Biomedical subjects

A M Bamberger

Publications and source records attributed to A M Bamberger.

At least 37 records · Page 2Linked to original sources

PubMed1999

[Molecular and clinical endocrinology of the endometrium].

The ovarian steroid hormones estradiol and progesterone exert their effect by interacting with their intracellular receptors, which, after ligand binding translocate to the nucleus and bind to the promoter regions of target genes. The consequence is a change in the transcription rate of the target genes, followed by a change in production of the corresponding proteins. Target genes of the sexual steroid hormones include cytokines and growth factors, among them CSF-1, TGF-beta and LIF. The rhythm and activity of steroidogenesis, receptor modulation and transcription are reflected by cycle-specific proliferation and differentiation processes in the endometrium. Quantitative and/or qualitative molecular endocrinology is of increasing interest for better definition of morphological changes, although, as yet, the pathological laboratory test is of much less practical consequence than a suspicious vaginal sonography. In spite of the high standard of ultrasound techniques, however, most cases with slightly increased endometrial thickness show histologically benign changes of the endometrium rather than endometrial precancer or cancer. This is especially true for perimenopausal women with no other clinical findings. Yet, the cancer risk is increased in women under tamoxifen therapy. Hence, as a rule, these cases, when endometrial thickness exceeds 5 mm, need a diagnostic biopsy or abrasio.

Biopsy↗

PubMed1999

P16/MTS1 and pRB expression in endometrial carcinomas.

p16MTS1/CDKN1 and the retinoblastoma protein Rb are both involved in negative regulation of G1/S progression in the mammalian cell cycle. Inactivation of one of these tumour suppressor genes is involved in many malignant tumours, and in some studies a negative correlation of p16 and Rb expression has been found. In order to study this interaction in endometrial carcinogenesis, we investigated 36 endometrial carcinomas, 11 cases of hyperplasia, 23 normal endometrial samples, and two uterine carcinoma cell lines by immunohistochemistry or RT-PCR. Rb was expressed in normal endometrial epithelium, hyperplasia, cell lines, and most carcinomas; negative immunostaining was only detected in 1 of 36 tumours. In contrast, p16 expression was weak in normal endometrium and increased in most cases of hyperplasia, but negative or minimally positive in 74% of the carcinomas and the Hec1B adenocarcinoma cell line, and there was no significant association with Rb immunostaining. Strikingly high p16 expression was found in foci of squamous metaplasia within hyperplastic or carcinomatous tissue. Deletion and mutation analysis of the p16 gene was performed in DNA from microdissected tumour samples and cell lines. No p16 deletion was found, and mutations were detected in only one tumour sample and Skut1B uterine mixed mesodermal tumour cells. Our data indicate that in spite of low or absent p16 expression, genetic alterations of the p16 and Rb tumour suppressor genes are rare in endometrial carcinogenesis.

Adult↗

PubMed1999

Strongly reduced expression of the cell cycle inhibitor p27 in endometrial neoplasia.

In the present study we investigated the expression of the cell cycle inhibitor p27 in endometrial neoplasia using immunohistochemistry with a p27-specific antibody. Expression of p27 in endometrial carcinomas was compared with expression in the normal endometrium throughout the cycle. Normal endometrial cells showed strong nuclear expression of p27. Expression was present throughout the cycle and was stronger during the secretory phase. We found strongly reduced or abolished expression of p27 in endometrial carcinoma (85.3% of cases). The 41 tumours analysed were classified according to p27 staining intensity and percentage of positive cells into the following categories of p27 expression: negative/very low (56.0%); low (29.3%); moderate (14.7%) and high (0.0%). All the p27-positive tumours were well-differentiated endometrioid carcinomas of malignancy grade G1. Comparison with the p53 status showed that all tumours with strong p53 expression had low/negative p27 staining, while those that were positive for p27 had negative/low p53 staining. Reduced or absent p27 levels were also observed by Western blot analysis both in tumour samples and in HEC-1B endometrial adenocarcinoma cells. It thus seems that p27 expression is essential for the control of normal endometrial proliferation, and reduced or absent p27 expression may be an important step in endometrial carcinogenesis.

Adenocarcinoma↗

PubMed1999

Telomerase activity in benign and malignant adrenal tumors.

Histological analysis of surgically removed adrenal masses often fails to differentiate between benign and malignant tumors. In normal cells, the telomeric ends of the chromosomes are shortened with each cell division, leading to chromosome destabilization and cellular senescence after a critical number of cell cycles. In tumor cells, telomere shortening is prevented by a specific DNA polymerase, called telomerase. In an effort to clarify the role of telomerase in the pathogenesis of adrenal tumors, and to test whether its activity could serve as marker of malignancy, we measured telomerase activity in 41 human adrenal tissue samples that were classified both by the clinical course and by histological examination. Telomerase activity was determined by TRAP ELISA and expressed as high (>50% of positive control telomerase activity), medium (31-50%), low (11-30%), very low (< or = 10%), or absent (0%). The 8 normal adrenal tissue samples showed very low levels of telomerase activity. Mean telomerase activity also very low in 3/3 incidentalomas, 6/6 Cushing adenomas, 6/6 Conn adenomas, 7/7 adrenocortical carcinomas, 8/8 benign pheochromocytomas, and 2/3 malignant pheochromocytomas. In contrast, one malignant pheochromocytoma showed high telomerase activity. These data indicate that telomerase activity may not be a suitable marker for malignancy in the adrenal gland. Our results also challenge the current dogma of close correlation between cell dedifferentiation and telomerase activity.

Adenoma↗

PubMed1999

Dissociative glucocorticoid activity of medroxyprogesterone acetate in normal human lymphocytes.

The immunosuppressive effects of glucocorticoids (GC) have led to their wide application in the treatment of inflammatory and autoimmune states. However, long term GC treatment is associated with severe side-effects. The development of agents displaying a more favorable ratio of wanted and unwanted GC effects, is, therefore, a major goal of pharmacological and clinical research. In this study, the progesterone receptor agonist medroxyprogesterone acetate (MPA), which also binds to the glucocorticoid receptor (GR), was tested with regard to its immunosuppressive properties. Using a recently established electroporation protocol, we show that MPA (but not progesterone) can suppress a human interleukin-2 (IL-2) promoter-luciferase construct to the same extent as the synthetic GC dexamethasone in normal human lymphocytes. MPA also markedly suppressed IL-2 (as well as IL-1 and IL-6) release, as assessed by specific enzyme-linked immunosorbent assays. In contrast, a highly dexamethasone-inducible glucocorticoid response element-driven promoter construct was only marginally stimulated by MPA in both normal human lymphocytes and HeLa cells. RT-PCR and Western blot analysis of normal human lymphocytes revealed that they do not express progesterone receptor messenger ribonucleic acid and protein, respectively. In contrast, the GR protein was clearly detectable in all samples and was shown to mediate the effects of MPA in transfected Jurkat T lymphoma cells. Our data indicate that 1) MPA can transrepress the human IL-2 gene in normal human lymphocytes in the absence of significant trans-activation; and 2) this effect is mediated by GR. Because of its dissociative GC activity, MPA is a highly promising substance for the treatment of inflammatory/autoimmune states.

Blotting, Western↗

PubMed1999

Reduced expression levels of the cell-cycle inhibitor p27Kip1 in human pituitary adenomas.

The molecular mechanisms leading to increased cellular proliferation rates and, thus, tumor formation in the anterior pituitary gland are poorly understood. The cyclin-dependent kinase inhibitor p27Kip1 is a key molecule regulating the G1 phase of the cell cycle in many cell types. Furthermore, it was shown that p27 knock-out mice develop pro-opiomelanocortin-positive pituitary tumors. In an effort to clarify the role of p27 in the normal and tumorous human pituitary, we studied the expression of p27 by immunohistochemistry, using a highly specific mouse monoclonal anti-human p27 antibody. Normal pituitaries and 54 pituitary adenomas (twelve somatotrope adenomas, nine prolactinomas, twelve corticotrope adenomas, three TSH-producing tumors, six gonadotrope adenomas, six null cell adenomas, and six oncocytomas) were analyzed. p27 expression was determined semiquantitatively with regard to both the percentage of positive cells and the intensity of the staining. Normal human pituitaries showed strong expression of p27 in most nuclei. In contrast, the levels of p27 were reduced in the majority of the tumors analyzed. Twenty-two tumors (six somatotrope adenomas, five prolactinomas, four corticotrope adenomas, two TSH-producing tumors, two gonadotrope adenomas, and three null cell adenomas) were completely p27-negative. In 18 tumors, p27 expression was found in < or = 10% of the cells. In the other ten tumors, 11-80% of the cells were p27-positive. In summary, we were able to demonstrate reduced expression levels of the cell-cycle inhibitor p27 in tumors derived from all pituitary cell types. Our data indicate that p27 may be an important regulator of cellular proliferation in the anterior pituitary, the underexpression of which could play a role in pituitary tumorigenesis.

Adenoma↗

PubMed1999

Relaxin-like factor (RLF): a new specific marker for Leydig cells in the ovary.

Relaxin-like factor (RLF), also known as the Leydig cell insulin-like factor (Ley-I-L), is a novel member of the insulin-IGF-relaxin family of hormones and growth factors that has recently been shown to be strongly expressed in testicular Leydig cells. Expression of the RLF peptide in the human ovary and in ovarian tumors has not been studied. In the present study, the expression of the RLF peptide in the human ovary was investigated by immunohistochemistry using a specific antibody raised against human RLF. By this method, RLF was found to be expressed in hilus (Leydig) cells and theca interna cells but absent in granulosa cells, ovarian stromal cells, and surface epithelium. RLF expression was also observed in the corpus luteum, although at a lower level than in theca cells. Thirty-seven sex cord-stromal tumors and five cases of hilar Leydig cell hyperplasia were also investigated for RLF expression. RLF was found to be strongly expressed in hilar Leydig cell hyperplasia and sex cord-stromal tumors with a component of Leydig or luteinized cells. Of the analyzed theca cell tumors, two displayed a diffuse staining pattern. As expected, RLF was not expressed in granulosa cell tumors. In conclusion, RLF appears to be a useful marker for Leydig cells in the human ovary and may be a diagnostic supplement in hyperplasias and tumors derived therefrom.

Biomarkers, Tumor↗

PubMed1998

Differential regulation of the human 'leukemia inhibitory factor' (LIF) promoter in T47D and MDA-MB 231 breast cancer cells.

Leukemia inhibitory factor (LIF) is a pleiotropic inflammatory cytokine. A potential role for LIF in the pathogenesis of human breast cancer was recently indicated by the finding that LIF is produced by MDA-MB 231 breast cancer cells and that it stimulates proliferation of the T47D and MCF-7 breast cancer cell lines. Despite its role as a possible therapeutic target in breast cancer, the transcriptional regulation of the LIF gene in breast cancer cells has not been investigated so far. In this context, we investigated the regulation of the human LIF promoter (human LIF666-luciferase) by ovarian steroids in transient transfection assays in MDA-MB 231 and T47D cells. Since the MDA-MB 231 cells are devoid of both estrogen (ER) and progesterone (PR) receptors, these cells were co-transfected with the respective receptor expression vector. Estradiol induced no stimulation in either T47D or ER-transfected MDA-MB 231 cells. Treatment with the progesterone agonist MPA (medroxy-progesterone acetate) resulted in induction of LIF transcription in PR-transfectant MDA-MB 231 cells, while it had no effect in T47D cells. Both PR isoforms (PR-B and PR-A) were effective in inducing the LIF promoter in MDA-MB 231 cells, and this effect was inhibited by the progestin antagonist RU 486. The stimulatory effect of MPA was maintained on deletion constructs (h274LIF-Luc, h148LIF-Luc and h82LIF-Luc), indicating that 82 bp are sufficient to mediate this effect. Our results indicate that the LIF promoter is transcriptionally active in human breast cancer cells and its activity can be modulated by progestins and anti-progestins in cells expressing the LIF protein, which might have therapeutic implications.

Breast Neoplasms↗

PubMed1998

Human lymphocytes produce urocortin, but not corticotropin-releasing hormone.

Hypothalamic corticotropin-releasing hormone (CRH) is the principal regulator of the hypothalamus-pituitary-adrenal axis in mammals. In addition, immunoreactive CRH is also present at peripheral sites, where it is thought to act as a proinflammatory peptide. However, the source of peripheral CRH has remained obscure. Human lymphocytes were shown to produce immunoreactive CRH, yet the data on CRH mRNA expression in these cells are equivocal. More recently, Vaughan et al. discovered a new member of the CRH family, termed urocortin. Urocortin was shown to act through the same receptors as CRH. The current study was designed to investigate both mRNA and protein expression of CRH and urocortin in human lymphocytes. Using a commercial CRH(1-41) radioimmunoassay, we demonstrate that normal human lymphocytes and Jurkat T lymphoma cells produce significant amounts of immunoreactive peptide. However, no CRH mRNA was detectable by RT-PCR in these cells. In contrast, a band of the correct size and sequence was amplified with urocortin-specific primers. Immunocytochemical analysis of human lymphocytes using antibodies that could distinguish between CRH and urocortin revealed significant expression of urocortin but not of CRH, consistent with our RT-PCR data. We conclude that human lymphocytes produce urocortin, but not CRH.

Cells, Cultured↗

PubMed1998

Dysregulated expression of CD66a (BGP, C-CAM), an adhesion molecule of the CEA family, in endometrial cancer.

CD66a (BGP, C-CAM) is an adhesion molecule of the carcinoembryonic antigen family that has been shown to be down-regulated in colorectal, prostate, and breast cancers. The purpose of the present study was to determine its expression pattern in the normal human endometrium and in endometrial neoplasia. For this purpose, we performed immunohistochemistry using the 4D1/C2 monoclonal antibody on a series of 24 normal endometrial samples and 47 endometrial carcinomas. Strong CD66a expression was observed in glandular and luminal epithelial cells of the normal endometrium with a consistent localization at the apical poles of these cells throughout the cycle. In late secretory (premenstrual) phase, loss of cellular polarity resulted in a membranous expression pattern in some glandular cells. In the analyzed tumor samples increasing areas with a complete loss of expression were observed with increasing malignancy grade. The apical expression pattern of the normal epithelium was changed to a membranous all-around pattern in 55% of the tumors, mostly in solid areas. This change correlated with malignancy grade and could be observed in 3 of 15 G1 tumors, 4 of 12 G2 tumors, 11 of 12 G3 tumors, and 8 of 8 serous-papillary carcinomas. Areas with membranous expression pattern could be observed along with areas with a normal apical expression pattern in lower grade carcinomas and with areas with complete loss of expression in high grade tumors. Northern blot analysis showed a loss of mRNA expression in tumor samples and HEC-1B endometrial adenocarcinoma cells. Loss of protein expression in the tumor samples was also observed by Western blot. In conclusion, CD66a protein expression is dysregulated in endometrial carcinomas, showing reduction or loss of expression with increasing malignancy grade and a change from the apical to a membranous localization.

Antigens, CD↗

PubMed1997

Regulation of the human interleukin-2 gene by the alpha and beta isoforms of the glucocorticoid receptor.

The immunosuppressive effects of glucocorticoids are largely due to transcriptional inhibition of immunologically relevant genes, such as the interleukin-2 (IL-2) gene. These effects are mediated by the intracellular glucocorticoid receptor (GR). In humans, alternative splicing of the GR precursor mRNA gives rise to two receptor isoforms, termed GRalpha and GRbeta. We previously demonstrated that GRbeta could antagonize GRalpha-mediated transactivation of a glucocorticoid-responsive element (GRE)-driven reporter gene in COS-7 cells. The present study was designed to analyze the roles of the two GR isoforms on glucocorticoid-mediated transrepression of the IL-2 gene. Using a recently developed transfection technique, we demonstrate that in primary human lymphocytes, stimulation of a 548 bp IL-2 promoter-luciferase reporter construct by phorbol ester and calcium ionophore is reversed by dexamethasone to a similar extent as in Jurkat T lymphoma cells transfected with a GRalpha expression vector. Transfection of a GRbeta expression vector alone did not result in IL-2 promoter repression in response to glucocorticoids. Furthermore, GRbeta did not antagonize the repressive effects of GRalpha on IL-2 promoter activity. Surprisingly, overexpression of GRbeta in Jurkat cells did not cause significant inhibition of GRalpha-induced transactivation of a GRE-dependent luciferase reporter gene either. We conclude that the transrepressive effect of glucocorticoids on IL-2 gene transcription is exclusively mediated by GRalpha. GRbeta can neither antagonize GRalpha-mediated transactivation nor transrepression in Jurkat cells, indicating a cell type-specific pattern of GRbeta-mediated antiglucocorticoid activity.

Calcium↗

PubMed1997

Inhibition of mineralocorticoid and glucocorticoid receptor function by the heat shock protein 90-binding agent geldanamycin.

The effects of mineralocorticoids and glucocorticoids are mediated by the intracellular mineralocorticoid glucocorticoid receptor (MR) and glucocorticoid receptor (GR), respectively. Several studies suggest that hormone binding and, thus, receptor activation depend on the association of both MR and GR with the 90-kDa heat shock protein (hsp 90). However, there are few reports analyzing the functional relevance of this association in vivo. The present study was designed to determine how the new hsp 90-binding agent geldanamycin, which was previously shown to disrupt the formation of steroid receptor/hsp complexes, interferes with MR- and GR-mediated transactivation in intact cells. We show that geldanamycin inhibits aldosterone-dependent transactivation of a mineralocorticoid-responsive reporter genes in a concentration-dependent manner. Similar effects were observed for the dexamethasone-activated GR. However, geldanamycin did not affect transcription from a retinoic acid-dependent reporter gene. Inhibition of GR-mediated transactivation was observed both in HeLa cells expressing endogenous GR and in COS-7 cells transfected with a GRa expression vector. Binding studies indicate that geldanamycin disrupts receptor function by reducing hormone binding affinity without lowering intracellular receptor protein levels. Our data support the current model of hsp 90-dependent steroid receptor activation. Furthermore, we show for the first time that MR function also depends on the interaction with hsp 90 in intact cells. Finally, we demonstrate that the function of endogenous is thought to keep the receptor protein in an inactive, yet ligand-activable state (9-17). Ligand binding induces a conformational change in the receptor molecule, which causes it to dissociate from the hsp complex, to translocate to the cell nucleus, and, finally, to interact with specific hormone response elements in the promoter regions of hormone-responsive genes (6-8). Both MR and GR bind as homodimers to identical palindromic sequences on the target DNA, termed glucocorticoid response elements (GREs) (18). The formation of GR/MR heterodimers has also been described (19,20) and may have profound functional consequences (21). The current model of MR and GR function holds that these receptors are unable to bind their respective hormones as long as they are not associated with the hsp complex (9-17). However, experimental support for this model is mainly based on in vitro work. There are few reports analyzing the functional relevance of GR/hsp interactions in mammalian cells. In the most recent study, Whitesell et al. showed that the hspE90-binding agent geldanamycin can specifically disrupt GR/hsp association, thus inhibiting glucocorticoid-mediated transcriptional activation (22). MR is even less well studied in this respect. To our knowledge, there have not been any data supporting a functional role for proper MR/hsp interaction in intact cells. In this study, we show for the first time that MR function depends on the interaction with hsp 90 in intact human cells. Furthermore, we demonstrate that geldanamycin inhibits GR-mediated transcriptional activation in two human cells lines, confirming the results by Whitesell et al. and extending them to transfected as opposed to endogenous GR.

Aldosterone↗

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

Progestin-dependent stimulation of the human leukemia inhibitory factor promoter in SKUT-1B uterine tumor cells.

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine which is essential for implantation in rodents and is expressed during the progesterone-dominated secretory phase of the menstrual cycle in the human endometrium. However, the effect of progestin on the transcriptional regulation of the LIF promoter has not been studied so far. In the present study, we used a luciferase reporter plasmid bearing 666 bp of the human LIF promoter (hLIF666-Luc) to investigate the effects of progestin on the transcriptional regulation of LIF in SKUT-1B uterine tumor cells. Jurkat T-lymphoma cells were used for comparison. Since both cell lines are devoid of functional progesterone receptors (PR), we co-transfected the cells with hLIF666-Luc and an expression vector for the human PR form B (PR-B) or A (PR-A). Addition of the progesterone agonist MPA (medroxy-progesterone acetate, 2.5 x 10(-7) M) resulted in induction of LIF transcription only in SKUT-1B cells, while it had no effect in Jurkat cells. Both PR forms were effective in inducing the LIF promoter in SKUT-1B cells when activated by MPA. However, the induction through PR-A was inhibited more efficiently by the progestin antagonist RU 486. We next investigated the stimulatory effect of MPA in SKUT-1B cells on deletion constructs (h274LIF-Luc, h148LIF-Luc and H82LIF-Luc) and found that it is maintained on these fragments. Thus, 82 bp are sufficient to mediate this effect. Our results show that the human LIF promoter is active in uterine tumor cells, and that it is differentially regulated by progestin in cells of uterine and lymphoid origin.

Female↗

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↗