Biomedical subjects

D Gagne

Publications and source records attributed to D Gagne.

At least 19 recordsLinked to original sources

PubMed2005

Microsphere intestinal blood flow analysis during pneumoperitoneum using carbon dioxide and helium.

BACKGROUND: Pneumoperitoneum has been associated with a decreased flow in the superior mesenteric artery and portal venous system. Intestinal blood flow was studied during a 2-h pneumoperitoneum with carbon dioxide (CO2) or helium in a porcine model using colored microspheres. METHODS: For this study, 12 pigs were divided into two groups (6 CO2 and 6 helium). Different colored microspheres were injected directly into the left ventricle before, 40, 80, and 120 min after insufflation with either gas at a pressure of 15 mmHg. Microsphere concentration was measured in the mucosa and muscularis/serosa layers of the jejunum, cecum, and sigmoid colon to calculate blood flow. RESULTS: Intestinal perfusion initially increases with insufflation and returns to near baseline levels during pneumoperitoneum of 2 h. The effect of helium on tissue perfusion is similar to that of carbon dioxide. CONCLUSIONS: Intestinal perfusion does not change significantly during prolonged pneumoperitoneum at a pressure of 15 mmHg with CO2 or helium.

Animals↗

PubMed2000

The third intracellular loop of the rat and mouse cholecystokinin-A receptors is responsible for different patterns of gene activation.

It has previously been reported that the cholecystokinin analog JMV-180 behaves differently on the rat and the mouse cholecystokinin-A receptor (CCK-AR). In mice this analog acts as an agonist on low- and high-affinity sites of the CCK-AR, whereas in rats this compound acts as an agonist on high-affinity sites and as an antagonist on low-affinity sites. In an attempt to understand why the same compound behaves differently on these two CCK-A receptors, we cloned the cDNA encoding the mouse CCK-AR. We then investigated a cellular model able to mimic the effect that was observed in rats and mice. HeLa cells were transiently cotransfected with plasmids leading to expression of the rat or mouse CCK-AR in the presence of pFos-Luc as reporter plasmid; such a plasmid placed the regulatory part of the human c-Fos gene upstream from the firefly luciferase structural gene (Luc). We then observed that the two CCK-A receptors behaved differently, not only in the presence of compound JMV-180 but also in the presence of cholecystokinin or even in absence of ligand; the rat CCK-AR was 2 to 3 times more potent than the mouse CCK-AR in inducing the reporter protein, whatever the ligand studied. This result was confirmed using the same kind of experiment with the reporter plasmid p(TRE)(3)-tk-Luc. Using various mutated receptors, we investigated the role of the putative third intracellular loop. We concluded that both the primary structure of the receptor and the cellular context are in part responsible for the differential behavior of these CCK-A receptors.

Animals↗

PubMed1997

CholecystokininB receptor from human Jurkat lymphoblastic T cells is involved in activator protein-1-responsive gene activation.

The aim of this study was to analyze the role of cholecystokinin (CCK(B)) receptor in human lymphoblastic Jurkat T cells. We investigated the trophic effect resulting from activation of such a receptor by using the reporter gene strategy. For this purpose, we transiently transfected Jurkat T cells with the reporter plasmid p[(TRE)3-tk-Luc] and found that CCK-8 was able to dose-dependently induce luciferase expression related to activator protein-1 (AP-1) activation with a maximal response identical to that obtained with compounds known to activate AP-1 complex (quantitatively, the same level of induction was obtained with 1 nM 12-O-tetradecanoylphorbol-13-acetate, 100 microM diacylglycerol, or 4 nM epidermal growth factor). The involvement of the CCK(B) receptor in such a stimulation was demonstrated by the inhibiting effect of the selective CCK(B) receptor antagonist PD-135,158. This effect was confirmed in COS-7 cells transfected with the cDNA of CCK(B) receptor cloned from Jurkat T cells. To better understand the AP-1-dependent luciferase expression in Jurkat T cells, we tested two specific inhibitors of serine/threonine phosphatases-1 and -2A: okadaic acid and calyculin A. These compounds strongly increased the phorbol-12-myristate-13-acetate response, whereas we have not observed a contribution of phosphatase inhibitors on a CCK-8-induced luciferase activity. To confirm that CCK(B) receptors are involved in AP-1 response, we investigated the CCK-8 effect on interleukin-2 expression, a natural endogenous gene regulated by several factors, including AP-1. In Jurkat T cells activated by phorbol-12-myristate-13-acetate and phytohemagglutinin, CCK-8 induced IL-2 expression. This induction was abolished by PD-135,158. Our results indicate that CCK-8 exerts a trophic effect in Jurkat T cells through stimulation of CCK(B) receptors by modulation of expression of AP-1-regulated genes.

Animals↗

PubMed1996

JMV641: a potent bombesin receptor antagonist that inhibits Swiss 3T3 cell proliferation.

The peptides of the bombesin family are involved in stimulation of mitogenesis in various cell lines, including cancerous cell lines. Bombesin receptor antagonists are of great interest to inhibit this proliferation. We have synthesized a potent bombesin receptor antagonist, e.g., compound JMV641 [H-DPhe-Gln-Trp-Ala-Val-Gly-His-NH-*CH[CH2-CH(CH3)2]-**CHOH- (CH2)3-CH3 [*(S); **92% of (S) isomer], in which a pseudopeptide bond mimicking the transition state analogue replaced the peptide bond between the two C-terminal residues. This compound was highly potent to dose-dependently inhibit binding of 125I-GRP to Swiss 3T3 cells (IC50 = 0.85 +/- 0.15 nM) and bombesin-stimulated Swiss 3T3 proliferation (pA2 = 8.78). However, compound JMV641 can inhibit bombesin-induced AP-1 regulated genes that are nuclear messengers mediating the actions of signal transduction pathways stimulated by growth factors.

3T3 Cells↗

PubMed1996

In vitro and in vivo interactions between nuclear receptors at estrogen response elements.

To study mechanisms involved in the antiestrogenic effect of retinoic acid (RA), previously described in mammalian cells, we used in vitro and in vivo approaches. One hypothesis was direct competition between nuclear receptors (ER, RAR and RXR) at the DNA level. We first showed in vitro that the RAR/RXR heterodimer could weakly bind an ERE and that retinoid receptors reduced binding of ER to an ERE. We next checked whether, in yeast, direct competition between receptors that recognize the same responsive element could be monitored in a reconstituted heterologous estrogen-responsive system, by determining the expression of a reporter gene. We then co-transformed RAR and RXR in an estrogenic responsive strain. This model demonstrated that, even though RAR/RXR was able to bind an ERE, the addition of retinoic acid had no inhibitory effect on estrogen-induced responses in this yeast system, unlike in mammalian cells. Interference between these receptors should require other factors than interactions at the ERE level. This model could be used to identify mammalian factors interacting with estrogen and retinoic acid receptors which could play a role in crosstalk between these receptors.

Animals↗

PubMed1995

Prolonged treatment of breast cancer cells with antiestrogens increases the activating protein-1-mediated response: involvement of the estrogen receptor.

At micromolar (pharmacological) concentrations, the action of tamoxifen on the proliferation of estrogen-dependent cells can be mediated not only by the estrogen receptor (ER), but also by other target molecules, such as protein kinase-C (PKC), which are easily inhibited by antiestrogens in cell-free experiments. By developing MTLN and MDT cell lines, in which any modulation of PKC activity is reflected by a variation of the expression of an activating protein-1 (AP-1)-controlled firefly luciferase gene, we investigated whether such antiestrogen inhibitory effects on PKC occurred in intact breast cancer cells. Firstly, in short term (4-h) treatment of both cell lines, antiestrogens only inhibited the 12-O-tetradecanoyl-phorbol-13-acetate-induced luciferase activity at very high concentrations (30 microM). A cytolytic effect was also observed. Secondly, in prolonged (4-day) treatments of MTLN (ER-positive) cells, low antiestrogen concentrations (nanomolar) decreased the basal AP-1 response by about 2 and increased the 12-O-tetradecanoyl-phorbol-13-acetate-stimulated AP-1 response by about 3-4. This stimulation was mediated by ER, because 1) dose-response curves established with tamoxifen and hydroxytamoxifen were in agreement with their affinity for ER; 2) when present with antiestrogens, estradiol abolished this phenomenon; and 3) this effect was not observed in MDT (ER-negative) cells. Such a latent activation of AP-1 pathway could appear in the course of breast cancer antiestrogen treatment, in conditions where natural PKC activators are abnormally produced with unexpected consequences on the results of a long term antiestrogen treatment.

Base Sequence↗

PubMed1995

[Applications of luminescence reactions in life science].

Bioluminescence and chemiluminescence have emerged in the last decade as a major tool for biochemical and biological studies. Several very sensitive assays have been developed in our laboratory such as enzymatic assays, immunoassays and detection of nucleic acids. The use of a new instrumentation allowing to dimensional photon counting has permit the emergence of new investigations at the cellular level. In addition to the advantages of sensitivity and the real-time non-invasive nature of this detection system, the imaging potential of using low-light and photon counting video cameras has been particularly influential in establishing its ascendence over more traditional systems. This review provides a reflection in this field through several applications in life sciences.

Adenosine Triphosphate↗

PubMed1995

[Modulation of cellular response expression during prolonged treatment with antiestrogens].

The effects of a prolonged antiestrogen treatment on two estrogen-dependent responses and an AP-1 response were studied on two cell lines derived from MCF-7 cells. 1) Hydroxytamoxifen specifically provoked an irreversible inactivation of a chimeric estrogen-dependent gene expression in less than 30 days. This process was estrogen receptor mediated and led to a cellular heterogeneity that was induced by the treatment and was not due to a cell selection process. A similar heterogeneity was also observed for the progesterone receptor expression but after a longer treatment time. The mechanism underlying this phenomenon is currently investigated. 2) After a four day treatment of cells with an antiestrogen, the phorbol ester inducible expression of a chimeric AP-1 response was stimulated by a factor 3-4. This stimulation was antiestrogen dose-dependent and suppressed by the presence of estradiol, which strongly suggested that estrogen receptor was involved. This was confirmed by the fact that the phenomenon was not observed in a cell line devoid of estrogen receptor. This result suggests a yet unknown mechanism by which an antiestrogen could have an agonistic property allowing hormone independence to appear. Both these phenomena show that new activities of antiestrogens may be evidenced after prolonged treatments with unexpected consequences on endocrine therapy.

Estrogen Antagonists↗

PubMed1994

Antiestrogenic effects of all-trans-retinoic acid and 1,25-dihydroxyvitamin D3 in breast cancer cells occur at the estrogen response element level but through different molecular mechanisms.

Most breast tumors show estrogen-dependent growth and are thus susceptible to antiestrogenic therapy. MCF-7 cells, obtained from a human estrogen-dependent breast carcinoma, are widely used for studying the modulation of estrogenic responses by different effectors. All-trans-retinoic acid (RA) and 1,25-dihydroxyvitamin D3 (Vit D3) inhibited estrogen-induced growth of MCF-7 cells and their effect was potentiated by the classical antiestrogen, hydroxytamoxifen. In MCF-7 cells, we found that RA and Vit D3 also inhibited estrogen-induced transcription; this was shown both for an endogenous gene (pS2) and for various exogenous transfected genes. Their inhibitory effect could not be reversed by increasing estradiol concentrations, showing that contrary to classical antiestrogens, they did not compete with estradiol to bind the estrogen receptor (ER). Analysis of the inhibitory mechanisms indicates that RA and Vit D3 receptors can directly or indirectly impair the binding of ER to the estrogen responsive element. The antagonist effect of RA would be found especially at DNA level since it seems to essentially involve an estrogen responsive element. The antagonist effect of Vit D3 would be found especially at the ER level since it seems to concern estrogen binding and dimerization domains of ER. We conclude that the antiestrogenic effects of RA and Vit D3 are similar since they can, via their receptors, interfere with estrogenic action at the estrogen responsive element level but that they are not identical since different molecular mechanisms are involved.

Breast Neoplasms↗

PubMed1994

The effects of therapeutic touch and relaxation therapy in reducing anxiety.

This study examines the effects of two noninvasive procedures on experienced anxiety. Thirty-one inpatients of a Veterans Administration psychiatric facility were randomly assigned to one of two treatment conditions, (therapeutic touch and relaxation therapy) or to a therapeutic touch placebo condition. An additional 13 patients were excluded because of failure to meet criteria for the study or failure to complete the procedures. Each subject completed a self-report anxiety measure and was rated for amount of motor activity before and after each of two 15-minute treatment sessions in a 24-hour period. Subjects' belief in the effectiveness of the intervention was measured. Expectancy did not correlate with outcome and was not analyzed further. Multivariate analysis of variance (MANOVA) showed that whereas relaxation therapy provided significant reduction of anxiety on the self-report measure and the movement measure, the nursing intervention of therapeutic touch resulted in significant reductions of reported anxiety. The control group showed small but nonsignificant effects. Results suggests that both relaxation and therapeutic touch are effective palliatives to experienced anxiety. Implications for nursing theory are discussed.

Adult↗

PubMed1993

MVLN cells: a bioluminescent MCE-7-derived cell line to study the modulation of estrogenic activity.

We previously established a stable expression system in MCF-7 cells for the detection of (anti)estrogenic activity by assaying the reporter enzyme activity of firefly luciferase. In this cell line (called MVLN), the bioluminescent response can be measured either in the cellular homogenate, or in intact living cells. Here we present various potential experimental uses of this cellular model. First, we used this cell line to screen natural or synthetic molecules classified as full or partial (anti)estrogens and observed that their behavior towards our model was identical to that expected. Moreover, the bioluminescent response was in agreement with the natural responses like cellular proliferation or stimulation of the progesterone receptor. We then demonstrated the inhibitory effects of retinoic acid and 1,25 dihydroxyvitamin D3, two molecules which do not compete with estradiol for its receptor. We thus deduced that with this cell line an "antiestrogenic" effect which occurred at any step of the estrogenic action, might be detected. Finally, we showed that detection of luciferase activity in intact living cells was particularly helpful for investigating the evolution of estrogenic activity. For instance, we observed that long-term treatment of MVLN cells with an antiestrogen irreversibly decreased the bioluminescent response by more than 90%. This phenomenon affected all cells equally and could not be reversed, even by long-term estradiol treatment. We therefore conclude that this chimeric response faithfully reflects estrogenic action in the cell and can be used to develop different aspects of the endocrine research.

Calcitriol↗

PubMed1992

Study of an antiestrogenic effect of retinoic acid in MCF-7 cells.

We recently developed the cellular model MVLN-15 in which estrogenic action can be detected by bioluminescence. Using this cellular model, we characterized the inhibitory effect of retinoic acid on the estrogen-dependent induction of luciferase transcription. We present evidence that i) the inhibitory effect of retinoic acid is not due to a simple competition between retinoic acid and estradiol for binding to the estrogen receptor, ii) a DNA sequence restricted to an estrogen-responsive element (ERE) was sufficient for the antiestrogenic effect of retinoic acid, and iii) retinoic acid does not act via a cryptic AP-1 binding site associated with this ERE. Therefore, we conclude that the antiestrogenic effect of retinoic acid is due to an inhibition of estrogen receptor activity, for example by altering the amount of estrogen receptor protein bound to the ERE or affecting the transcriptional efficiency of this complex.

Breast Neoplasms↗

PubMed1992

[Bioluminescence and creation of chimeric cellular models].

The response of a chimeric cellular model to a hormone has to be more specific, more sensitive, more easily measurable and quantifiable than the natural response. Hence, it can be used either in fundamental and applied studies, for example to analyse hormonal interactions, or to screen new hormone molecules. In the field of steroid hormones, we took benefit from the advantages of the firefly luciferase as receptor gene (simplicity and rapidity of the measurement, high sensitivity, handiness ...) to construct such chimaeric models. Such cell lines were obtained by developing another original use of this reporter gene based on an easy detection of the luciferase activity in intact living cells and using a photon counting camera. This was successfully adapted to obtain and to detect stable transfected cells.

Cell Line↗

PubMed1992

[Using examples of firefly luciferase gene for the study of biological activities (estrogens, retinoids, phorbol esters)].

Some possible applications of chimeric cellular models, specifically responding to an effector through firefly luciferase induction are presented with the help of examples in relation with the biological activity of estradiol or retinoic acid, or phorbol ester. A comparison of experiments on either chimeric or natural responses shows that: i) the responses of both type of cellular models are effector concentration-dependent; ii) these concentrations are in the same order of magnitude; partial agonist compounds and antagonist compounds; iv) potencies (EC50) of test-compounds are similarly classified. Moreover we show that a chimeric cellular model allows the observation of interactions between hormone or effector pathways: it allows readily performed kinetic studies and long-term experiments in intact cells that permit to investigate the effect of a given effector, its reversibility and time-dependent action. Therefore, various steps of a cellular signalling pathway involved in the action of an effector may be observed with such a valuable tool.

Cell Line↗

PubMed1991

[Retinoic acid has an antiestrogenic effect on different regulated estrogen genes in different cellular types].

In this paper, we confirmed that retinoic acid is an antiestrogenic compound with respect to different chimaeric estrogenic responses and with respect to different cellular types. This was shown by transient transfection of MCF-7 cells with plasmids driving the chloramphenicol acetyl-transferase gene via different estrogenic regulatory part (pS2) and the first promotor of the progesterone receptor gene (PR1); an identical conclusion was obtained in HeLa cells by cotransfecting a plasmid expressing the estrogen receptor. In addition, the inhibitory effect of retinoic acid was not observed for genes regulated by the progesterone receptor and the glucocorticoid receptor. As the antiestrogenic effect of retinoic acid was increased by cotransfecting acid receptor(s) RAR alpha, beta, gamma, we concluded that RAR(s) is(are) involved in the antiestrogenic effect of retinoic acid.

Estrogen Antagonists↗

PubMed1991

[Antiestrogenic effect of retinoic acid in MCF-7 cells].

We recently developed the cellular model MVLN-15 in which an estrogenic action can be detected by bioluminescence. Using this cellular model, we characterized the inhibitory effect of retinoic acid on the estrogen-dependent induction of luciferase transcription. We present evidence that i) the inhibitory effect of retinoic acid was not due to a simple competition between retinoic acid and estradiol for the estrogen receptor binding site, ii) retinoic acid does not significantly modify the parameters of the estradiol binding to the estrogen receptor, iii) retinoic acid does not act at a post-transcriptional level of the estrogenic action, iv) a DNA sequence restricted to an estrogen responsive element (ERE) was sufficient to observe the antiestrogenic effect of retinoic acid, and v) retinoic acid does not act via a cryptic AP-1 binding site associated with this ERE. We concluded that the antiestrogenic effect of retinoic acid is due to an inhibition of estrogen receptor activity, for example by altering the amount of estrogen receptor protein bound to the ERE or affecting the transcriptional efficiency of this complex.

Estrogen Antagonists↗

PubMed1990

A new cellular model of response to estrogens: a bioluminescent test to characterize (anti) estrogen molecules.

With the aim of quickly and easily characterizing new estrogen or anti-estrogen molecules, we developed a cellular model in which estrogenic action can be detected by bioluminescence. This model is based on MCF-7 cells stably transfected with a receptor gene which allows expression of the firefly luciferase enzyme under control of the estrogen regulatory element of the Xenopus vitellogenin A2 gene. A stably transfected cell line (cultured for more than eight months without loss of the chimeric estrogenic response) was established by cotransfection of a neomycin resistance gene and cloning under selective pressure. Subcloning luminescent clones was accomplished by using a single-photon detecting camera. This cellular model allowed the study of an estrogenic activity either in whole-cell or in cell-free experiments by detection of the induced luciferase. Estradiol induced the luciferase activity in a dose-dependent manner at subnanomolar concentrations. The induced luciferase activity reached a maximum level as early as 24 hours after the cells were incubated with estradiol. The antiestrogen 4-hydroxy-tamoxifen inhibited the luciferase activity induced by estradiol. The cross-reactivity of ligands, such as dexamethasone, progesterone, testosterone, aldosterone, calcitriol, oxysterol and retinoic acid, were also studied, showing an estradiol specificity for a 24-hour incubation time.

Animals↗

PubMed1988

A model of antiglucocorticoid action for designing a potent glucocorticoid antagonist.

We have previously shown that the biological efficacy of an antiglucocorticoid is directly related to its affinity for the glucocorticoid receptor in whole cells at 37 degrees C. We have also shown that RU 486-receptor complexes differ from other antiglucocorticoid-receptor complexes in so far as their affinity is as high at 37 degrees C in whole cells as at 0 degree C in a cell-free system, whereas a decrease by a factor of 5-10 is observed with the other antagonists. The aim of the present paper was to evaluate the contributions of temperature and cellular integrity (or the biological events linked to temperature and cellular integrity) to the affinity of a steroid for its receptor for the purpose of determining the parameters favorable to high affinity, which is the prerequisite of a potent antagonist. We provide evidence showing that: (1) an increase in temperature has an unfavorable effect on the affinity of a glucocorticoid for its receptor (4-6-fold decrease between 0 and 37 degrees C), (2) RU 486, like an agonist, forms a complex with the cytosolic glucocorticoid receptor, which satisfies the criteria for an "activated" complex under "in vitro activating treatment", (3) these biological post-binding events (either agonistic or otherwise nature), which change the nature of the complexes, contribute to compensating for the negative effect of rising temperatures on their apparent dissociation constant. We conclude that potent antiglucocorticoids must have a chemical structure allowing them to induce biological post-binding events, such as receptor activation, but in an abortive form which thus effectively "traps" the receptor in a non-functional state.

Animals↗