Apoptosis and aging: breast cells and apoptosis.
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Aging↗
Biomedical subjects
P Forgez
Publications and source records attributed to P Forgez.
Transcriptional regulation of the tyrosine hydroxylase gene by neurotensin in human neuroblastoma CHP212 cells.
The human neuroblastoma cell line CHP212 was found to express functional high affinity neurotensin (NTS-1) receptor subtype. Based on the functional interactions between neurotensin and dopamine transmission, we have used this cell line to investigate the short- and long-term modulation of tyrosine hydroxylase gene expression by the stable neurotensin agonist JMV 449. After exposure of the cells to 1 microM JMV 449 for 5 or 72 h, tyrosine hydroxylase protein and mRNA levels were significantly increased as detected by western blot analysis and quantitative RT-PCR, respectively. Transfection of CHP212 cells with a plasmid containing the luciferase reporter gene under the control of a limited proximal region of the cloned tyrosine hydroxylase promoter, revealed that the effect of JMV 449 results from an increase in the transcriptional activity of the TH gene. These results indicate that modulation of tyrosine hydroxylase gene expression may constitute one of the mechanisms involved in the control of dopamine transmission by neurotensin. Such neurotensin-mediated changes in tyrosine hydroxylase expression may also participate in multiple adaptation processes within the central nervous system to environmental conditions where neurotensin is released such as stress and food intake.
Blotting, Western↗
Regulation of human corneal epithelial cell proliferation and apoptosis by dexamethasone.
PURPOSE: To investigate whether human corneal epithelial cells express the glucocorticoid receptor (GR) and to assess the influence of dexamethasone (DEX) on these cells. METHODS: Human corneal epithelial cells were cultured in medium supplemented with various concentrations of DEX (ranging from 10(-10) to 10(-4) M). Cell proliferation was analyzed by 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfop henyl) -2H-tetrazolium inner salt (MTS) assay at 2, 4, and 6 days of culture. Apoptosis was studied by nucleus labeling using a fluorescent dye and immunostaining by APO 2.7 at 6 days of culture. GR mRNA was detected in corneal epithelium and cultured corneal epithelial cells by means of reverse transcription-polymerase chain reaction (RT-PCR). Immunocytochemical staining of the epithelial cells was performed with a monoclonal anti-human GR. RESULTS: RT-PCR and immunocytochemistry showed the expression of GR (mRNA and protein) in corneal epithelial cells. DEX significantly increased corneal epithelial cell proliferation with concentrations ranging from 10(-10) to 10(-6) M, with a maximum effect at 10(-7) M (P < 0.005). However, DEX also induced apoptosis of cultured corneal epithelial cells at any concentration used. CONCLUSIONS: These results indicate that human corneal epithelial cells express the GR and proliferate in response to DEX stimulation which also induces corneal epithelial cell apoptosis.
Apoptosis↗
In vitro effects of dexamethasone on human corneal keratocytes.
PURPOSE: To investigate whether cultured human keratocytes express the glucocorticoid receptor (GR) and to assess the influence of dexamethasone (DEX) on these cells. METHODS: Human keratocytes were cultured in medium supplemented with various concentrations of DEX (ranging from 10(-10) to 10(-4) M). Cell proliferation was analyzed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-s ulfophenyl)-2H-tetrazolium inner salt (MTS) assay at 2, 4, and 6 days of culture. Some experiments were performed in the presence of mifepristone (RU38486), an antiglucocorticoid molecule. The early phase of apoptosis was studied by means of keratocyte staining with a fluorescein conjugate of annexin V and propidium iodide, and cells were analyzed by flow cytometry. Glucocorticoid receptor mRNA was detected in keratocytes by means of reverse transcription-polymerase chain reaction (RT-PCR). Immunocytochemical staining of the cells was performed with a monoclonal anti-human GR. RESULTS: RT-PCR and immunocytochemistry showed the expression of GR (mRNA and protein) in cultured keratocytes. Dexamethasone significantly increased keratocyte proliferation with concentrations ranging from 10(-9) to 10(-5) M, with a maximum effect at 10(-7) M (P < 0.005). Dexamethasone's proproliferative effect was inhibited by RU38486. However, DEX also induced apoptosis of cultured keratocytes at any concentration used. CONCLUSIONS: These results indicate that cultured human keratocytes express the GR and proliferate in response to DEX stimulation (10(-9)-10(-5) M), which also induces keratocyte apoptosis.
Apoptosis↗
Activation of receptor gene transcription is required to maintain cell sensitization after agonist exposure. Study on neurotensin receptor.
Neurotensin (NT) acts through specific G protein-coupled receptors to induce effects in the central nervous system and periphery. In this study we have shown that in the human neuroblastoma cell line CHP 212, an NT agonist, JMV 449, induced high affinity neurotensin receptor (NTR) gene activation. 125I-NT binding of cells challenged with JMV 449 rapidly decreased then reappeared and subsequently stabilized at 50% of the control values after 48 h of agonist exposure. These receptors, which reappeared at the cell surface, are as active as those found in control cells as demonstrated by Ca2+ mobilization. Furthermore, the tyrosine hydroxylase (TH) gene, a known NT target gene, remained activated after prolonged NT agonist exposure in this cell line. In the murine neuroblastoma cell line, N1E-115, NT did not stimulate NTR gene activation but induced NTR mRNA destabilization after long term agonist exposure. In this cell line, NT binding dropped to 15% of control values and remained at this value after agonist treatment. The TH expression, which was originally activated upon NT agonist exposure, decreased to control values after prolonged agonist exposure. These observations combined with the data obtained from a complementary study with HT-29 cells (Souazé, F., Rostène, W., and Forgez, P. (1997) J. Biol. Chem. 272, 10087-10094) revealed the crucial role of agonist-induced receptor gene transcription in the maintenance of cell sensitivity. A model for G protein-coupled receptor regulation induced by prolong and intense agonist stimulation is proposed.
Animals↗
Distinct functional characteristics of levocabastine sensitive rat neurotensin NT2 receptor expressed in Chinese hamster ovary cells.
Neurotensin has been shown to produce pharmacological effects both in brain and periphery. Several of these effects are mediated by a high-affinity neurotensin NT1 receptor. On the other hand, a low-affinity levocabastine-sensitive neurotensin NT2 receptor was molecularly cloned from rodent brain recently. In this study, in contrast to NT1 receptor, levocabastine (a histamine H1 receptor antagonist) and SR48692 (an antagonist for NT1 receptor) strongly stimulated intracellular Ca2+ mobilization in transfected Chinese hamster ovary cells expressing rat NT2 receptor, thus acting as potent NT2 receptor. Furthermore, despite of their affinities for NT2 receptor, the Ca2+ responses to potent NT1 agonists, neurotensin or JMV449 ([Lys8-(CH2NH)-Lys9]Pro-Tyr-Ile-Leu, a peptidase resistant analogue of neurotensin) were much smaller than that observed with SR48692. These findings suggest that NT1 and NT2 receptors present distinct functional characteristics and that SR48692 may act as a potent agonist for NT2 receptor.
Animals↗
Neurotensin agonist induces differential regulation of neurotensin receptor mRNA. Identification of distinct transcriptional and post-transcriptional mechanisms.
The binding of neurotensin (NT) to specific receptors triggers the multiple functions that NT exerts in both periphery and brain. By studying the effect of the concentration and time of NT agonist exposure, two separate regulatory mechanisms were detected for the neurotensin receptor (NTR) gene in human colonic adenocarcinoma cells (HT-29). The incubation of cells for 6 h with the NT agonist, JMV 449, resulted in an increase of 270% in NTR mRNA levels. These changes were the direct result of new NTR gene transcription, as indicated by run-on and half-life experiments. In addition, the transcriptional activation of the NTR gene was dependent on NT-receptor complex internalization and de novo protein synthesis. A second response was detected with prolonged exposure to JMV 449. In this case, a decrease of 70% was detected in NTR mRNA levels. Unlike the initial phase, this change was mediated by a post-transcriptional event as the half-life of NTR mRNA from treated cells decreased by 50% as compared with control cells. NT agonist appears to regulate the synthesis of NTR mRNA. In HT-29 cells, this feedback is exerted by a biphasic response. These phases are apparently independent and mediated by two separate mechanisms.
Analgesics↗
High affinity neurotensin receptor mRNA distribution in rat brain and peripheral tissues. Analysis by quantitative RT-PCR.
Neurotensin (NT) is widely distributed in the central nervous system (CNS) and peripheral tissues, and its actions are mediated by a specific family of G protein-coupled receptors. In this study, the authors have measured the levels of gene expression of the high-affinity neurotensin receptor (NTR) with quantitative reverse-transcriptase-polymerase chain reaction (RT-PCR). In the rat brain, the highest quantities of NTR mRNA were found in the ventral mesencephalon and in the hypothalamus. Surprisingly, almost identical quantities were detected in both structures, despite results from in situ hybridization studies revealing a low expression of NTR mRNA in the hypothalamus. The RT-PCR data suggest that large scale NTR mRNA synthesis is occurring in restrictive hypothalamic nuclei. Intermediate levels of expression were detected in the prefrontal cortex and striatum, and scant levels in the cerebellum. In peripheral tissues, the highest levels of NTR mRNA were detected in the colon, followed by the liver, and then duodenum and pancreas. In this study, the sensitivity and the accuracy of the quantitative RT-PCR method provided the means to estimate the relative distribution of NTR mRNA between brain structures and peripheral tissues. Therefore, this study promotes a better understanding of the localization of NTR synthesis in relationship with the various physiological effects of NT.
Animals↗
Quantitative RT-PCR: limits and accuracy.
In this paper we determine the limits and accuracy of quantitative reverse transcription (RT)-PCR using a modification of the original protocol. The quantification of mRNA with this procedure requires a preliminary estimation of the target molecule (TM) concentration, established from experiments with an internal control molecule (ICM). A definitive quantification is then attained from serial dilutions of the reverse transcription reaction. The success of this latter step is dependent on maintaining an equivalent number of TM and ICM in the reaction. The purpose of our study was to evaluate the influence of the deviation between the TM and the ICM on the result. We show here that we can control the accuracy of the assay by fixing the limit of the TM/ICM ratio. Indeed, when the TM/ICM ratio is between 0.66 and 1.5 (i.e., the difference between TM and ICM is 1.5-fold), the final result has an error of approximately 10%. Exceeding this limit produces errors approaching 60%, as in the case of TM/ICM = 2. When the above conditions are respected, a difference as small as 20% between two samples can be determined with an accuracy of 95%.
Animals↗
Neurotensin receptor down-regulation induced by dexamethasone and forskolin in rat hypothalamic cultures is mediated by endogenous neurotensin.
Neurotensin (NT) has been shown to be involved in neuroendocrine regulation, and the presence of both the peptide and its receptors has been demonstrated in the hypothalamus. In the present study, we show that hypothalamic neurons in primary cultures express the neurotensin receptor (NTR) and we examined a possible regulation of this receptor by glucocorticoids and activators of adenylate cyclase. In the hypothalamic cultures, 125I-NT bound to a single class of binding sites, presenting a selectivity similar to that observed for the high-affinity NTR previously described in the adult rat brain. Radioautographic studies demonstrated that these 125I-NT binding sites were present on 3% of the neurons. A 48-h treatment with forskolin (fsk) decreased 125I-NT binding by 30%. No effect of dexamethasone (dex) alone was found on that parameter. However, a combined treatment with both agents led to a 40% decrease in 125I-NT binding, corresponding to a reduced number of binding sites, and to a 68% decrease in the amount of NTR mRNA. In parallel, the dex plus forsk treatment increased NT release in the incubation medium. Moreover, the decreases in 125I-NT binding and NTR mRNA induced by this treatment were abolished in the presence of an anti-NT antibody or SR 48692, a non-peptidic antagonist of NTR, suggesting that the down-regulation of NTR observed after dex plus fsk treatment was mediated by the release of endogenous NT. Agonist-induced down-regulation of the NTR in this system was confirmed by the application of an exogenous NT analogue, JMV 449. The present findings indicate that, in hypothalamic cultures, dex and fsk indirectly down-regulate NTR expression via the release of endogenous NT.
Analysis of Variance↗
Characterization of apolipoproteins B-100, AI and C from plasma lipoprotein in the goose, Anser anser. Evidence for a genetic polymorphism in ApoC-like apolipoproteins.
In this study we have characterized four of the principle goose apolipoproteins and compared their physicochemical properties with human and avian counterparts. Goose ApoB-100 and ApoAI amino acid compositions were very similar to their chicken and human homologous proteins. The partial N-terminal sequence from goose ApoAI was 91% and 82% similar to the corresponding duck and chicken proteins, respectively. Most of the observed amino acid changes detected between the ApoAI sequences were amino acid replacements having the same characteristics and could be the result of a single base mutation. The N-terminal portion of two ApoC-like apolipoproteins were also studied. Goose ApoCa had an electrophoretic mobility of 0.31 and exhibited a nine-residue motif that was well conserved between ApoCIII sequences from different species. We therefore suggest that ApoCa is the equivalent of mammalian ApoCIII. The N-terminal portion of goose ApoCb, the second major ApoC in high-density apolipoprotein, showed no similarity to proteins previously described in the literature. This protein displayed two isomorphs in alkaline urea gel electrophoresis called ApoCb1 and ApoCb2 with Rf values of 0.36 and 0.39, respectively. A genetic polymorphism was detected in the population whereby 25% of the animals carried only one isomorph and 50% exhibited both ApoCb isomorphs. These frequencies were similar in females and males. The transmission mode of these ApoCb isomorphs was consistent with two segregating alleles from a single codominantly expressed gene.
Amino Acid Sequence↗
Immunological recognition of different forms of the neurotensin receptor in transfected cells and rat brain.
In this work, the molecular forms of the rat neurotensin receptor (NTR) expressed in transfected Chinese hamster ovary (CHO) cells, in infected Sf9 insect cells and in rat cerebral cortex were immunologically detected by means of an anti-peptide antibody raised against a fragment of the third intracellular loop of the receptor. Immunoblot experiments against a fusion protein indicated that the anti-peptide antibody recognized, under denaturing conditions, the corresponding amino acid sequence within the NTR. In immunoblot analysis of membranes from NTR-transfected CHO cells, high levels of immunoreactivity were observed between 60 and 72 kDa, while only a faint labelling was observed at 47 kDa, the molecular mass deduced for the rat NTR cDNA. The bands of high molecular mass were no longer observed after deglycosylation of membrane proteins by peptide N-glycosidase F, indicating that they represented glycosylated forms of the receptor. Extracts of membranes derived from baculovirus-infected Sf9 insect-cells expressing the NTR provided a quite different immunoblot pattern, since the major band detected in that case was at 47 kDa, the molecular size of the non-glycosylated receptor. Taken together, these data show that, while most of the NTR protein was glycosylated in CHO cells, it was unglycosylated in Sf9 insect-cells. In addition, molecular sizes of the receptor proteins observed in these two cell lines differed from those obtained for the NTR endogenously expressed in the rat cerebral cortex of 7 day-old rats, where bands at 56 and 54 kDa were detected. Binding experiments carried out on membrane preparations obtained from baculovirus-infected Sf9 cells demonstrated that the immunogenic sequence was still accessible to the antibody when the receptor was embedded in the cell membrane. Immunohistochemical studies carried out on both transfected CHO cells and infected Sf9 cells confirmed this interpretation and further indicated that the antibody could be applied in the visualization of the receptor.
Amino Acid Sequence↗
Characterization of the effect of SR48692 on inositol monophosphate, cyclic GMP and cyclic AMP responses linked to neurotensin receptor activation in neuronal and non-neuronal cells.
1. Neurotensin stimulated inositol monophosphate (IP1) formation in both human colonic carcinoma HT29 cells and in mouse neuroblastoma N1E115 cells with EC50 values of 3.5 +/- 0.5 nM (n = 4) and 0.46 +/- 0.02 nM (n = 3), respectively. Neurotensin also stimulated cyclic GMP production with an EC50 of 0.47 +/- 1.2 nM and inhibited cyclic AMP accumulation induced by forskolin (0.5 microM) with an IC50 of 1.33 +/- 1.5 nM (n = 3) on the N1E115 cell line. 2. The competitive antagonism by the non-peptide neurotensin receptor antagonist, SR48692 of neurotensin-induced IP1 formation revealed pA2 values of 8.7 +/- 0.2 (n = 3) for HT29 and 10.1 +/- 0.2 (n = 3) for N1E115 cells. SR48692 also antagonized the cyclic GMP and cyclic AMP responses induced by neurotensin in the N1E115 cell line with pA2 values of 10.7 +/- 0.7 (n = 3) and 9.8 +/- 0.3 (n = 3), respectively. 3. In CHO cells transfected with the rat neurotensin receptor, neurotensin stimulated IP1 and cyclic AMP formation with EC50 values of 3.0 +/- 0.5 nM (n = 3) and 72.2 +/- 20.7 nM (n = 3), respectively. Both effects were antagonized by SR48692, giving pA2 values of 8.4 +/- 0.1 (n = 3) for IP1 and 7.2 +/- 0.4 (n = 3) for cyclic AMP responses. 4. Radioligand binding experiments, performed with [125I]-neurotensin (0.2 nM), yielded IC50 values of 15.3 nM (n = 2) and 20.4 nM (n = 2) for SR48692 versus neurotensin receptor binding sites labelled in HT29 and N1E115 cells, respectively. 5 In conclusion, SR48692 appears to be a potent, species-independent antagonist of the signal transduction events triggered by neurotensin receptor activation in both neuronal and non-neuronal cell systems.
Animals↗
Effects of dexamethasone and forskolin on neurotensin production in rat hypothalamic cultures.
In the present study, the effects of glucocorticoids and forskolin, an activator of adenylate cyclase, were examined on neurotensin (NT) production from rat hypothalamic neurons in primary culture. Treatment with dexamethasone induced a dose-dependent increase in NT content. The maximum was reached at 1 microM dexamethasone, which induced a 100% increase in NT levels. The effect of dexamethasone was mimicked by the glucocorticoid agonist RU28362 and blocked by the antiglucocorticoid RU38486, suggesting that this effect was mediated through the glucocorticoid receptor. The treatment with dexamethasone also enhanced the number of immunoreactive NTergic cells (92% increase). In contrast to dexamethasone, forskolin affected neither the NT content nor the number of immunoreactive NTergic cells. However, when cells were treated with both dexamethasone and forskolin, a 285% increase in NT content and a 430% increase in the number of immunoreactive NTergic cells were observed, representing 2.8- and 4.7-fold increases, respectively, compared to the effect of dexamethasone alone. Moreover, this combined treatment increased the accumulation of NT in the culture medium (160% increase) as well as the abundance of NT messenger RNA. We conclude from the present findings that dexamethasone and forskolin act synergistically to enhance NT production in hypothalamic neurons.
Amino Acid Sequence↗
Effect of dexamethasone and forskolin on neurotensin expression in hypothalamic cells in culture.
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Animals↗
Automated image analyzing system for the quantitative study of living cells in culture.
A fully automated image analyzing system was developed for the quantitative study of cells in culture. It was able to count cells, to classify cells according to their morphological characteristics and to follow cell culture development. A specific procedure was designed to process Hoffman modulation contrast images. It detects local gray level differences while using conditional dilation techniques. We were able to successfully detect aggregated unstained cells, presently a technical limit in image segmentation. Living cells can be studied in a noninvasive and nondestructive way with this system. An improved automatic focusing algorithm was developed which ensured an accurate prediction of the optimal focus position. A strictly defined sampling procedure was applied to estimate unbiasedly cell density and obtain precisely cell contours. The evaluation of the system was carried out on Chinese hamster ovary (CHO-NTR) cell cultures treated with a newly developed neurotensin agonist JMV449. Chinese hamster ovary cell division was found to be retarded 20 hours after the JMV449 treatment, while the morphology of CHO-NTR cells has already undergone significant changes 12 hours after the treatment. This image analyzing system provides the possibility to follow cell culture development (e.g., cell density evolution, cell morphological changes) under various experimental conditions.
Animals↗
Chromosomal localization of mouse and human neurotensin receptor genes.
Neurotensin is a tridecapeptide that plays several neurotransmitter or neuromodulatory roles both in the central nervous system and in the periphery. These actions are mediated by a high-affinity receptor (Ntsr). Both rat and human cDNAs encoding high-affinity receptors have been recently cloned. The availability of Ntsr probes allowed us to localize the corresponding genes on the mouse and human chromosomes. The present data demonstrate that the Ntsr gene is assigned to the H region of the mouse Chromosome (Chr) 2 and to the long arm of the human Chr 20.
Animals↗
Expression of the human apolipoprotein E gene suppresses steroidogenesis in mouse Y1 adrenal cells.
The lipid transport protein, apolipoprotein E (apoE), is expressed in many peripheral tissues in vivo including the adrenal gland and testes. To investigate the role of apoE in adrenal cholesterol homeostasis, we have expressed a human apoE genomic clone in the Y1 mouse adrenocortical cell line. Y1 cells do not express endogenous apoE mRNA or protein. Expression of apoE in Y1 cells resulted in a dramatic decrease in basal steroidogenesis; secretion of fluorogenic steroid was reduced 7- to greater than 100-fold relative to Y1 parent cells. Addition of 5-cholesten-3 beta,25-diol failed to overcome the suppression of steroidogenesis in these cells. Cholesterol esterification under basal conditions, as measured by the production of cholesteryl [14C]oleate, was similar in the Y1 parent and the apoE-transfected cell lines. Upon incubation with adrenocorticotropin or dibutyryl cAMP, production of cholesteryl [14C]oleate decreased 5-fold in the Y1 parent cells but was unchanged in the apoE-transfected cell lines. These results suggest that apoE may be an important modulator of cholesterol utilization and steroidogenesis in adrenal cells.
Adrenal Glands↗