Biomedical subjects

L E Chalifour

Publications and source records attributed to L E Chalifour.

At least 19 recordsLinked to original sources

PubMed2001

Cloning and characterization of a novel gene that is regulated by estrogen and is associated with mammary gland carcinogenesis.

Estrogens play a role in mammary gland function and are implicated in mammary carcinogenesis. We report the cloning of a novel gene [steroid-sensitive gene 1 (SSG1)] that is regulated by E(2) in the rat uterus and mammary gland. The full-length SSG1 complementary DNA has an open reading frame of 1158 nucleotides encoding a putative protein of 385 amino acids. A SSG1-specific antibody recognizes a 40-kDa protein localized to myoepithelial cells of normal mammary tissue and to endothelial cells of 7,12-dimethylbenz(a)antracene-induced mammary tumors. Treatment of rats with E(2) at 1.2 or 2.4 microg/kg.day for 21 days increases SSG1 protein levels in mammary tissue by 16-fold compared with controls. Removal of E(2) after a 14-day treatment decreases SSG1 protein levels 6-fold and 3-fold at 120 and 144 h, respectively. Treatment of rats with the estrogen antagonists tamoxifen or ICI 182,780 did not affect SSG1 protein levels compared with controls. SSG1 protein levels in 7,12-dimethylbenz(a)antracene-induced rat mammary tumors were 23-fold greater than SSG1 levels in resting mammary tissue, and 8-fold higher than protein levels expressed in lactating mammary glands. We propose that SSG1 plays a role in estrogen functions, and its overexpression is correlated with mammary carcinogenesis.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed2001

Steroid-sensitive gene-1 is an androgen-regulated gene expressed in prostatic smooth muscle cells in vivo.

Steroid-sensitive gene-1 (SSG1) is a novel gene we cloned, found regulated by 17beta-estradiol in the rat uterus and mammary gland, and over-expressed in 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors. We show here that SSG1 mRNA and protein expression are regulated by androgens in the rat ventral prostate. Increases in SSG1 mRNA levels were detected by Northern blotting after 24 h and reached a 27-fold peak 96 h following castration, relative to SSG1 mRNA expression in sham-operated rats. Dihydrotestosterone or testosterone supplementation of castrated rats prevented this rise in SSG1 mRNA. In contrast with SSG1 mRNA expression, SSG1 protein was decreased 16-fold 2 weeks following castration but was at control levels in the prostates of castrated rats receiving dihydrotestosterone or testosterone. Although SSG1 is regulated by androgens in vivo, treatment of LnCap cells with dihydrotestosterone, cyproterone acetate or flutamide did not result in the regulation of SSG1 protein levels in vitro. Immunofluorescence studies show that SSG1 is mainly expressed in prostatic smooth muscle cells. These results indicate that SSG1 is an androgen-regulated gene that is expressed in the smooth muscle component of the rat ventral prostate in vivo.

Amino Acid Sequence↗

PubMed2001

Diminished molecular response to doxorubicin and loss of cardioprotective effect of dexrazoxane in Egr-1 deficient female mice.

Doxorubicin (DOX) and VP16 are DNA topoisomerase II inhibitors yet only DOX induces an irreversible cardiotoxicity, likely through DOX-induced oxidative stress. Egr-1 is overexpressed after many stimuli that increase oxidative stress in vitro and after DOX-injection into adult mice in vivo. To investigate Egr-1 function in the heart, we compared the molecular and histological responses of wild type (+/+) and Egr-1 deficient (-/-) female mice to saline, DOX, VP16, the cardioprotectant dexrazoxane (DZR), or DOX+DZR injection. DOX, and to a lesser extent VP16, induced characteristic increases in cardiac muscle and non-muscle genes typical of cardiac damage in +/+ mice, whereas only beta-MHC and Sp1 were increased in -/- mice. DZR-alone treated +/+ mice showed increased cardiomyocyte transnuclear width without a change to the heart to body weight (HW/BW) ratio. However, DZR-alone treated -/- mice had an increased HW/BW, increased cardiomyocyte transnuclear width, and gene expression changes similar to DOX-injected +/+ mice. DZR pre-injection alleviated DOX-induced gene changes in +/+ mice; in DZR+DOX injected -/- mice the increases in cardiac and non-muscle gene expression were equal to, or exceeded that, detected after DOX-alone or DZR-alone injections. We conclude that Egr-1 is required for DOX-induced molecular changes and for DZR-mediated cardioprotection.

Animals↗

PubMed2000

Altered molecular response to adrenoreceptor-induced cardiac hypertrophy in Egr-1-deficient mice.

Unmanipulated early growth response-1 (Egr-1)-deficient -/- mice have similar heart-to-body weight ratios but express lower amounts of atrial natriuretic factor (ANF), beta-myosin heavy chain (beta-MHC), skeletal actin, NGF1-A binding protein (NAB)-2, Sp1, c-fos, c-jun, GATA-4, and Nkx2.5 than +/+ or +/- mice. alpha-MHC, tubulin, and NAB-1 expression was similar. Isoproterenol (Iso) and phenylephrine (PE) infusion into +/+ and -/- mice increased heart weight, ANF, beta-MHC, skeletal actin, Sp1, NAB-2, c-fos, and c-jun expression, but induction in -/- mice was lower. Only Iso + PE-treated +/+ mice showed induction of NAB-1, GATA-4, and Nkx2.5. Foci of fibrosis were found in Iso + PE-treated -/- and +/+ mice. Surprisingly, vehicle-treated -/- mice displayed fibrosis and increased Sp1, skeletal actin, Nkx2.5, and GATA-4 expression without hypertrophy. Minipump removal caused the agonist-treated hearts and gene expression to regress to control or near-control levels. Thus Egr-1 deficiency caused a blunted catecholamine-induced hypertrophy response and increased sensitivity to stress.

Actins↗

PubMed1999

Expression of immediate early genes, GATA-4, and Nkx-2.5 in adrenergic-induced cardiac hypertrophy and during regression in adult mice.

Adrenoreceptor agonists induce a hypertrophic phenotype in vitro and in vivo. To investigate the molecular remodeling in chronic cardiac hypertrophy we infused adult male mice with vehicle. isoproterenol, phenylephrine or both agonists for 3, 7 or 14 days. All drugs increased cardiac mass. After minipump removal cardiac mass regressed to control levels within 7 days after PE and ISO treatment whereas ISO + PE treated hearts were incompletely regressed. ANF and beta-MHC, but not alpha-MHC, expression were increased by agonists at all time points. GATA-4, Nkx-2.5, Egr-1, c-jun and c-fos expression were increased after 3, 7 and 14 days of treatment. Expression was greatest after ISO+PE> >ISO>PE>vehicle infusion suggesting a synergistic effect of adrenoreceptor stimulation and indicating a greater effect of beta- than alpha-adrenergic action in vivo. After PE or ISO drug withdrawal the HW/BW was normal and Egr-1, c-jun, c-fos and GATA-4, but not Nkx2.5, expression dropped to control levels. HW/BW regression was incomplete after ISO+PE and elevated levels of Egr-1, c-jun and Nkx2.5 expression remained. A hydralazine-mediated reduction in blood pressure had no effect on the agonist-induced cardiac hypertrophy or gene expression. In conclusion, we found that continued agonist stimulation, and not blood pressure. is responsible for the maintained increase in gene expression. Further, we found the decrease in gene expression in the regression after drug withdrawal was gene specific.

Animals↗

PubMed1999

TAFII250, Egr-1, and D-type cyclin expression in mice and neonatal rat cardiomyocytes treated with doxorubicin.

Differential display identified that gene fragment HA220 homologous to the transcriptional activator factor II 250 (TAFII250) gene, or CCG1, was increased in hypertrophied rodent heart. To determine whether TAFII250 gene expression is modified after cardiac damage, we measured TAFII250 expression in vivo in mouse hearts after injection of the cardiotoxic agent doxorubicin (DXR) and in vitro in DXR-treated isolated rat neonatal cardiomyocytes. In vivo atrial natriuretic factor (ANF), beta-myosin heavy chain (beta-MHC), Egr-1, and TAFII250 expression increased with dose and time after a single DXR injection, but only ANF and beta-MHC expression were increased after multiple injections. After DXR treatment of neonatal cardiomyocytes we found decreased ANF, alpha-MHC, Egr-1, and TAFII250 expression. Expression of the TAFII250-regulated genes, the D-type cyclins, was increased after a single injection in adult mice and was decreased in DXR-treated cardiomyocytes. Thus expression of Erg-1, TAFII250, and the D-type cyclins is modulated after cardiotoxic damage in adult and neonatal heart.

Amino Acid Sequence↗

PubMed1996

Embryo infiltration by maternal macrophages is associated with selective expression of proto-oncogenes in a murine model of spontaneous abortion.

The causes and precise mechanisms leading to early embryo loss in mammals remain largely unknown, especially from a molecular point of view. Using the CBA/J x DBA/2 murine model of early spontaneous embryo loss (25-30% embryo loss), we have previously demonstrated the involvement of infiltrating activated macrophages and their cytolytic products such as nitric oxide and tumor necrosis factor alpha (TNF alpha) in the etiology of early embryo loss. On the other hand, far fewer of the CBA/J x Balb/c conceptuses (5-10% embryo loss) displayed significant cellular infiltration and nitric oxide and TNF alpha. Having used probes for cellular activation markers, we now present evidence indicating that significantly increased expression of AP-1 family members, Ha-ras, Ki-ras, v-erbA, v-raf, v-abl, and c-myc was present in 24.4% of the CBA/J x DBA/2 embryonic units that also harbored significant Mac-1, F4/80, and class II major histocompatibility complex (MHC) molecule cellular infiltration. In contrast, only 7% of CBA/J x Balb/c conceptuses displayed increased proto-oncogene expression and increased cellular infiltration. Therefore, macrophage infiltration, cellular activation as identified by the increased expression of proto-oncogenes, and the production of cytotoxic macrophage products are closely linked to early embryo loss. These data add to the evidence that activated maternal macrophages may be directly responsible for spontaneous pregnancy failure.

Abortion, Spontaneous↗

PubMed1996

Analysis of gene expression during experimental uveitis in the rabbit.

OBJECTIVE: To analyse the pattern of gene expression at the messenger RNA level during ocular inflammation in the rabbit. DESIGN: A gene screen assay was used to quantify specific binding over background (expression index) of various activation markers and cytokines in rabbit iris-ciliary body obtained during active experimental uveitis induced by injection of porcine lens protein (two animals) and in a control group (two animals). OUTCOME MEASURES: Expression indices corresponding to the activation markers and cytokines assayed. RESULTS: Compared with the control eyes, analysis of triplicate samples from the inflamed eyes showed a significantly higher expression index corresponding to the proto-oncogenes c-fos, c-jun and Ha-ras, interleukin-2 and heat shock protein Hsp27 and a significantly lower index corresponding to transforming growth factor-beta (TGF-beta) (p < 0.05). CONCLUSIONS: Active experimental lens-induced uveitis is associated with a significant rise in the gene expression of cellular activation factors and a decrease in an immunoprotective factor (TGF-beta) in the iris and ciliary body of the rabbit.

Animals↗

PubMed1995

Molecular remodelling in hypertrophied hearts from polyomavirus large T-antigen transgenic mice.

Polyomavirus large T-antigen transgenic mice develop cardiac hypertrophy characterized by an increase in atrial natriuretic factor and beta-myosin heavy chain isoform expression. The aim of this study was to examine changes in proto-oncogene expression in hypertrophied hearts from the transgenic mice. Expression of early growth response-1 (Egr-1) mRNA was detected in hearts from all 15 transgenic mice, but was not detectable in 13 control mice. Reverse transcriptase-polymerase chain reaction experiments using Egr-1-specific primers confirmed the increase in Egr-1 mRNA in enlarged hearts from the transgenic mice. Expression of c-jun, junD and Ha-ras mRNAs was increased in the transgenic hearts 3, 17 and 2.8-fold respectively. Western blots showed an increase in c-myc, c-jun and ras protein in hypertrophied transgenic hearts. Immunofluorescence analyses confirmed an increase in Egr-1 and c-jun protein in transgenic cardiomyocytes. Proliferating cell nuclear antigen, Ki-ras and HSP 90 mRNAs were decreased 22, 2.7 and 3-fold, respectively in the transgenic hearts. Not altered in most hypertrophied hearts was expression of c-fos, junB, p53, c-neu, c-myc, HSP70, HSP27, TGF-beta or IGF 1 mRNAs. Proto-oncogene and growth factor gene expression in hypertrophy induced by PVLT expression is modulated with some proto-oncogenes increased and others decreased in expression.

Animals↗

PubMed1995

Differential effects of cysteamine on heat shock protein induction and cytoplasmic granulation in astrocytes and glioma cells.

The sulfhydryl agent, cysteamine (CSH), promotes the accumulation of autofluorescent, peroxidase-positive cytoplasmic granules in cultured astroglia akin to those which naturally accumulate in astrocytes of the aging periventricular brain. Both in vitro and in situ, CSH rapidly induces various heat shock proteins (HSP) in astrocytes long before granulation occurs. In the present study, we determined that CSH treatment resulted in an increase in HSP 27, HSP 90 and heme oxygenase (HO-1) at both the protein and mRNA level. We also showed that C6 glioma cells, unlike primary astrocytes, constitutively express HSP 27, HSP 90 and HO-1 at low levels. Moreover, CSH is incapable of eliciting further HSP expression or inducing granulation in the glioma cells. Our results support the hypothesis that the biogenesis of redox-active astrocytic inclusions in CSH-treated glial cultures and in the aging periventricular brain is dependent on an antecedent cellular stress response.

Animals↗

PubMed1995

A transgenic mouse line harboring a smooth muscle alpha-actin promoter polyomavirus middle T antigen transgene develops an epithelial hyperplasia in the rectum and distal stomach.

BACKGROUND: Transgenic mice are the product of the microinjection of foreign DNA directly into the pronuclei of a one-cell embryo. The foreign DNA can cause insertional inactivation or activation of the flanking genetic locus. EXPERIMENTAL DESIGN: We isolated five lines of transgenic mice harboring the chicken alpha-actin vascular smooth muscle enhancer/promoter linked to the polyomavirus middle T antigen using a standard microinjection protocol. The expression of the transgene was assessed in RNA prepared from affected and nonaffected tissue by RNase protection and reverse transcriptase-polymerase chain reaction analyses. Cell morphology was determined in stained sections from fixed tissues. RESULTS: In this article, we document the development of epithelial hyperplasia in the rectum and distal stomach together with female infertility in a single line of transgenic mice harboring the transgene. We were unable to demonstrate the expression of the transgene in any tissue examined, regardless of the degree of hyperplasia. The phenotype was present in the heterozygous state in both males and females. CONCLUSIONS: In the absence of the expression of the transgene, we conclude that the insertion of the transgene may have caused the epithelial hyperplasia directly or may have contributed to a condition that promotes hyperplasia. The transgene may have activated a dominant-acting neighboring gene.

Actins↗

PubMed1994

A method for analysis of gene expression patterns.

mRNA can be copied into cDNA with the use of reverse transcriptase so that the relative abundance of individual mRNAs is reflected in the cDNA product. With further manipulation a replica of the mRNA expression pattern can be duplicated into a radioactive double-stranded DNA probe. DNA from a series of genes inserted into plasmids can be fixed to a membrane using a slot blot manifold and probed with the RNA-derived DNA probe. The intensity of the hybridization signal for a given gene is a result of its relative abundance in the RNA-derived DNA probe. Quantitation can be achieved through the use of housekeeping genes as baseline monitors. Inclusion of vector sequences can negate any spurious hybridization to vector rather than insert sequences. We have successfully used this method to obtain gene expression patterns for RNA isolated from diverse sources including rodent tissues, various cell lines, and Drosophila and Caenorhabditis elegans samples. Northern blots have verified the results obtained. The pattern of expression of many genes can be determined from as little as 10 micrograms of total RNA, making this method ideally suited for studies in which RNA is rare or in short supply.

Base Sequence↗

PubMed1993

Immortal cell lines isolated from heart differentiate to an endothelial cell lineage in the presence of retinoic acid.

Previously, we isolated a single line of transgenic mice which develop an enlarged heart due to the expression of the immortalizing gene, polyomavirus large T antigen. Immortal cell lines were isolated from adult transgenic but not from nontransgenic hearts. All of the 24 cell lines expressed vimentin and fibronectin but not desmin or myosin heavy chain. We conclude that the cell lines are of non-muscle origin. Six cell lines were chosen for further study. All six cell lines demonstrate profound morphological and biochemical effects when incubated with 10(-4) M to 10(-7) M retinoic acid. The retinoic acid-treated cell lines showed arrested cellular proliferation and aligned to form rows and vesicle-like structures. Cycloheximide inhibited these retinoic acid-induced changes, indicating a need for continued protein synthesis. Retinoic acid-treated, but not untreated, cells lost expression of vimentin and fibronectin, gained the ability to incorporate acetylated low density lipoprotein, and expressed Factor VIII-related antigen. Retinoic acid did not induce expression of desmin or myosin heavy chain. Incubation of the cell lines with transforming growth factor beta 1, dimethyl sulfoxide, or phorbol esters had no biochemical or morphological effect. We conclude that these cell lines differentiate to an endothelial lineage in the presence of retinoic acid.

Animals↗

PubMed1992

Testicular adenoma and seminal vesicle engorgement in polyomavirus large-T antigen transgenic mice.

Six lines of transgenic mice harboring the cDNA for polyomavirus large-T antigen (PVLT) linked to the mouse metallothionein-1 promoter were isolated. The transgene was expressed in testes in all lines isolated and in testes and seminal vesicles in two lines. Three lines developed enlarged testes and seminal vesicles. Development of the phenotype was divided into three stages separable by age and pathology. In stage 1, birth to 6 mo, PVLT was expressed in testes but no pathology was noted; in stage 2, 6-10 mo, PVLT was expressed solely in testes and not in seminal vesicles, yet the seminal vesicles were enlarged; and in stage 3, 10 mo and older, both testes and seminal vesicles expressed PVLT and both were enlarged. Testes were up to sevenfold heavier and increased up to fourfold to fivefold in each dimension. Seminal vesicles were enlarged up to 20-fold as the result of an accumulation of seminal vesicle fluid. In addition to the four major proteins of seminal vesicle fluid, extra proteins, initially found in stage 2, were increased in stage 3 seminal vesicle fluid. The Leydig cell was the dominant cell type in affected testes; there were few or no normal Sertoli cells or seminiferous tubules remaining by stage 3. The Leydig cells were physiologically active, as indicated by a 8.5-fold higher testosterone level in sera from stage 3 affected mice compared with sera from age-matched normal males. PVLT was present in the nuclei of the Leydig cells and was able to confer an immortal phenotype in vitro. Formation of the Leydig cell adenoma was dependent on PVLT expression, but since PVLT expression occurred much earlier than did pathology, additional secondary factors must determine the delay in phenotype development.

Adenoma↗

PubMed1990

Polyomavirus large T-antigen expression in heart of transgenic mice causes cardiomyopathy.

One of the early genes of polyomavirus, large T-antigen (PVLT), has been classified in vitro as an immortalizing gene. In order to determine the ability of PVLT to cause the formation of hyperplasia or tumors in vivo, we generated transgenic mice harboring the cDNA for PVLT linked to the heavy-metal responsive metallothionein-1 promoter (MT). The transgene was primarily expressed in testes and seminal vesicles, but expression was also detected in heart of a single transgenic line. The expression of the transgene in the heart of MT-PVLT line 8 mice was correlated with cardiomyopathy and atrial thrombus formation leading to premature death at approximately 160 days due to cardiac failure. The heart of affected animals was from 1.5 to 5.2 fold greater in weight and 2 fold greater in dimensions than normal nontransgenic mice. Affected hearts fell short of frank tumor phenotype and no macroscopic nor microscopic focal growth was found. Histologically the heart has a heterogenous cardiomyocyte population with markedly enlarged cells mixed with relatively normal cells. Both cell types express PVLT protein. The primary cell type affected is the cardiomyocyte however, as heart proportions are maintained, interstitial and non-myocyte cells must be affected either directly or indirectly. Expression of PVLT has upset normal strict control of cell growth in these hearts to result in a new model of congestive cardiomyopathy.

Animals↗

PubMed1989

Microinjection of metallothionein-oncomodulin DNA into fertilized mouse embryos is correlated with fetal lethality.

Oncomodulin (ONCO) is an oncodevelopmental protein expressed in placental and extraembryonic tissue and re-expressed in a wide variety of tumors. The metallothionein promoter (MT) is active in numerous adult tissues, in parietal and visceral extraembryonic endoderm, and developing liver. To study the function of oncomodulin we microinjected MT-ONCO DNA into one-cell embryos and examined tissues of fetal and adult mice. Analysis of implant sites from embryos, microinjected with MT-ONCO DNA then placed into pseudopregnant females, indicated a greater than three-fold increase in empty and necrotic implant sites relative to SV2NEO-microinjected embryos and a seven-fold rise relative to non-microinjected embryos. The striking feature of the lethality was the presence of a normal placenta but absence of fetal tissue. Few MT-ONCO DNA transgenic mice were isolated (3.5%) and none were able to express oncomodulin protein or RNA in any tissue examined, even after prolonged heavy metal stimulation of the MT promoter. Fetal mortality is best correlated with expression of oncomodulin causing an interruption of either cellular differentiation or organogenesis before day 9 in development.

Animals↗

PubMed1987

cis- and trans-acting sequences required for expression of simian virus 40 genes in mouse oocytes.

To determine the requirements for gene expression in mammalian germ cells, circular double-stranded simian virus 40 (SV40) DNA molecules containing deletions in sequences controlling transcription and replication were injected into the nucleus of mouse oocytes. Expression of large (T-Ag) and small (t-Ag) tumor antigens ("early gene products") required at least three GGGCGG boxes, but did not require either the origin of viral DNA replication (ori) or a TATA box. Expression of capsid antigen VP1 ("late gene products") required at least three GGGCGG boxes, sequences between nucleotides 197 and 273 in the 72-bp repeat region, and transactivation by T-Ag. These results are consistent with the requirements for expression of the same genes in differentiated mammalian cells. Surprisingly, however, the 72-bp repeats ("enhancer elements") that are required for expression of T-Ag and t-Ag genes in differentiated cells were not required in mouse oocytes. Similarly, expression of both the early and late genes was unaffected in mouse oocytes by the absence of either DNA replication or an intact ori sequence, components required for maximum expression of late genes in differentiated cells. Thus, mammalian oocytes effectively utilize promoters that are fully active in mammalian differentiated cells only when associated with either enhancer elements or DNA replication. Furthermore, requirements for expression of SV40 genes in mouse oocytes are distinctly different from those reported for Xenopus oocytes. This suggests that caution should be exercised when extrapolating conclusions drawn from experiments with amphibian germ cells to mammalian germ cells.

Animals↗

PubMed1986

Expression of simian virus 40 early and late genes in mouse oocytes and embryos.

Simian virus 40 (SV40) large- and small-tumor antigens (T-Ag, t-Ag) are normally synthesized early after infection of either permissive (monkey) or nonpermissive (mouse) fibroblasts, whereas an equivalent amount of viral coat protein (V-Ag) is observed late after infection of permissive cells and only after viral DNA replication has occurred. To determine whether or not expression of these genes is regulated in the same manner during early mammalian development, SV40 DNA was injected into the nuclei of mouse oocytes and one- and two-cell embryos. In oocytes, about three times more V-Ag was produced than T-Ag, and both were synthesized concomitantly in the same cells. Viral mRNA and proteins synthesized in oocytes comigrated during gel electrophoresis with the same products synthesized in SV40-infected monkey cells. Viral gene expression required circular DNA molecules injected into the nuclei of transcriptionally and translationally active cells. Injected DNA was stable and underwent conformational changes consistent with chromatin assembly. Oocytes did not replicate either polyomavirus or SV40 DNA. Thus, the temporal order of viral gene expression is circumvented in mouse germ cells, allowing these proteins to be expressed concurrently and in equivalent amounts with no requirement for DNA replication. However, in preimplantation embryos, neither T-Ag nor V-Ag was detected by immunoprecipitation although T-Ag synthesis was demonstrated as a specific requirement for SV40 DNA replication. Thus, viral gene expression in mouse embryos as early as the one-cell stage was reduced at least 500-fold relative to that in oocytes. Similarities between SV40 gene expression in mouse oocytes and that in Xenopus oocytes suggest that germ cells in higher animals share common regulatory mechanisms.

Animals↗