimulatory effects, RU 486 has limited chronic utility as an antiglucocorticoid. To date, the application of RU 486 can only be recommended in inoperable patients with ectopic ACTH secretion or adrenal carcinoma who have failed to respond to other treatments. The unusually long half-life of this drug also poses problems with titrating its dose within a therapeutic range. The development of a short-acting, selective glucocorticoid antagonist is therefore, a desirable goal of future research.

Adrenocorticotropic Hormone↗
PubMed1995

Growth hormone secretion in response to the new centrally acting antihypertensive agent moxonidine in normal human subjects: comparison to clonidine and GHRH.

It is well established that the central alpha 2-adrenergic agonist clonidine can enhance growth hormone (GH) secretion in humans. This effect is most likely due to stimulation of hypothalamic growth hormone releasing hormone (GHRH) release. To determine the potency of the new I1-imidazoline receptor agonist moxonidine to release pituitary hormones, 12 normal volunteers received clonidine (0.3 mg), moxonidine (0.3 mg), or placebo orally according to a randomized, double-blind protocol. Blood was drawn prior and up to 180 min after drug administration for determination of GH, adrenocorticotropic hormone (ACTH), prolactin, thyrotropin (TSH), luteinizing hormone (LH), follicle-stimulating hormone (FSH), glucose, clonidine, and moxonidine concentrations. The results were compared to those obtained in a standard GHRH stimulation test (1 microgram/kg i.v.). Serum GH levels increased significantly in response to GHRH, clonidine, and moxonidine. However, the increase was less pronounced in response to clonidine and moxonidine as compared to GHRH (mean +/- SEM): after clonidine, GH increased from 0.2 +/- 0.1 to 5.4 +/- 1.5 ng/ml, p < 0.05; moxonidine increased GH levels from 0.1 +/- 0.04 to 4.8 +/- 1.9 ng/ml (p < 0.05); GHRH caused an increase from 0.01 +/- 0.05 to 14.8 +/- 2.5 ng/ml (p < 0.05). No significant change was observed in the concentration of any other pituitary hormone. We conclude that the new I1-imidazoline receptor agonist moxonidine stimulates GH release to a similar extent as clonidine.

Administration, Oral↗
PubMed1995

Glucocorticoid receptor beta, a potential endogenous inhibitor of glucocorticoid action in humans.

Alternative splicing of the human glucocorticoid receptor (hGR) pre-mRNA generates two highly homologous isoforms, termed hGR alpha and hGR beta. hGR alpha is a ligand-activated transcription factor which, in the hormone-bound state, modulates the expression of glucocorticoid-responsive genes by binding to specific glucocorticoid response element (GRE) DNA sequences. In contrast, hGR beta does not bind glucocorticoids and is transcriptionally inactive. We demonstrate here that hGR beta is able to inhibit the effects of hormone-activated hGR alpha on a glucocorticoid-responsive reporter gene in a concentration-dependent manner. [3H]-Dexamethasone binding studies indicate that hGR beta does not alter the affinity of hGR alpha for its hormonal ligand. The presence of hGR beta in nuclear extracts and its ability to bind to a radiolabeled GRE oligonucleotide suggest that its inhibitory effect may be due to competition for GRE target sites. Reverse transcription-PCR analysis shows expression of hGR beta mRNA in multiple human tissues. These results indicate that hGR beta may be a physiologically and pathophysiologically relevant endogenous inhibitor of glucocorticoid action, which may participate in defining the sensitivity of target tissues to glucocorticoids. They also underline the importance of distinguishing between the two receptor isoforms in all future studies of hGR function and the need to revisit old data.

Alternative Splicing↗
PubMed1995

Expression of pit-1 messenger ribonucleic acid and protein in the human placenta.

It is well established that the human placenta produces a wide range of hormones similar to those secreted by the pituitary and hypothalamus. However, the physiological role and regulation of placental hormone synthesis and release are still largely unknown. GH (GH-N) is expressed in the pituitary, where it requires the tissue-specific transcription factor Pit-1. Chorionic somatomammotropin A (CS-A) and CS-B as well as the placental GH variant (GH-V), which also belong to the GH gene family and are located in the same chromosomal cluster, are expressed in the placental syncytiotrophoblast. The presence of Pit-1-binding sites in the CS-A and GH-V promoter regions predicts that Pit-1 may be expressed in the placenta. However, this has not yet been demonstrated. To examine possible similarities in the regulation of these genes in the pituitary and placenta, we studied the expression of pit-1 messenger ribonucleic acid (mRNA) in the human placenta, transformed human placental cells, and the JEG-3 choriocarcinoma cell line. Polymerase chain reaction (PCR) products of the expected size were amplified from first and third trimester placentas, transformed placental cells, and JEG-3 complementary DNA by reverse transcription-PCR. The pit-1-specific sequence was confirmed by restriction endonuclease digestion, Southern hybridization, and DNA sequencing. Human pituitary tissue was used as a positive control; no PCR product was obtained from hippocampus (negative control). In situ hybridization of placental tissue sections revealed the presence of pit-1 mRNA in first and third trimester syncytiotrophoblast. Pit-1 protein was localized by immunohistochemistry with the same tissue distribution and a nuclear localization pattern. These data demonstrate expression of pit-1 mRNA and Pit-1 protein in the human placenta, thus questioning its role as a pituitary-specific regulator of GH-N gene transcription. The expression of Pit-1 in the placenta, together with its previously demonstrated capability to bind to and activate the CS-A and the GH-V promoters, suggests that it may play a role in the regulation of hormones belonging to the GH gene family in both pituitary and placenta.

Base Sequence↗
PubMed1994

Systemic interleukin-1 alpha and interleukin-2 secretion in response to acute stress and to corticotropin-releasing hormone in humans.

Acute stress results in activation of the hypothalamic-pituitary-adrenal (HPA) axis. ACTH and cortisol secretion is stimulated by corticotropin-releasing hormone (CRH). It has also been shown that activation of the HPA axis during stress is accompanied by changes in the immune response. However, little is known about the influence of acute stress on the release of cytokines such as interleukin-1 (IL-1) or interleukin-2 (IL-2). In this study, we determined serum IL-1 alpha and IL-2 levels in 19 patients undergoing the acute stress of angioplasty for coronary artery disease. A second protocol was devised to determine serum IL-1 alpha and IL-2 concentrations as well as lymphocyte subpopulations in 10 normal volunteers receiving 1 microgram kg-1 human CRH intravenously. Finally, IL-1 alpha concentrations were measured in CRH-incubated mononuclear cell (MNC) and monocyte cultures. In response to the stress of angioplasty, ACTH and cortisol as well as IL-1 alpha and IL-2 concentrations were clearly above baseline levels (IL-1 alpha, mean +/- SEM, baseline: 1.39 +/- 0.34 ng ml-1, after angioplasty: 2.64 +/- 0.73 ng ml-1, P < 0.05; IL-2, baseline: 1.2 +/- 0.13 ng ml-1, after angioplasty: 2.8 +/- 1.14 ng ml, P < 0.05). A similar pattern was obtained in normal subjects in response to CRH (Il-1 alpha, baseline: 0.8 +/- 0.2 ng ml-1, after angioplasty: 3.7 +/- 1.4 ng ml-1, P < 0.05; IL-2, baseline: 1.9 +/- 0.4 ng ml-1, after angioplasty: 5.4 +/- 2.2 ng ml-1, P < 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗
PubMedDate not supplied

Expression patterns of the cell-cycle inhibitor p27 and the cell-cycle promoter cyclin E in the human placenta throughout gestation: implications for the control of proliferation.

The rapid development of the placenta necessitates a high proliferative potential and cell-division rate. This, coupled with a high capacity for invasion, could confer on the placental tissue a tumour-like character. To exclude this, tight mechanisms of control are necessary for both proliferation and invasiveness. Despite their importance, very little is known about the molecular basis of these mechanisms. The present study was thus designed to investigate the molecular mechanisms implicated in the control of proliferation in the human placenta. We used immunohistochemistry to study the expression of two cell-cycle controlling molecules with opposing effects: the cell-cycle inhibitor, p27, which belongs to the Kip/Cip family of CDK inhibitors and can mediate G1 arrest, and cyclin E, a G1-cyclin esential for G1/S progression. Expression was studied throughout pregnancy in a total of 41 normal human placental samples. In addition, immunohistochemistry for Ki-67 was performed as a control for proliferation. The cell-cycle inhibitor p27 was expressed in the differentiated, non-dividing syncytiotrophoblast, while expression of cell-cycle promoter cyclin E was localized to the nuclei of the cytotrophoblast and correlated well with expression of Ki-67. No cyclin E expression was observed in the syncytiotrophoblast. In conclusion, strong expression of the cell-cycle inhibitor p27 and absence of expression of cyclin E in the syncytiotrophoblast might represent an important control mechanism in placental proliferation. This differentiates it from the proliferation of malignant tumours, where p27 has been shown to be frequently downregulated while cell cycle promoters are overexpressed.

Cell Cycle↗
PubMedDate not supplied

Molecular mechanisms of proliferation in endometrial tumour cells.

The human endometrium normally undergoes a cyclic proliferation process followed by differentiation under the influence of ovarian steroids and locally produced growth and differentiation factors. Understanding of the molecular mechanisms involved in controlling these processes is of great interest, since imbalances between proliferation- and differentiation-promoting signals can have pathophysiological consequences ranging from infertility to endometrial hyperplasia and tumour formation. The present work reviews aspects of the role played by oncogenes and ovarian steroid receptors in modulating proliferation of endometrial tumour cells. The expression pattern and possible roles of protein kinase C (PKC) subunits are discussed in the context of response-specificity of endometrial tumour cells to tumour-promoting agents such as 12-O-tetradecanoyl-phorbol acetate (TPA) and possible implications for anti-tumour therapy.

Cell Division↗