Biomedical subjects

Y de Launoit

Publications and source records attributed to Y de Launoit.

At least 73 records · Page 4Linked to original sources

PubMed1991

Structure and expression of a new complementary DNA encoding the almost exclusive 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase in human adrenals and gonads.

The 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta HSD) enzyme catalyzes the oxidation and isomerization of delta 5-3 beta-hydroxysteroid precursors into delta 4-ketosteroids, thus leading to the formation of all classes of steroid hormones. In addition, 3 beta HSD catalyzes the interconversion of 3 beta-hydroxy- and 3-keto-5 alpha-androstane steroids. Clinical observations in patients with 3 beta HSD deficiency as well as our recent data obtained by Southern blot analysis using a human placental 3 beta HSD cDNA (type I) as probe suggested the existence of multiple related 3 beta HSD isoenzymes. We now report the isolation and characterization of a second type of cDNA clone (arbitrarily designated type II) encoding 3 beta HSD after screening of a human adrenal lambda gt22A library. The nucleotide sequence of 1676 basepairs of human 3 beta HSD type II cDNA predicts a protein of 371 amino acids with a calculated molecular mass of 41,921 daltons, which displays 93.5% and 96.2% homology with human placental type I and rhesus macaque ovary 3 beta HSD deduced proteins, respectively. To characterize and compare the kinetic properties of the two isoenzymes, plasmids derived from pCMV and containing type I or type II 3 beta HSD full-length cDNA inserts were transiently expressed in HeLa human cervical carcinoma cells. In vitro incubation with NAD+ and 3H-labeled pregnenolone or dehydroepiandrosterone shows that the type I protein possesses a 3 beta HSD/delta 5-delta 4 isomerase activity higher than type II, with respective Km values of 0.24 vs. 1.2 microM for pregnenolone and 0.18 vs. 1.6 microM for dihydroepiandrosterone, while the specific activity of both types is equivalent. Moreover, incubation in the presence of NADH of homogenates from cells transfected with type I or type II 3 beta HSD indicates that dihydrotestosterone is converted into 5 alpha-androstane-3 beta, 17 beta-diol, with Km values of 0.26 and 2.7 microM, respectively. Ribonuclease protection assay using type I- and type II-specific cRNA probes revealed that type II transcripts are the almost exclusive 3 beta HSD mRNA species in the human adrenal gland, ovary, and testis, while type I transcripts correspond to the almost exclusive 3 beta HSD mRNA species in the placenta and skin and represent the predominantly expressed species in mammary gland tissue. The present data show for the first time that adrenals and gonads express a type of 3 beta HSD isoenzyme that is distinct from the type expressed in the placenta.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenal Glands↗

PubMed1990

Characterization of human 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase gene and its expression in mammalian cells.

Three beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD) catalyze the oxidative conversion of delta 5-3 beta-hydroxysteroids to the delta 4-3-keto configuration and is therefore essential for the biosynthesis of all classes of hormonal steroids, namely progesterone, glucocorticoids, mineralocorticoids, androgens, and estrogens. Using human 3 beta-HSD cDNA as probe, a human 3 beta-HSD gene was isolated from a lambda-EMBL3 library of leucocyte genomic DNA. A fragment of 3 beta-HSD genomic DNA was also obtained by amplification of genomic DNA using the polymerase chain reaction. The 3 beta-HSD gene contains a 5'-untranslated exon of 53 base pairs (bp) and three successive translated exons of 232, 165, and 1218 bp, respectively, separated by introns of 129, 3883, and 2162 bp. The transcription start site is situated 267 nucleotides upstream from the ATG initiating codon. DNA sequence analysis of the 5'-flanking region reveals the existence of a putative TATA box (ATAAA) situated 28 nucleotides upstream from the transcription start site while a putative CAAT binding sequence is located 57 nucleotides upstream from the TATA box. Expression of a cDNA insert containing the coding region of 3 beta-HSD in nonsteroidogenic cells shows that the gene encodes a single 42-kDa protein containing both 3 beta-hydroxysteroid dehydrogenase and delta 5-delta 4-isomerase activities. Moreover, all natural steroid substrates tested are transformed with comparable efficiency by the enzyme. In addition to its importance for studies of the regulation of expression of 3 beta-HSD in gonadal as well as peripheral tissues, knowledge of the structure of the human 3 beta-HSD gene should permit investigation of the molecular defects responsible for 3 beta-HSD deficiency, the second most common cause of adrenal hyperplasia in children.

3-Hydroxysteroid Dehydrogenases↗

PubMed1990

Characterization of human colorectal mucosa, polyps, and cancers by means of computerized morphonuclear image analyses.

Fifty-eight Feulgen-stained imprint smears from freshly resected colorectal tissue were analyzed by means of a SAMBA 200 cell image processor to establish a quantitative grading of their evolution from normal to malignant mucosa on the basis of 15 morphonuclear parameters relative to morphometry (nuclear size), densitometry (DNA content), and texture (chromatin pattern). The colorectal samples belonged to six groups: normal mucosa from patients without (Group 1) or with (Group 2) colorectal cancer, adenomas (Group 3), and cancers corresponding to the three stages of the Dukes' classification (Groups 4 to 6). Results indicated that analysis of the morphonuclear parameters computed on 250 to 300 nuclei/cases objectively and quantitatively showed the adenoma-cancer sequence. This need for only a small number of nuclei to assess a highly efficient analysis created a preoperative investigative tool using cytologic smears during endoscopy. The authors also made preliminary data banks for objective and reproducible grading of unknown cases by comparisons (discriminant analyses) with their contents. This approach must be validated prospectively on a large series of cases to furnish a system for colorectal malignancy diagnosis.

Cell Nucleus↗

PubMed1990

Influence of smear preparation and fixatives on the DNA ploidy and the morphonuclear features of the MXT mammary tumor and normal tissues in the mouse.

We compared cytomorphonuclear parameters--related to DNA histogram and chromatin distribution--on MXT mouse mammary tumor and murine normal cells from fresh squash smears or from deparaffinized tissue smears fixed in several fluids. We used the SAMBA 200 cell image processor with software allowing for the discrimination of parameters computed on Feulgen-stained nuclei. The spectrophotometric results--assessed by integrated optical density values--indicate that the nuclei from deparaffinized tissue fixed in Bouin's fluid are around 50% less stained than those fixed in formalin or ethanol-formalin-acetic acid (EFA). The fresh smears of nuclei fixed in formalin contain a less well-defined and more homogeneous chromatin than after Bouin's or EFA fixation. This has led to the conclusion that morphonuclear parameter comparisons performed on tissues differently processed or from different origins present severe limitations.

Adenocarcinoma↗

PubMed1990

Characterization of the morphonuclear features and DNA ploidy of prostatic disease.

The relationship between several morphonuclear parameters related to nuclear densitometry (DNA content and ploidy), and the chromatin pattern of Feulgen-stained nuclei is confronted to the histopathological grading of 39 prostatic samples obtained from surgical adenomatous or transurethral resection. We use a SAMBA 200 cell image processor with software allowing for the setting up of preliminary data banks that enable carrying out an objective and reproducible grading of unknown cases obtained from core biopsies of fine-needle aspirations. The present approach must be validated prospectively on a large series of cases in order to create an expert system for diagnosing such fine-needle aspirations from prostate.

Analysis of Variance↗

PubMed1990

Computerized morphonuclear cell image analyses of malignant disease in bladder tissues.

We analyzed the relationship between several morphonuclear parameters related to nuclear size, densitometry (deoxyribonucleic acid content and ploidy) and the chromatin pattern versus the histopathological grading of 46 bladder cancer samples graded according to the World Health Organization classification. We used a SAMBA 200 cell image processor with software allowing for the discrimination of 15 different parameters on Feulgen-stained imprint smears. In addition, we set up preliminary data banks that enable objective and reproducible grading of unknown cases. This approach must be validated in a large series of cases to create an expert system for bladder malignancy diagnosis.

Cell Division↗

PubMed1990

Characterization of the morphonuclear features and DNA ploidy of typical and atypical carcinoids and small cell carcinomas of the lung.

The authors analyzed several cytomorphonuclear parameters related to chromatin distribution and DNA ploidy in typical and atypical carcinoids and in small cell lung cancers. Nuclear measurements and analysis were performed with a SAMBA 200 (TITN, Grenoble, France) cell image processor with software allowing the discrimination of parameters computed on cytospin preparations of Feulgen-stained nuclei extracted from deparaffinized tumor tissues. The authors' results indicate a significant increase in DNA content--assessed by integrated optical density (IOD)--from typical carcinoids to small cell lung carcinomas, with atypical carcinoids showing an intermediate value. Parameters related to hyperchromatism (short and long run length and variance of optical density) also characterize the atypical carcinoids as being intermediate between typical carcinoids and small cell lung cancers. The systematic measurement of these cytomorphonuclear parameters seems to define an objective, reproducible "scale" of differentiation that helps to define the atypical carcinoid and may be of value in establishing cytologic criteria for differential diagnosis.

Carcinoid Tumor↗

PubMed1989

Correlation between nuclear cytomorphometric parameters and estrogen receptor levels in breast cancer.

The authors studied the relationships existing between various cytomorphonuclear parameters recorded on 25 primary breast cancers and their estrogen receptor (ER) content. Cell image analyses of Feulgen-stained imprint smears, allowing determination of morphologic, densitometric, as well as textural parameters, were assessed by using the SAMBA 200 system (TITN, France). The ER levels were measured by the conventional dextran-coated charcoal assay. The authors then divided the 25 cancers into three categories: (1) "ER-negative or poorly positive tumors," i.e., those having less than 50 fmol ER/mg protein; (2) "ER-positive tumors," i.e., those containing between 50 and 150 fmol ER/mg protein; and (3) "ER highly positive tumors," i.e., those having more than 150 fmol ER/mg protein. The authors' results show that ER-negative or poorly positive breast cancers possess cells with bigger nuclei and higher DNA content, related to higher proliferation index than ER-rich tumors. Furthermore, the chromatin pattern of cells from ER-negative or poorly positive breast cancers is significantly more condensed than the thinly textured chromatin of ER highly positive tumors. Cell image analysis of Feulgen-stained imprints is proposed as an additional tool for grading malignancy.

Breast Neoplasms↗

PubMed1989

Influence of pituitary grafts or prolactin administrations on the hormone sensitivity of ovarian hormone-independent mouse mammary MXT tumors.

The sensitivity of MXT mouse mammary tumors to ovarian hormones was assessed by the following parameters: growth in vivo; presence or absence of estrogen receptors and progesterone receptors; and histological differentiation. A spontaneous evolution from hormone sensitivity (HS) to hormone independence (HI) was observed when the tumors underwent monthly transplantation onto intact recipient mice, with the tumors fulfilling the criteria of HI tumors after the 12th transplantation. In contrast, the tumors recovered most of the criteria of hormone sensitivity when pituitary isografts were placed under the kidney capsules of HI tumor-bearing animals or when these animals received daily administrations of prolactin over several months. Sensitivity to 17 beta-estradiol, progesterone, or prolactin was further assessed by actinomycin binding on the nucleus and thymidine labeling index, both measured by autoradiography. These technical approaches revealed that 17 beta-estradiol and prolactin stimulated the thymidine labeling index of both HI (despite the lack of detectable estrogen receptors) and HS MXT tumors whereas progesterone influenced only that of HS cancers. The three hormones significantly stimulated [3H]actinomycin D binding within HS tumors but not within HI ones. However, such "HI" tumors were characterized by increased actinomycin binding and thymidine labeling index in comparison with HS neoplasms. Thus, all the data presently reported strongly suggest that prolactin is able to restore the hormone-sensitive phenotype within so-called MXT hormone-independent tumors.

Adenocarcinoma↗

PubMed1989

In vivo influence of androgens on the cell kinetics and chromatin pattern of the MXT mouse mammary tumor treated or not by aminoglutethimide.

We characterized the influence of androstenedione, 5-alpha-dihydrotestosterone and 17-beta-estradiol on the chromatin organization and the cell kinetics (distribution of the cells into the G0-G1, S and G2+ M fractions) of MXT mouse mammary tumors grafted onto animals that were left intact or that were oophorectomized and/or treated with aminoglutethimide. The cell kinetics and chromatin pattern were monitored by analyzing Feulgen-stained MXT imprint smears through a cell image processor, the SAMBA 200 system. We have also assayed the estrogen and progesterone receptors of these MXT tumors, which appeared to possess both of these biological markers. Our results show that ovariectomy or aminoglutethimide treatment slowed down the MXT tumor growth without any additive effect between them. Androstenedione exerted a stimulating influence on the cell kinetics, which were very similar to those observed after estradiol administration; the treatment of castrated animals with aminoglutethimide completely abolished this androstenedione-induced stimulating influence, however. This androgen lacked any apparent efficiency to transform cell nuclei from a state of castration-induced chromatin condensation into a thinly textured and decondensed state, as did estradiol. Dihydrotestosterone was able to activate the cell kinetics of MXT tumor grafted onto castrated animals as well as those of MXT neoplasms grafted on oophorectomized mice treated with aminoglutethimide. Dihydrotestosterone also possesses the property to transform condensed chromatin into decondensed chromatin. It thus appeared that androstenedione and dihydrotestosterone could activate MXT cell proliferation, as did estradiol, although it appeared that their mechanisms of action were quite distinct from each other.

Aminoglutethimide↗

PubMed1989

Influence of L-thyroxine, L-triiodothyronine, thyroid stimulating hormone, or estradiol on the cell kinetics of cultured mammary cancer cells.

We describe the in vitro influence of 3,5,3'-triiodo-L-thyronine (T3), L-thyroxine (T4), a thyroid-stimulating hormone (TSH), and/or estradiol (E2: chosen as the control of the methodology) on the cell kinetics (cell distribution in the S + G2 + M phases) of mouse MXT and human MCF-7 mammary cancer cells. Experiments were performed by means of a cell image processor, analyzing MCF-7 or MXT cells that had been grown on glass cover slips and whose nuclei had been stained by the Feulgen reaction, which is selective and quantitative (stoichiometric) with respect to DNA. We show that T3, T4, and TSH at 0.01 microM dramatically stimulate the cell kinetics of the MXT mouse and the MCF-7 human mammary cancer cell lines. Indeed, the three hormones bring about a significant transient increase in the S + G2 + M fraction as does E2. Furthermore, our data indicate that E2 and TSH are antagonistic with regards to MXT or MCF-7 cell kinetics.

Animals↗

PubMed1989

Inhibitory action of androstenedione on the proliferation and cell cycle kinetics of aromatase-free MXT and MCF-7 mammary tumour cell lines.

The effects of androstenedione (AD) on cell proliferation and kinetics have been measured in MXT mouse and MCF-7 human mammary cancer cell lines using SAMBA 200 cell image analysis of Feulgen-stained nuclei. At a concentration of 0.01 microM AD inhibited the proliferation of both cell lines whereas a higher dose (1 microM) was inhibitory on MCF-7 cell proliferation but stimulatory in MXT cells. It is unlikely that these effects are due to aromization of AD into oestrogen since (a) both cell lines were devoid of aromatase and (b) both cell lines were similarly affected by oestradiol (E2), being stimulated at low concentrations and inhibited at high doses. Furthermore, inhibition by AD seems to occur, at least in part, by blockade of the cell cycle whereas that by E2 appears to be cell cycle independent.

Androstenedione↗

PubMed1989

Influence of fetal calf serum in combination with pharmacological doses of progesterone or estradiol on proliferation and cell cycle kinetics of cultured mammary cancer cells.

We describe an original method to monitor clonal cell density (hyperplasia) and the cell cycle kinetics of neoplastic cells simultaneously. We thus characterize the in vitro influence of two different types of fetal calf serum (FCS), defined as FCS-S and FCS-I, on the progesterone- or estradiol-induced effect on proliferation and cell cycle kinetic parameters of the MXT mouse and MCF-7 human mammary cancer cell lines. The two sera were treated with dextran-coated charcoal. The FCS-S serum showed a stimulatory influence on MXT and MCF-7 growth, whereas FCS-I was devoid of any clear-cut influence. The cells were cultured on glass coverslips, placed in Petri dishes containing either a control or a hormone-added medium, fixed for histology and submitted to the Feulgen reaction, which allows selective and quantitative (stoichiometric) staining of DNA. Proliferation and cell cycle kinetics were analyzed on the same sample of cells by means of the SAMBA 200 cell image processor. Our results show that steroid-mediated effects were dramatically modulated according to the type of serum used. Furthermore, they also show that pharmacological doses of progesterone or estradiol decrease MXT cell growth by at least two different mechanisms: the first is related to cell cycle kinetics, i.e. an inhibition of the cells into the S phase, while the second remains unknown but seems to be cell cycle independent. High dose estradiol, but not progesterone, induced the same inhibitory influence on the MCF-7 cells.

Animals↗

PubMed1989

[Application of nuclear image analysis to diagnosis in oncology].

Cell image analysis applied to Feulgen stained cells allows description of each nucleus according to 15 parameters which measure DNA content, shape and size of the nucleus, as well as chromatin texture and distribution. We have studied the effects of hormones or chemotherapy on human and murine mammary cancer cell lines and on an in vivo murine mammary tumor. The method was then applied to different kinds of human cancers and proved to bring consistent results with anatomopathological grading (breast, colon, bladder), with estrogen receptor content (breast) and with molecular tumor marker (hybridization to the 16 or 18 papilloma virus in uterine cervix dysplasia).

Animals↗

PubMed1989

In vivo relationship between morphonuclear parameters and hormone sensitivity of the murine MXT mammary tumor.

The initially hormonosensitive (HS) MXT mouse mammary tumor spontaneously evolved to hormonoin-dependence (HI). Using a SAMBA 200 cell image processor, we compared the DNA content and the chromatin structure of HS and HI tumor cells squashed onto histologic slides; the nuclei were colored by the Feulgen reaction. We compared HI and HS nuclei by a discriminant analysis using the 15 parameters obtained on each nucleus. We show that the percentage of well-classified granulocytes (2n DNA content control) versus HS or HI nuclei exceeded 99. On the other hand, this percentage did not reach 70 when we compared HS and HI. The cell cycle analysis revealed that the percentage of cells in S and G2 + M phases were significantly higher in HI tumors than in the HS. Hence, HI and HS MXT tumor nuclei seem to be morphologically identical, but are significantly different if we refer to cell proliferation rates.

Animals↗

PubMed1989

Detection of human papillomaviruses 16-18 in cervical cells by molecular hybridization: relationship with morphonuclear cell image analyses.

Forty-one Feulgen-stained cervical imprint smears were analyzed by means of the SAMBA 200 cell image processor in order to quantitatively score human papillomavirus (HPV) 16-18-induced morphonuclear modifications as assessed by morphometric, densitometric, and textural parameters. Molecular hybridization technology using 16 and 18 type specific genetic probes made it possible to divide our series into three groups: Group 1, containing noninfected smears; Group 2, containing "suspicious", i.e., borderline positive, smears; and Group 3, those related to infected patients. Our results show that nuclei from infected smears are much more hyperchromatic and bigger than those arising from noninfected smears. This quantitative description of HPV 16-18-induced chromatin modifications enabled us to create preliminary data banks which could lead to an objective and reproducible grading of unknown cases. This approach is now being prospectively assessed on a large series of cases because the value of the current study is limited until the data bank is tested against unknown specimens with a broader spectrum of HPV infection.

Adult↗

PubMed1989

Relationship between computerized morphonuclear image analysis and histopathologic grading of breast cancer.

A pilot study analyzed the relationship between several morphonuclear parameters and the Bloom-Richardson score for 37 invasive, not-otherwise-specified (NOS) ductal breast carcinomas. The SAM-BA 200 cell image processor and its software were used to measure the nuclear features on Feulgen-stained imprint smears. Two parameters representing the numbers of large and dense chromatin clots and two parameters describing the heterogeneity of the chromatin among nuclei in a specimen evolved in a continuous manner parallel with the Bloom-Richardson score from stages NOS-4 to NOS-8. The systematic measurement of these four parameters on a large series of breast cancers may be able to define an objective and reproducible "scale" of differentiation that could be a helpful tool for pathologists and clinicians.

Adult↗

PubMed1988

Influences of dihydrotestosterone, testosterone, estradiol, progesterone, or prolactin on the cell kinetics of human hyperplastic prostatic tissue in organ culture.

In an effort to characterize the hormone sensitivity of human benign prostatic hyperplasia (BPH) maintained in organ cultures for 12-72 h, the influence of 5-alpha-dihydrotestosterone (DHT), testosterone (T), 17-beta-estradiol (E2), progesterone (Pg), or prolactin (PRL) was assessed on the cell proliferation rate of 25 BPH specimens by the use of tritiated thymidine incorporation followed by autoradiography. Significant increases in the thymidine-labeling index (TLI: percentage of labeled nuclei) were observed in glandular tissue after a 36-h incubation period in presence of DHT, E2, Pg, or PRL in 52%, 44%, 28%, and 60% of BPH cases, respectively. Nonparametric statistics (Spearman and Kendall rank correlation tests) have shown that 1) the steroid-induced TLI increases are dependent on the basal rate of cell proliferation, while the PRL-induced effect is independent of it, and 2) all the steroid-mediated effects on BPH TLI are correlated together, whereas they seem to be independent of the PRL-induced TLI increase. When T was compared with DHT on nine BPH specimens, three were found to be sensitive to both hormones, and two responded to DHT only. We propose that our study methods are suitable as a means to assess the hormone sensitivity of individual cases of BPH and possibly prostatic tumors.

Aged↗