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Y de Launoit

Publications and source records attributed to Y de Launoit.

At least 55 records · Page 3Linked to original sources
PubMed1994

Molecular cloning and characterization of human ERM, a new member of the Ets family closely related to mouse PEA3 and ER81 transcription factors.

The ets-related transcription factors PEA3 and ER81 have recently been isolated and characterized in the mouse. They share 95% identity in a 85 amino acid (AA) domain termed the ETS domain which is responsible for DNA binding, and therefore they form an Ets family group. By screening a human testis cDNA library with a probe containing the mouse PEA3 ETS domain, we isolated a 2.2 kb clone containing a 510 AA open reading frame. Since the ETS domain, which is localized in the carboxy terminal region of the encoded protein, is 95% and 96% identical to that of PEA3 and ER81, respectively, we named this new member 'Ets Related Molecule PEA3-like' (ERM). Although the first 120 AA in the amino-terminal region of ERM share 47% identity with PEA3 and 66% with ER81, ERM contains a central region of approximately 35 AA not found in the two mouse proteins. Gel shift analysis indicates that the full-length ERM protein is able to bind specifically to an oligonucleotide containing the consensus nucleotide core sequence GGAA recognized by the Ets proteins. Moreover, in vitro translation of 83 AA of the ERM ETS domain led to the production of a truncated protein which also binds to DNA. Though differential expression is observed in primary tumors and normal lymphocytes do not express ERM, this gene is almost ubiquitously expressed in human normal tissues. ERM mRNA is highly expressed in brain as well as in placenta and, to a lesser degree, in lung, pancreas, and heart. Moreover, almost all human cell lines tested express it at varying levels. In mouse tissues, we showed that PEA3 and ER81 mRNAs display restricted expression, whereas ERM is almost ubiquitously expressed as observed for human tissues. Altogether these results indicate that ERM is clearly a new ets family member and not the human equivalent of PEA3 or ER81.

Amino Acid Sequence↗
PubMed1994

Computer assisted image analysis of bladder tumour nuclei for morphonuclear and ploidy assessment.

Morphonuclear analysis using a quantitative image analysis system has been demonstrated to be a potentially useful technique in the prognostic evaluation of bladder carcinoma. The integrated optical density parameter permits DNA content evaluation in addition to 15 other morphonuclear parameters. We assessed the reliability of morphonuclear analysis by image analysis and flow cytometry for 46 bladder carcinomas and 14 normal bladder specimens. Frozen sample material was obtained from endoscopic resection, radical cystectomy and from cadaveric donors. The grade and staging of the tumours according to the World Health Organization was as follows: 8 G1, 18 G2, 20 G3 and 28 T1, 7 T2, 7 T3, 4 T4. Quantitative image analysis was made on imprint smears stained by the Feulgen method. Simultaneously cell suspensions were obtained by mechanical dissociation and stained with propidium iodide for Flow cytometry analysis. There was a good agreement between quantitative image analysis and flow cytometry for DNA content measurement indicating that image analysis is a reliable method for the quantitation of DNA content (P = 0.001). Moreover we found a good correlation between five of the morphonuclear parameters: surface (P < 0.001), chromatin clumps distribution (P < 0.001), frequency of small (P < 0.001) to large chromatin clumps (P < 0.001) and the grade of bladder tumours. These results indicate that morphonuclear analysis may be a valuable method of quantitating DNA and morphonuclear parameters in a single analysis to provide information which may have some prognostic significance for patients with bladder carcinoma.

Cell Nucleus↗
PubMed1993

Predicted common structural features of DNA-binding domains from Ets, Myb and HMG transcription factors.

The Ets family of transcription factors shares a 85 amino acid domain, named the ETS domain, which appears responsible for their DNA binding activity. This domain did not show any clear similarity with already known DNA binding motifs. Hydrophobic Cluster Analysis (HCA), a sensitive method able to detect protein structural relationships even at low sequence identity, was chosen in order to compare the ETS domain with other conventional DNA binding motifs. HCA analysis combined with known three-dimensional NMR data, suggests that the ETS domain may be structurally related to the Myb DNA binding domain and possibly to the HMG one. Indeed, the ETS domain is likely to contain two helix-loop-helix motifs.

Amino Acid Sequence↗
PubMed1993

Molecular basis of congenital adrenal hyperplasia due to 3 beta-hydroxysteroid dehydrogenase deficiency.

Congenital adrenal hyperplasia is the most frequent cause of adrenal insufficiency and ambiguous genitalia in newborn children. In contrast to congenital adrenal hyperplasia due to 21-hydroxylase and 11 beta-hydroxylase deficiencies, which impair steroid formation in the adrenal cortex, exclusively, classical 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) deficiency affects steroid biosynthesis in the gonads as well as in the adrenals. The structures of the highly homologous type I and II 3 beta-HSD genes have been analyzed in three male pseudohermaphrodite 3 beta-HSD deficient patients from unrelated families in order to elucidate the molecular basis of classical 3 beta-HSD deficiency from patients exhibiting various degrees of severity of salt losing. The nucleotide sequence of DNA fragments generated by selective polymerase chain reaction amplification that span the four exons, the exon-intron boundaries, as well as the 5'-flanking region of each of the two 3 beta-HSD genes have been determined in the three male patients. The five point mutations characterized were all detected in the type II 3 beta-HSD gene, which is the gene predominantly expressed in the adrenals and gonads, while no mutation was detected in the type I 3 beta-HSD gene, predominantly expressed in the placenta and peripheral tissues. The two male patients suffering from severe salt-losing 3 beta-HSD deficiency are compound heterozygotes, one bearing the frame-shift mutation 186/insC/187 and the missense mutation Y253N, while the other bears the nonsense mutation W171X and the missense mutation E142K. The influence of the detected missense mutations on enzymatic activity was assessed by in vitro expression analysis of mutant recombinant enzymes generated by site-directed mutagenesis in heterologous mammalian cells. Recombinant mutant type II 3 beta-HSD enzymes carrying Y253N or E142K substitutions exhibit no detectable activity. On the other hand, the nonsalt-losing patient is homozygous for the missense mutation A245P. This mutation decreases 3 beta-HSD activity by approximately 90%. The present findings, describing the first missense mutations in the human type II 3 beta-HSD gene, provide unique information on the structure-activity relationships of the 3 beta-HSD superfamily. Moreover, the present findings provide a molecular explanation for the enzymatic heterogeneity responsible for the severe salt-losing form to the clinically inapparent salt-wasting form of classical 3 beta-HSD deficiency.(ABSTRACT TRUNCATED AT 400 WORDS)

3-Hydroxysteroid Dehydrogenases↗
PubMed1992

Expression of liver-specific member of the 3 beta-hydroxysteroid dehydrogenase family, an isoform possessing an almost exclusive 3-ketosteroid reductase activity.

We have recently characterized two types of rat 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) isoenzymes expressed in adrenals and gonads. In addition, we have cloned a third type of cDNA encoding a predicted type III 3 beta-HSD protein specifically expressed in the male rat liver which shares 80% similarity with the two other isoenzymes. Transient expression in human HeLa cells of the cDNAs reveals that the type III 3 beta-HSD protein does not display oxidative activity for the classical substrates of 3 beta-HSD, in contrast to the type I 3 beta-HSD isoenzyme. However, in the presence of NADH, type III isoenzyme, in common with the type I isoform, converts 5 alpha-androstane-3,17-dione (A-dione) and 5 alpha-dihydrotestosterone (DHT) to the corresponding 3 beta-hydroxysteroids. In fact, the type I and the type III isoenzymes have the same affinity for DHT with Km values of 5.05 and 6.16 microM, respectively. When NADPH is used as cofactor, the affinity for DHT of the type III isoform becomes higher than that of the type I isoform with Km values of 0.12 and 1.18 microM, respectively. The type III isoform is thus a 3-ketoreductase using NADPH as preferred cofactor which is responsible for the conversion of 3-keto-saturated steroids such as DHT and A-dione into less active steroids.

3-Hydroxysteroid Dehydrogenases↗
PubMed1992

Structure and tissue-specific expression of 3 beta-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase genes in human and rat classical and peripheral steroidogenic tissues.

The enzyme 3 beta-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase (3 beta-HSD) catalyzes the oxidation and isomerization of 5-ene-3 beta-hydroxypregnene and 5-ene-hydroxyandrostene steroid precursors into the corresponding 4-ene-ketosteroids necessary for the formation of all classes of steroid hormones. We have recently characterized two types of human 3 beta-HSD cDNA clones and the corresponding genes which encode deduced proteins of 371 and 372 amino acids, respectively, and share 93.5% homology. The human 3 beta-HSD genes containing 4 exons were assigned by in situ hybridization to the p11-p13 region of the short arm of chromosome 1. We have also recently elucidated the structure of three types of rat 3 beta-HSD cDNAs as well as that of one type of 3 beta-HSD from bovine and macaque ovary lambda gt11 cDNA libraries which all encode 372 amino acid proteins. The human type I 3 beta-HSD is the almost exclusive mRNA species detected in the placenta and skin, while the human type II is the predominant mRNA species in the adrenals, ovaries and testes. The predicted rat type I and type II 3 beta-HSD proteins expressed in adrenals, gonads and adipose tissue share 94% homology while they share 80% similarity with the liver-specific type III 3 beta-HSD. Transient expression of human type I and type II as well as rat type I and type II 3 beta-HSD cDNAs in HeLa human cervical carcinoma cells reveals that 3 beta-ol dehydrogenase and 5-ene-4-ene isomerase activities reside within a single protein and these cDNAs encode functional 3 beta-HSD proteins that are capable of converting 3 beta-hydroxy-5-ene-steroids into 3-keto-4-ene derivatives as well as the interconversion of 3 beta-hydroxy and 3-keto-5 alpha-androstane steroids. We have found that the rat type III mRNA species was below the detection limit in intact female liver while, following hypophysectomy, its accumulation increased to 55% of the levels measured in intact or HYPOX male rats, an increase which can be blocked by administration of ovine prolactin (oPRL). In addition, in female rats, treatment with oPRL for 10 days starting 15 days after HYPOX, markedly decreased ovarian 3 beta-HSD mRNA accumulation accompanied by a similar decrease in 3 beta-HSD activity and protein levels. Treatment with the gonadotropin hCG reversed the potent inhibitory effect of oPRL on these parameters and stimulated 3 beta-HSD mRNA levels in ovarian interstitial cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1992

Expression of human 17 beta-hydroxysteroid dehydrogenase in mammalian cells.

The insert of 1278 bp containing the entire coding region of cDNA encoding human 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) was inserted into a pHS1 vector and expressed in HeLA human cervical carcinoma cells and COS-1 monkey kidney tumor cells. Western blot analysis indicated that the expressed protein migrates at the same position as the purified enzyme and is recognized by the antibody raised against purified human placental 17 beta-HSD. The expressed enzyme efficiently catalyzes the interconversion of estrone and estradiol while dehydroepiandrosterone and 5-androstene-3 beta,17 beta-diol are interconverted at a lower rate. The present data suggest the existence of two 17 beta-HSDs.

17-Hydroxysteroid Dehydrogenases↗
PubMed1992

Androgenic 17 beta-hydroxysteroid dehydrogenase activity of expressed rat type I 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase.

Transient expression in nonsteroidogenic mammalian cells of the rat wild type I and type II 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD) cDNAs shows that the encoded proteins, in addition to being able to catalyze the oxidation and isomerization of delta 5-3 beta-hydroxysteroid precursors into the corresponding delta 4-3-ketosteroids, interconvert 5 alpha-dihydrotestosterone (DHT) and 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol). When homogenate from cells transfected with a plasmid vector containing type I 3 beta-HSD is incubated in the presence of DHT using NAD+ as cofactor, a somewhat unexpected metabolite is formed, namely 5 alpha-androstanedione (A-dione), thus indicating an intrinsic androgenic 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activity of this 3 beta-HSD isoform. Although the relative Vmax of 17 beta-HSD activity is 14.9-fold lower than that of 3 beta-HSD activity, the Km value for the 17 beta-HSD activity of type I 3 beta-HSD is 7.97 microM, a value which is in the same range as the conversion of DHT into 3 beta-diol which shows a Km value of 4.02 microM. Interestingly, this 17 beta-HSD activity is highly predominant in unbroken cells in culture, thus supporting the physiological relevance of this "secondary" activity. Such 17 beta-HSD activity is inhibited by the classical substrates of 3 beta-HSD, namely pregnenolone (PREG), dehydroepiandrosterone (DHEA), delta 5-androstene-3 beta,17 beta-diol (delta 5-diol), 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol) and DHT, with IC50 values of 2.7, 1.0, 3.2, 6.2, and 6.3 microM, respectively. Although dual enzymatic activities have been previously reported for purified preparations of other steroidogenic enzymes, the present data demonstrate the multifunctional enzymatic activities associated with a recombinant oxidoreductase enzyme. In addition to its well known 3 beta-HSD activity, this enzyme possesses the ability to catalyze DHT into A-dione thus potentially controlling the level of the active androgen DHT in classical steroidogenic as well as peripheral intracrine tissues.

17-Hydroxysteroid Dehydrogenases↗
PubMed1992

Additive stimulatory action of glucocorticoids and androgens on basal and estrogen-repressed apolipoprotein-D messenger ribonucleic acid levels and secretion in human breast cancer cells.

Recent elucidation of the amino acid sequence of the progesterone-binding protein GCDFP-24, the major protein found in human breast gross cystic disease fluid, reveals that this glycoprotein corresponds to apolipoprotein-D (apo-D), a member of the alpha 2-microglobulin superfamily which binds small hydrophobic ligands. The present study describes the multiple hormonal control of apo-D mRNA levels, intracellular protein content, as well as secretion compared to cell proliferation in human ZR-75-1 breast cancer cells. In these cells, exposure to the synthetic glucocorticoid dexamethasone (DEX) markedly decreases basal as well as 17 beta-estradiol (E2)-induced cell proliferation while causing a maximal 10-fold stimulation of apo-D secretion in the presence or absence of E2. Incubation with 500 nM DEX or 10 nM dihydrotestosterone (DHT), alone and in combination, markedly increased apo-D mRNA/actin mRNA ratios by 16-, 22-, and 28-fold, respectively. Exposure to 1 nM E2 decreased the apo-D mRNA/actin mRNA ratio by 65%. In E2-treated cells, simultaneous exposure to DHT, DEX, and DHT plus DEX markedly increased the apo-D mRNA/actin mRNA ratios by 50-, 35-, and 105-fold, respectively. The stimulatory effect of DEX on intracellular apo-D content and secretion was also additive to that of the androgen DHT in the presence or absence of E2. The present study provides the first data describing the hormonal regulation of apo-D mRNA levels and intracellular protein content and demonstrates the effect of glucocorticoids alone as well as their interaction with androgens and estrogens on these parameters as well as on apo-D secretion. As shown in the present data, the effects of steroids on apo-D gene expression, intracellular apo-D protein content, and secretion are opposite their respective specific effects on cell proliferation in human ZR-75-1 breast cancer cells.

Actins↗
PubMed1992

Androgens and breast cancer.

We have recently demonstrated that physiological levels of androgens exert direct and potent inhibitory effects on the growth of human breast cancer ZR-75-1 cells in vivo in nude mice as well as in vitro under both basal and estrogen-stimulated conditions. The inhibitory effect of androgens has also been confirmed on the growth of dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma in the rat. Such observations are in close agreement with the clinical data showing that androgens and the androgenic compound medroxyprogesterone acetate (MPA) have beneficial effects in breast cancer in women comparable to other endocrine therapies, including tamoxifen. Although the inhibitory action of androgens on cell proliferation in estrogen-induced ZR-75-1 cells results, in part, from their suppressive effect on expression of the estrogen receptor, the androgens also exert a direct inhibitory effect independent of estrogens. Androgens cause a global slowing effect on the duration of the cell cycle. These observations support clinical data showing that androgenic compounds induce an objective remission after failure of antiestrogen therapy as well as those indicating that the antiproliferative action of androgens is additive to that of antiestrogens. We have also recently demonstrated in ZR-75-1 human breast cancer cells the antagonism between androgens and estrogens on the expression of GCDFP-15 and GCDFP-24 which are two major proteins secreted in human gross cystic disease fluid. The effects of androgens and estrogens as well as those of progestins and glucocorticoids on GCDFP-15 and GCDFP-24 mRNA levels and secretion are opposite to those induced by the same steroids on cell growth in ZR-75-1 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Androgens↗
PubMed1991

Characteristics of the biphasic action of androgens and of the potent antiproliferative effects of the new pure antiestrogen EM-139 on cell cycle kinetic parameters in LNCaP human prostatic cancer cells.

The most potent steroid in human prostatic carcinoma LNCaP cells, i.e., dihydrotestosterone (DHT), has a biphasic stimulatory effect on cell proliferation. At the maximal stimulatory concentration of 0.1 nM DHT, analysis of cell kinetic parameters shows a decrease of the G0-G1 fraction with a corresponding increase of the S and G2 + M fractions. In contrast, concentrations of 1 nM DHT or higher induce a return of cell proliferation to control levels, reflected by an increase in the G0-G1 fraction at the expense of the S and especially the G2 + M fractions. Continuous labeling for 144 h with the nucleotide analogue 5'-bromodeoxyuridine shows that the percentage of cycling LNCaP cells rises more than 90% after treatment with stimulatory concentrations of DHT, whereas in control cells as well as in cells treated with high concentrations of the androgen, this value remains below 50%. Although LNCaP cells do not contain detectable estrogen receptors, the new pure steroidal antiestrogen EM-139 not only reversed the stimulation of cell proliferation and cell kinetics induced by stimulatory doses of DHT but also inhibited basal cell proliferation.

Androgens↗
PubMed1991

Evidence for distinct dehydrogenase and isomerase sites within a single 3 beta-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase protein.

Complementary DNA encoding human 3 beta-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase (3 beta-HSD) has been expressed in transfected GH4C1 with use of the cytomegalovirus promoter. The activity of the expressed protein clearly shows that both dehydrogenase and isomerase enzymatic activities are present within a single protein. However, such findings do not indicate whether the two activities reside within one or two closely related catalytic sites. With use of [3H]-5-androstenedione, the intermediate compound in dehydroepiandrosterone (DHEA) transformation into 4-androstenedione by 3 beta-HSD, the present study shows that 4MA (N,N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxamide) and its analogues inhibit DHEA oxidation competitively while they exert a noncompetitive inhibition of the isomerization of 5-androstenedione to 4-androstenedione with an approximately 1000-fold higher Ki value. The present results thus strongly suggest that dehydrogenase and isomerase activities are present at separate sites on the 3 beta-HSD protein. In addition, using 5 alpha-dihydrotestosterone (DHT) and 5 alpha-androstane-3 beta, 17 beta-diol as substrates for dehydrogenase activity only, we have found that dehydrogenase activity is reversibly and competitively inhibited by 4MA. Such data suggest that the irreversible step in the transformation of DHEA to 4-androstenedione is due to a separate site possessing isomerase activity that converts the 5-ene-3-keto to a much more stable 4-ene-3-keto configuration.

Androgen Antagonists↗
PubMed1991

Characterization of the structure-activity relationships of rat types I and II 3 beta-hydroxysteroid dehydrogenase/delta 5 -delta 4 isomerase by site-directed mutagenesis and expression in HeLa cells.

The membrane-bound enzyme 3 beta-hydroxysteroid dehydrogenase/delta 5 -delta 4 isomerase (3 beta-HSD) catalyzes the conversion of delta 5 -3 beta-hydroxysteroid precursors into delta 4-ketosteroids, thus representing an essential step in the biosynthesis of all classes of hormonal steroids. We have recently characterized two types of cDNA clones encoding rat 3 beta-HSD proteins, the rat type I protein being much more active than type II. In order to characterize further the functional difference between these two 3 beta-HSD types, transient expression of type I and type II 3 beta-HSD cDNAs was performed in HeLa human cervical carcinoma cells. The present study demonstrates that the type I 3 beta-HSD protein has a relative specificity 64- and 46-fold higher than type II protein for pregnenolone (PREG) and dehydroepiandrosterone (DHEA) as substrates, respectively. The Km values of type I and type II enzymes were calculated at 0.74 and 14.3 microM, respectively, using PREG as substrate whereas the respective Km values were 0.68 and 12.9 microM when DHEA was used, thus showing that their different relative specificity results largely from a different affinity for substrates. Since the change of 4 amino acid residues in type II could prevent the formation of a putative membrane-spanning domain (MSD) predicted between amino acid residues 75 and 91, chimeric cDNAs containing either type I MSD in type II (II + MSD) or an absence of this MSD in type I (I-MSD) were constructed and transiently expressed. The addition of MSD intype II 3 beta-HSD markedly increased the affinity leading to Km values similar to those found in type I 3 beta-HSD, namely 0.36 and 0.40 microM for PREG and DHEA, respectively. II + MSD chimera thus encodes a protein having a relative specificity for PREG and DHEA of 58 and 73%, respectively, to that of native type I 3 beta-HSD. Moreover, removal of MSD in the type I protein (I-MSD chimera) decreased the relative specificity of type I 3 beta-HSD protein for PREG and DHEA to only 0.37 and 0.48%, with respective Km values of 11.7 and 11.0 microM, thus strongly indicating the functional importance of this putative MSD which is predicted in wild type rat type I as well as in macaque and human 3 beta-HSD proteins.

Amino Acid Sequence↗
PubMed1991

Stimulation of apolipoprotein D secretion by steroids coincides with inhibition of cell proliferation in human LNCaP prostate cancer cells.

Although steroid hormones are known to play a predominant role in the regulation of cell growth in hormone-sensitive cancers, their mechanisms of action, especially their interaction with growth factors and/or growth inhibitors, is poorly understood. We have recently observed that the effects of androgens and estrogens on the expression of the major protein found in human breast gross cystic disease fluid, protein-24, are opposite to their respective action on cell proliferation in human breast cancer cell lines. Somewhat surprisingly, the recent elucidation of the amino acid sequence of this progesterone binding protein reveals that this tumor marker is apolipoprotein D (apo D), a member of a superfamily of lipophilic ligand carrier proteins. The present study was designed to determine whether apo D is secreted by human prostate cancer cells and could thus be a new marker of steroid action in these cancer cells, and whether the sex steroid-induced stimulation of apo D secretion coincides with inhibition of cell proliferation. We took advantage of the biphasic pattern of the effect of steroids on the proliferation of the human prostate cancer LNCaP cell line, which offers the opportunity to discriminate between positive and negative steroid receptor-regulated cell growth processes. A 10-day exposure to low concentrations of dihydrotestosterone and testosterone caused a potent stimulation of LNCaP cell proliferation, whereas incubation with higher concentrations of these androgens led to a progressive decrease in cell proliferation towards basal levels. The biphasic action of androgens was also observed on apo D secretion, the effects on apo D secretion being inversely related to their action on LNCaP cell proliferation. Similar opposite biphasic effects were also observed with 9 other steroids, thus indicating that the stimulation of secretion of this new biochemical marker coincides with inhibition of cell proliferation in LNCaP human prostatic cancer cells.

Apolipoproteins↗
PubMed1991

Inhibition of cell cycle kinetics and proliferation by the androgen 5 alpha-dihydrotestosterone and antiestrogen N,n-butyl-N-methyl-11-[16' alpha-chloro-3',17 beta-dihydroxy-estra-1',3',5'-(10')triene-7' alpha-yl] undecanamide in human breast cancer ZR-75-1 cells.

We have investigated the effects of the pure antiestrogen EM-139 and the nonaromatizable androgen dihydrotestosterone (DHT) alone or in combination with estradiol (E2) on cell proliferation and cell kinetic parameters in human ZR-75-1 breast cancer cells. Following a 24- to 30-h exposure to E2, a decrease in the proportion of G0-G1 cells was observed, this effect being accompanied by the well-known stimulatory effect of the estrogen on cell proliferation at later time intervals. By contrast, DHT or EM-139 alone inhibited basal cell proliferation without a significant influence on cell cycle distribution. Moreover, pretreatment with DHT for 8 days, while decreasing ZR-75-1 cell number, did not cause a loss in E2 sensitivity. In fact, as early as after 24 h of E2 treatment, a decrease in the G0-G1 cell fraction accompanied by a corresponding increase of the S-phase was observed in both control and DHT-pretreated cells. When added concomitantly with E2, DHT or EM-139 inhibited the E2 stimulatory effect on cell proliferation, but only EM-139 significantly reversed the G0-G1 decrease induced by E2. Although DHT and EM-139 did not affect the distribution of ZR-75-1 cells between the different phases of the cell cycle, continuous labeling with 5'-bromodeoxyuridine showed that EM-139 and DHT had a global slowing effect on the duration of the cell cycle, thus explaining the potent inhibitory effect of these compounds on cell proliferation. The present data demonstrate that DHT and EM-139 are both potent inhibitors of the stimulatory effect on E2 on cell proliferation, their main action being related to a general increase in the duration of the cell cycle.

Breast Neoplasms↗
PubMed1991

Characterization of rat 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase cDNAs and differential tissue-specific expression of the corresponding mRNAs in steroidogenic and peripheral tissues.

The conversion of 3 beta-hydroxy-5-ene steroids by the enzyme complex 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) is an essential step in the biosynthesis of all classes of hormonal steroids. We report the characterization of two types of cDNA clones encoding rat 3 beta-HSD isolated from a rat ovary lambda gt11 cDNA library with a human 3 beta-HSD cDNA probe. Both type I and type II cDNAs encode proteins of 372 amino acids having 94% homology. Transient expression of the type I and the type II 3 beta-HSD cDNAs in HeLa human cervical carcinoma cells reveals that both proteins possess 3 beta-hydroxysteroid dehydrogenase as well as delta 5-delta 4 isomerase activities for both delta 5-pregnene and delta 5-androstene precursors, although the type I 3 beta-HSD protein is more active than the type II. RNA blot analysis using type I 3 beta-HSD cDNA identifies major mRNA transcripts of 1.7 kilobase in rat ovary, testis, and adrenal poly(A)+ RNA. RNase protection assay using type I- and type II-specific cRNA probes revealed the existence of the two corresponding mRNAs in male and female rat adrenals and gonads as well as in female adipose tissue while only type I mRNA is present in male and female kidney. Moreover, in situ hybridization performed using type-specific labeled 24-mer oligonucleotides confirms that type I is the major mRNA species in the ovary and further indicates that both mRNA species have a similar cellular distribution in the ovarian tissue with the highest level of expression found in corpora lutea. Immunoblot analysis using polyclonal antibodies raised against purified human placental 3 beta-HSD identified a single 42-kDa band in rat ovary, testis, and adrenal, which agrees with the calculated molecular masses of 41,911 and 42,150 daltons for the type I and II proteins, respectively. Determination of 3 beta-HSD enzymatic activity using [14C]pregnenolone and [14C]dehydroepiandrosterone as substrates shows that 3 beta-HSD activity is present not only in the gonads and adrenals of animals of both sexes, but also in many peripheral tissues including adipose tissue, mammary gland, kidney, liver, prostate, seminal vesicle, uterus, skin, brain, heart, thymus, pancreas, lung, and spleen. The present data indicate the existence of two mRNAs encoding rat 3 beta-HSD and their differential tissular distribution in both steroidogenic and peripheral tissues.(ABSTRACT TRUNCATED AT 400 WORDS)

Adipose Tissue↗
PubMed1991

Influence of fetal bovine serum and hormones on primary vs. long-term cultures of human breast cancers.

We investigated the influence of fetal bovine serum, complemented or otherwise with estradiol or insulin or both, on the proliferation of mammary cancer cells from long-term and primary cultures. The long-term culture corresponded to mouse MXT and MCF-7 cell lines whereas the primary culture corresponded to primitive breast cancers squashed onto histologic slides and maintained in cultures for between 12 and 48 h. Cell proliferation was evaluated by means of digital cell image analysis of Feulgen-stained nuclei. Our results show that the addition of estradiol and insulin slightly but nevertheless significantly increases the proportion of cells still living at Hour 48 of culture. Fetal bovine serum, necessary for the growth of MXT and MCF-7 mammary cells, was highly cytotoxic with respect to the primary cultures of the 20 breast cancers under study. We are now conducting new experiments using chemically defined media to study the influence of new antineoplastic compounds on primary cultures of breast cancers.

Animals↗