The management of endoscopic retrograde cholangio- pancreatography-related infections risk: results of an italian survey at regional level.

Background and aim: Among the Endoscopic retrograde cholangiopancreatography (ERCP) adverse events, an increasingly arising problem is the transmission of Multi Drug Resistant (MDR) Bacteria through duodenoscopes. The aim of this survey was to evaluate the current clinical practice of management of ERCP associated infections in Emilia-Romagna, Italy. Methods: An online survey was developed including 12 questions on management of ERCP associated infections risk. The survey was proposed to all 12 endoscopy centers in Emilia Romagna that perform at least > 200 ERCPs per year. Results: 11 centers completed the survey (92%). Among all risk factors of ERCP infections, hospitalization in intensive care units, immunosuppressant therapies, and previous MDR infections have achieved a 80 % minimum of concurrence by our respondents. The majority of them did not have a formalized document in their hospital describing categories and risk factors helpful in the detection of patients undergoing ERCP with an high-level infective risk (9/11, 82%). Most centers (8/11, 72%) do not perform screening in patients at risk of ERCP infections. Post procedural monitoring is performed by 6 of 11 centers (55%). Conclusion: Our survey showed that, at least at regional level, there is a lack of procedures and protocols related to the management of patients at risk of ERCP infections.

Influence of genetic and environmental factors in peripheral arterial disease natural history: Analysis from six years follow up.

BACKGROUND: Peripheral arterial disease (PAD) is a disease affecting million of patients worldwide. Though traditional cardiovascular risk factors have been associated with the development of PAD, the possible existence of an inherited genetic predisposition to PAD has been investigated in few familial aggregation studies. A link between genetics and PAD may open new avenues for the prevention of this morbid and mortal disorder. AIM: The aim of this study is to investigate a possible role of some genetic determinant involving into coagulation and homocysteine metabolism in the progression of PAD. MATERIALS AND METHODS: We follow one-hundred patients affected by PAD for six years. We evaluated Ankle-Brachial Index (ABI) two times; first at the time of recruitment and then after six years, in order to assess the progression of disease. Genotypes for the genes of Factor V Leiden, Prothrombin or Factor II G20210A, Cystathionine Beta-Synthase 844ins68bp and Methylenetetrahydrofolate Reductase C677T was ascertained after taking blood samples. Chi-square test was performed to determinate the possible correlation of these genes and the most common environmental factors in the progression of PAD. RESULTS: Genetic disorders resulting in high level of homocysteina or thrombophilic phenotype are not so frequent. None among the genetic factors we considered were correlated with PAD. CONCLUSION: PAD is a chronic disease whose course can be slowed down especially with the control of environmental risk factors. Genetic analyses are not useful to determine the disease progression or its tendency to remain stable.

The environmental chemical tributyltin chloride (TBT) shows both estrogenic and adipogenic activities in mice which might depend on the exposure dose.

Exposure during early development to chemicals with hormonal action may be associated with weight gain during adulthood because of altered body homeostasis. It is known that organotins affect adipose mass when exposure occurs during fetal development, although no knowledge of effects are available for exposures after birth. Here we show that the environmental organotin tributyltin chloride (TBT) exerts adipogenic action when peripubertal and sexually mature mice are exposed to the chemical. The duration and extent of these effects depend on the sex and on the dose of the compound, and the effects are relevant at doses close to the estimated human intake (0.5µg/kg). At higher doses (50-500µg/kg), TBT also activated estrogen receptors (ERs) in adipose cells in vitro and in vivo, based on results from acute and longitudinal studies in ERE/luciferase reporter mice. In 3T3-L1 cells (which have no ERs), transiently transfected with the ERE-dependent reporter plus or minus ERα or ERß, TBT (in a dose range of 1-100nM) directly targets each ER subtype in a receptor-specific manner through a direct mechanism mediated by ERα in undifferentiated preadipocytic cells and by ERß in differentiating adipocytes. The ER antagonist ICI-182,780 inhibits this effect. In summary, the results of this work suggest that TBT is adipogenic at all ages and in both sexes and that it might be an ER activator in fat cells. These findings might help to resolve the apparent paradox of an adipogenic chemical being also an estrogen receptor activator by showing that the two apparently opposite actions are separated by the different doses to which the organism is exposed.

TARDBP mutations in frontotemporal lobar degeneration: frequency, clinical features, and disease course.

The 43-kD transactive response (TAR)-DNA-binding protein (TARDBP) mutations have been demonstrated to be causative of sporadic and familial forms of amyotrophic lateral sclerosis. More recently, these mutations have been reported in cases of frontotemporal lobar degeneration (FTLD). The aim of this study was to evaluate the role of TARDBP genetic variations in a large sample of consecutive patients with FTLD. A total of 252 FTLD patients were investigated. Each subject had a clinical and neuropsychological evaluation and a brain imaging study. The clinical diagnosis was confirmed by at least 1 year of follow up. The entire TARDBP gene, the intronic flaking regions, and the 5'-untranslated region (5'-UTR) were screened. Six genetic variations were identified in patients with behavioral variant frontotemporal dementia (FTD) and FTD with motor neuron disease phenotypes. Two of these mutations, namely N267S and M359V, lead to amino acid changes within exon 6. We further identified three genetic variations, i.e., Y214Y, IVS-IV + 45C/T, and 5'-UTR G/A, that could potentially affect the normal splicing process as predicted by in silico analyses. None of these genetic variations was found in healthy age-matched controls. Moreover, we identified a previously described benign variant, A66A, in 5 patients. Our study has confirmed and extended the list of pathogenetic mutations in the TARDBP gene in both apparently sporadic and familial FTLD patients. This work further supports the need for TARDBP screening in FTLD. Also intronic splicing that affects mutations should be considered as well.

Estrogen receptor-mediated transcriptional activity of genistein in the mouse testis.

Here we show that genistein, through an estrogen receptor-mediated action, modulates gene expression in the mouse testis throughout development. Genistein passed from the lactating mother to the suckling offspring at levels sufficient to activate gene expression in the testis of the pups. Testis are already responsive to genistein as well as to estradiol at day 14.5 of fetal development. Activation of luciferase correlates with an activation of cell proliferation. In conclusion, our results show that genistein affects reproductive organs of male mice at all developmental ages.

Colonic lymphoid follicles and NOD2/CARD15 mutational status in Crohn's disease.

BACKGROUND: Interactions between the colonic lymphoid system and the genetic background in Crohn's disease are unexplored. This study analysed variations of colonic lymphoid follicles (CLFs) according to the nucleotide-binding oligomerization domain 2 (NOD2) and caspase recruitment domain-containing protein 15 (CARD15) gene in patients with Crohn's disease. METHODS: CLFs were characterized by histology and immunohistochemistry in the specimens of 41 patients undergoing colonic resection for Crohn's disease. Variants of the NOD2/CARD15 gene were assessed by denaturing high performance liquid chromatography and confirmed by DNA sequencing. RESULTS: Eleven patients had a heterozygous variant of the NOD2/CARD15 gene. The uninvolved colon of mutants had significantly lower CLF density (0.9 versus 2.7 follicles per cm(2); P < 0.001) and proportion of those with a germinal centre (9 versus 22 per cent; P = 0.040) than in non-mutants. In active disease, CLF density increased similarly in patients with and without the mutation. The proportion of extramucosal CLFs was higher in mutants than in non-mutants (34 versus 22 per cent; P = 0.030). No significant difference between groups was recorded for cellular profile and proliferation. CONCLUSION: Patients with Crohn's disease and the NOD2/CARD15 mutation show a remodelling of CLFs in both uninvolved and actively inflamed intestines. These subjects may have a defective immune response by organized lymphoid structures.

Modelling tissues in 3D: the next future of pharmaco-toxicology and food research?

The development and validation of reliable in vitro methods alternative to conventional in vivo studies in experimental animals is a well-recognised priority in the fields of pharmaco-toxicology and food research. Conventional studies based on two-dimensional (2-D) cell monolayers have demonstrated their significant limitations: the chemically and spatially defined three-dimensional (3-D) network of extracellular matrix components, cell-to-cell and cell-to-matrix interactions that governs differentiation, proliferation and function of cells in vivo is, in fact, lost under the simplified 2-D condition. Being able to reproduce specific tissue-like structures and to mimic functions and responses of real tissues in a way that is more physiologically relevant than what can be achieved through traditional 2-D cell monolayers, 3-D cell culture represents a potential bridge to cover the gap between animal models and human studies. This article addresses the significance and the potential of 3-D in vitro systems to improve the predictive value of cell-based assays for safety and risk assessment studies and for new drugs development and testing. The crucial role of tissue engineering and of the new microscale technologies for improving and optimising these models, as well as the necessity of developing new protocols and analytical methods for their full exploitation, will be also discussed.

Genistein affects adipose tissue deposition in a dose-dependent and gender-specific manner.

The soy isoflavone genistein targets adipose tissue and elicits physiological effects that may vary based on dietary intake. We hypothesized that the adipose effects of genistein are dose and gender dependent. Four-week-old C57BL/6 male and female mice received daily oral doses of genistein (50-200,000 microg/kg.d) or 17beta-estradiol (E2) (5 microg/kg.d) for 15 d or a diet containing 800 ppm genistein. Genistein increased epididymal and renal fat pad and adipocyte size at doses up to 50,000 microg/kg.d or at 800 ppm in the diet in males but not in females. The alteration in adipocity correlated with changes in peripheral insulin resistance. These treatments increased genistein serum concentrations from 35+/-6 to 103+/-26 nM 12 h after treatment and lowered plasma triglycerides and cholesterol levels. The 200,000 microg/kg.d genistein dose decreased adipose tissue weight similarly to E2. This genistein dose down-regulated estrogen receptor (beta more than alpha) and progesterone receptor expression and induced estrogen-dependent adipose differentiation factors; it did not change expression of the minimal consensus estrogen-responsive element in ERE-tK-LUC mice, which was positively modulated in other tissues (e.g. the lung). E2 down-regulated almost all examined adipogenic factors. Gene microarray analysis identified factors in fat metabolism and obesity-related phenotypes differentially regulated by low and high doses of genistein, uncovering its adipogenic and antiadipogenic actions. The lower dose induced the phospholipase A2 group 7 and the phospholipid transfer protein genes; the 200,000 microg/kg.d dose inhibited them. The antiadipogenic action of genistein and down-regulation of adipogenic genes required the expression of ERbeta. In conclusion, nutritional doses of genistein are adipogenic in a gender-specific manner, whereas pharmacological doses inhibited adipose deposition.

Activation of brain estrogen receptors in mice lactating from mothers exposed to DDT.

The insecticide dichlorodiphenyltrichloroethane (DDT) interferes with physiological endocrine processes modulating estrogens receptor activity. Most of the data describing the DDT mechanism of action have been collected in vitro or in reproductive tissues in vivo. Here we use a new transgenic mouse model to investigate the DDT effects on estrogens receptor activation in vivo in non-reproductive tissues. In particular, we demonstrate that DDT is able to activate estrogen receptors in the brain and the liver of adult mice after acute administration, and it is active in lactating mice when accumulated in the mother's milk. Furthermore, we demonstrate that the acute administration of DDT activates estrogen receptors with a different kinetics with respect to 17beta-estradiol. Experiments with a breast cancer cell line engineered to express luciferase under the transcriptional control of activated estrogen receptors reveal that the microsomal metabolization of DDT is required for its full activity on estrogen receptors. Taken together these data lead to hypothesize that the delayed DDT time course on estrogen receptor activation in vivo might be due to a necessary step of metabolism of the compound.

Target-specific action of organochlorine compounds in reproductive and nonreproductive tissues of estrogen-reporter male mice.

Organochlorines are lipophylic molecules that accumulate in the fat where they remain for years. During weight loss, they are mobilized and their concentration increases in blood. The present work tests, in transgenic estrogen-reporter mice (ERE-tK-LUC), whether this increase is sufficient to modulate the estrogen receptors (ERs) in the whole body. Three weak estrogens were studied: p,p'DDT [1,1,1-trichloro2,2-bis(p-chlorophenyl) ethane], p,p'DDE [1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene], and betaBHC [beta-benzene-hexachloride]. Dose-dependent analysis of reporter expression (luciferase) were performed in tissues of acutely treated mice. A body map of ER activation was obtained. All these chemicals modulated the reporter, although with a different efficiency and depending upon the tissue analyzed. Induction was confirmed in the liver by determining the expression of the endogenous progesterone receptor (PR) gene, at the dose and time point at which the luciferase gene was maximally induced. After experimental accumulation in the fat tissue, followed by a 48-h period of fasting, we tested whether these compounds could be mobilized to reach sufficient levels to activate the ERs in selected reproductive and nonreproductive tissues (testicle, prostate, liver, and lung). This experimental setting produced results that were different than those obtained following acute treatments. In loaded mice, fasting induced betaBHC mobilization resulted in strong ER activation in the liver and the lung, which was blocked by ICI-182780. p,p'DDT mobilization had no effect in these tissues, but it acted efficiently in the prostate and testis. betaBHC inhibited the ERE-mediated reporter in the testicle and induced the reporter in the prostate. In this tissue, betaBHC action was not inhibited by the anti-estrogen ICI-182780. During fasting, betaBHC, p,p'DDT, and metabolite p,p'DDE increased in blood concentration, from 2.25 +/- 0.25, 0.51 +/- 0.09, and 0.38 +/- 0.06 microg/ml to 8.24 +/- 0.95, 4.52 +/- 0.68, and 5.06 +/- 0.57 microg/ml, respectively. The effect produced by these organochlorines in the liver correlates with the modulation of the ERalpha protein. We conclude that these organochlorines modulate differently the expression of estrogen-regulated genes in male mice. Their effect is tissue- and compound-specific and is dependent on the energetic balance.

Whole body action of xenoestrogens with different chemical structures in estrogen reporter male mice.

The present work tested the estrogenic activity of three weak environmental estrogens p,p'DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane], p,p'DDE [1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene] and betaBHC [beta-benzene-hexachloride] in the transgenic estrogen-reporter mouse model (ERE-tK-LUC). By a time dependent analysis of the transgenic reporter expression (luciferase), we showed that all these chemicals modulated the estrogen receptors (ERs) in the whole body, although with a different efficacy and depending upon the tissue analyzed. Peak activity was registered at 16 h of treatment with 5000 microg/kg of each compound. Organochlorines are lipophylic molecules that accumulate in fat. During weight loss they are mobilized and their concentration increases in blood. We tested whether after experimental accumulation in fat tissue, followed by a 48 h period of fasting, these compounds could be modulated to reach sufficient levels to activate the ERs in target tissues. This experimental setting produced results that were different from those obtained following acute treatments. In loaded mice, fasting induced betaBHC mobilization resulted in strong ER activation in the liver, lung, eye, cerebellum, hypothalamus and cortex. p,p'DDT mobilization had no effect in these tissues, but efficiently acted in the testis, where, on the contrary, betaBHC inhibited reporter expression. During fasting, betaBHC, p,p'DDT and the metabolite p,p'DDE increased in blood concentration, from 2.7 +/- 0.36, 0.65 +/- 0.01 and 0.48 +/- 0.06 microg/ml to 9.51 +/- 1.1, 4.98 +/- 0.77 and 6.0 +/- 0.71 microg/ml, respectively. We conclude that these organochlorines modulate differently the expression of estrogen regulated genes in a tissue- and compound-specific manner and that their action is dependent on the energy balance. Moreover, we show that this mouse model is suitable to detect the estrogenic activity of chemicals with variable structures such as alkyl phenols and polychlorobiphenyls.

Isomer-specific activity of dichlorodyphenyltrichloroethane with estrogen receptor in adult and suckling estrogen reporter mice.

We investigated the tissue-specific effects of dichlorodyphenyltrichloroethane (DDT) isomers in adult and suckling newborn mice, using a novel mouse line engineered to express a reporter of estrogen receptor transcriptional activity (ERE-tkLUC mouse). The DDT isomers p,p'-DDT [1,1,1-trichloro2,2-bis(p-chlorophenyl) ethane] and o,p'-DDT [1,1,1-trichloro-2(p-chlorophenyl)-2-(o-chlorophenyl) ethane] were specifically selected as a weak and a strong estrogen, respectively. In adult male mice, p,p'-DDT induced luciferase activity in liver, brain, thymus, and prostate but not in heart and lung. The effect of p,p'-DDT was dose-dependent, maximal at 16 h after sc treatment, and completely blocked by the estrogen receptor antagonist ICI-182,780. In all the organs analyzed, except the liver, administration of o,p'-DDT showed a pattern of luciferase induction superimposable to that of its isomer p,p'-DDT. In liver, o,p'-DDT significantly decreased basal luciferase activity and blocked the reporter induction by 17beta-estradiol. These data lead us to hypothesize that a modulation of ER activity may be involved in the toxic effects of DDT demonstrated by epidemiological and experimental studies. Luciferase activity was also studied in 4-d-old mice lactating from a mother injected with either p,p'-DDT or o,p'-DDT. Both isomers induced a 2-fold increase in the newborn brain. An opposite effect was observed in liver, where p,p'-DDT increased and o,p'-DDT decreased luciferase, thus indicating that these compounds modulate ER activity in adult and newborn tissues by use of a similar mechanism. The ERE-tkLUC mouse proves to be a suitable tool to functionally assess the tissue specificity of estrogenic/antiestrogenic compounds in adult (as well as in suckling) mice.

Identification of estrogen-responsive genes in neuroblastoma SK-ER3 cells.

To evaluate the role of estrogen receptor in the differentiation of cells of neural origin, we developed a molecular approach aimed at the identification of estrogen target genes by mRNA differential display PCR (ddPCR) in human neuroblastoma SK-ER3 cells. More than 3000 RNAs were examined, a few of which displayed a differential regulation pattern in response to 17beta-estradiol (E2). Sequence analysis of three differentially amplified ddPCR products showed homology with the growth-associated nuclear protein prothymosin-alpha (PTMA), the Bcl2-interacting protein Nip2, and one mRNA previously described by others in fetal human brain. Two ddPCR products, referred to as P4 and P10, corresponded to new DNA sequences. Northern analysis confirmed that estrogen treatment of SK-ER3 cells resulted in the upregulation and downregulation of expression of these messages. In particular, PTMA was found to accumulate at both 1 and 17 hr after E2 treatment, whereas P10 product accumulated only at 1 hr. Conversely, P4, Nip2, and the fetal brain-related mRNAs were significantly decreased by the treatment. Further time course analysis of PTMA and Nip2 mRNAs levels indicated that the hormone exerted a marked biphasic regulatory effect on expression of both messages during the course of cell differentiation. In the present study we report for the first time the identification of a panel of estrogen target genes in neural cells that provide new insights in the molecular mechanism of action of E2 in cells of neural origin.

The glucocorticoid receptor regulates the binding of C/EPBbeta on the alpha-1-acid glycoprotein promoter in vivo.

A complex interaction between the Glucocorticoid Receptor (GR), C/EBPbeta, and other transcription factors activate the Alpha-1 Acid Glycoprotein (AGP) promoter in HTC(JZ-1) rat hepatoma culture cells. This effect is mediated by the so-called Steroid Responsive Unit (SRU) of the AGP promoter that contains several binding sites for C/EBP transcription factors, some of which overlap with the Glucocorticoid Responsive Element (GRE). Our in vivo footprinting experiments revealed that the GRE- and the C/EBP-binding sites were already occupied glucocorticoid dependently in HTC(JZ-1) cells 10 min after dexamethasone administration (10(-6) M). Furthermore, local changes in the chromatine structure shown by the appearance of DNAse I hypersensitive sites (HS sites) also took place. These changes were probably dependent on a tissue-specific organization of the chromatine at the SRU because they were not detectable in a different glucocorticoid-responsive cell line (PC12) that did not express AGP. Here, we have also shown that withdrawal of dexamethasone or addition of the anti-glucocorticoid RU486 were able to revert the pattern induced by dexamethasone in vivo. The disappearance of the protected region and the hypersensitive sites, typical of the hormone activated promoter, confirmed the necessity of the GR to be bound by the agonist and the inability of the GR-antagonist complex to bind the DNA. By functional assays, we showed that the occupancy of the SRU by these transcriptional proteins in vivo correlated with the activation of the AGP gene transcription. With these results, we have shown that one of the functions of the GR to activate transcription of the AGP gene is to recruit C/EBPbeta and to maintain it bound at its target DNA sequences (SRU). This process was not accomplished by RU486.

Manganese effects on the human neuroblastoma cell line SK-ER3.

SK-ER3 cells were recently demonstrated to represent a valuable model for the study of estrogen-inducible differentiation of neural cells in culture. This system may constitute an important tool also for the analysis of the effects of neurotoxic drugs. The present study demonstrates that short term exposure to Mn causes increased proliferation rate of SK-ER3 cells regardless of their differentiation. Long term treatment causes cell death in undifferentiated cells at concentrations of the metal as low as 100 nM. When the cells are differentiated with estrogens, death is observed only with a Mn concentration two orders of magnitude higher. Measurement of neurite extension and quantitation of tyrosine hydroxylase content after long-term exposure to the metal allow the conclusion that Mn does not alter the state of differentiation of SK-ER3 cells induced by the treatment with the hormone. The study underlines the importance of studying the effect of Mn in proliferating neural cells and demonstrates the toxic role of micromolar concentrations of the metal in fully differentiated neural cells. Since other authors produced evidence of effects of the metal on cell death and proliferation only at millimolar concentrations, and none described its proliferative activity, the model utilized in the present study seems to be of particular interest.

A glucocorticoid receptor-independent mechanism for neurosteroid inhibition of tumor necrosis factor production.

We investigated the effect of two neurosteroids, pregnenolone and dehydroepiandrosterone sulfate on lipopolysaccharide-induced tumor necrosis factor (TNF) production in vivo and in vitro. Dehydroepiandrosterone sulfate (0.3-30 mg/kg, i.p.) inhibited serum TNF induced by lipopolysaccharide (2.5 micrograms/mouse, i.p.), without affecting the induction of serum corticosterone. Intracerebroventricular (i.c.v.) administration of dehydroepiandrosterone sulfate (0.2-5 micrograms/mouse) also inhibited brain TNF induced by i.c.v. lipopolysaccharide (2.5 micrograms/mouse). Dehydroepiandrosterone sulfate and pregnenolone (10(-6)-10(-4) M) inhibited TNF production in vitro by lipopolysaccharide-stimulated human peripheral blood mononuclear cells or by the human THP-1 cell line, suggesting that this action might also be relevant in humans. We obtained two lines of evidence that neurosteroids do not inhibit TNF via the glucocorticoid receptor. (1) Dehydroepiandrosterone sulfate and pregnenolone did not activate the alpha 1-acid glycoprotein promoter, a typical effect of glucocorticoids mediated by the glucocorticoid receptor, while strong activation of this promoter was observed with dexamethasone. (2) The inhibitory effect of dehydroepiandrosterone sulfate and pregnenolone on TNF production was not reversed by the glucocorticoid receptor antagonist, mifepristone (RU38486). On the contrary the inhibitory effect of dexamethasone, a classical glucocorticoid and inhibitor of TNF synthesis, was completely reversed by RU38486.

Role of IL-1 beta and corticosteroids in the regulation of the C/EBP-alpha, beta and delta genes in vivo.

We have examined the regulatory effects of interleukin 1 beta (IL-1 beta) on the activation of three different isoforms of the C/EBP family of transcription factors (alpha, beta and delta), in hepatocytes of normal and adrenalectomized (ADX) rats. C/EBP-beta and delta mRNA levels were enhanced by IL-1 beta, whereas that of C/EBP-alpha was not affected by treatment with this interleukin in both normal and adrenalectomized rats. The magnitude of the induction was strikingly higher for C/EBP delta in adrenalectomized animals, indicating a suppressive effect of corticosteroids in the IL-1 beta regulatory pathway. The pattern of C/EBP protein synthesis did not always reflect the mRNA findings. For C/EBP-alpha the protein synthesis was higher than expected in IL-1 beta treated ADX animals compared to normal rats. The pattern of C/EBP synthesis was the one that better reflected the pattern of the mRNA transcription. Differently, the induction of C/EBP-delta was not as pronounced as that of the corresponding mRNA in IL-1 beta treated ADX rats. Hormonal modulation of C/EBP transcription factors was studied in parallel with the hormonal induction of the Alpha-1-Acid Glycoprotein (AGP) gene, which is known to be highly induced in rat liver during the acute phase response. This short report also indicates an important role of corticosteroids in the regulation of transcription factors involved in IL-1 beta signalling during the acute phase response.

Specificity of action of a herpes virus VP16/tetracycline-dependent trans-activator in mammalian cell cultures.

In this work, we have studied the activity of a tetracycline modulatable trans-activator (tTA) generated by fusing the DNA binding domain of the tetracycline repressor to the trans-activation domain of the Herpes simplex virus protein 16 (HSV VP16) (plasmid pUHD15-1Neo). In the three different cell lines studied (HTC, rat hepatoma; T47D, human breast cancer; SK-N-BE, human neuroblastoma), the expression of the luciferase gene under the control of a tetracycline operator sequence (plasmid pUHC13-3) was used as a control of the incorporation and the functionality of the trans-activator. Clones selected from these cells responded in a time and dose-dependent manner to the withdrawal of tetracycline. In all these clones, the tTA trans-activator not only modulates the activity of the luciferase gene, but also modulates the activity of a number of endogenous proteins, including C/EBP beta, the glucocorticoid receptor (GR), and SP1. In the transfected cells, the level of these transcription factors was strongly inhibited in the presence of tetracycline and was highly increased after tetracycline removal. Electrophoresis mobility shift assay (EMSA) and footprint experiments proved that the induced proteins are perfectly efficient in binding the DNA. Their transcriptional activity was also determined. In HTC/A9 cells, the level of the chloramphenicol acetyltransferase (CAT) expression driven by the promoter of the alpha 1-glycoprotein (AGP) gene was strongly enhanced at 72-84 hr following removal of tetracycline from the growth media. The accumulation of the endogenous AGP mRNA also increased at 84 hr. In the T47D/TA11 and SK-N-BE/C2.6 cells, a general activation of protein synthesis was also evidenced.

Progesterone agonists and antagonists induce down- and up-regulation of estrogen receptors and estrogen inducible genes in human breast cancer cell lines.

The effects of the synthetic progestin R5020 and the antiprogestin RU486 on the cellular content of estrogen receptors (ER) and on cell responsiveness to estrogens, have been investigated in the sex hormone-sensitive human breast cancer cell lines MCF-7 and T47D. When T47D cells were treated with R5020 (Promegestone) (10(-8) M), ER was down-regulated to about 50% of the control level in a time-dependent manner. Maximum down-regulation was observed after 24 hours and remained at this level for the next 24 hours. Dihydrotestosterone (DHT) or dexamethasone (DEX) had no effect on ER sites. R5020 also down-regulated, although to a lesser extent, ER in the MCF-7 cells which contain fewer progesterone receptor (PR) sites. When MCF-7 cells were transfected with a progesterone receptor expression vector (tMCF-7) to increase the number of PR sites, R5020 down-regulated the ER to a level similar to that reached in T47D cells. In both cell lines ER down-regulation was completely inhibited by a 10-fold molar excess of the antiprogestin RU486 (Mifepristone) (10(-7) M). Surprisingly, when incubated with RU486 alone, T47D cells responded by up-regulating ER 2-4 fold. The functional relevance of inhibition and up-regulation of ER for the estrogen responsiveness of hormone-sensitive human breast cancer cells was tested by assaying the synthesis of an estrogen-regulated product, the PS2 protein. Estrogen induction of this protein was inhibited by at least 70% in T47D cells exposed to R5020 for 24 hours before estrogen administration and by about 25% in MCF-7 cells under the same conditions.(ABSTRACT TRUNCATED AT 250 WORDS)