Hormonal regulation of H19 gene expression in prostate epithelial cells.

The H19 gene is transcribed in an mRNA-like noncoding RNA. When tumors of various organs or cell types are considered, H19 oncogene or tumor-suppressor status remains controversial. To address the potential regulation of H19 gene expression by an androgen steroid hormone (DHT: dihydrotestosterone) or by a peptidic hormone (PRL: prolactin), we performed experiments in rats systemically treated with chemical mediators. This range of in vivo experiments demonstrated that chronic hyperprolactinemia upregulated the H19 expression in epithelial and stromal cells whereas DHT downregulated the gene. PRL and DHT appeared to be opposite mediators in the H19 RNA synthesis. We investigated these hormonal effects in three human prostate epithelial cell lines. In LNCaP cancer cells, the opposite effect of PRL and DHT was corroborated. However, in normal cells (PNT1A), H19 remained insensitive to the hormones in fetal calf serum (FCS) medium but became responsive in a serum-stripped medium. In the DU-145 cancer cell line, tested for its androgen-independence and aggressiveness, the hormones had no effect on H19 expression whatever the culture conditions. Finally, we demonstrated that PRL upregulated the H19 expression in LNCaP cells by the JAK2-STAT5 transduction pathway. We conclude that H19 expression is regulated by both a peptidic and a male steroid hormone.

Steroid hormones modulate H19 gene expression in both mammary gland and uterus.

H19 is an imprinted and developmentally regulated gene whose product remains apparently untranslated. In a previous study on breast adenocarcinomas, we reported that overexpression of the H19 gene was significantly correlated with the presence of steroid receptors, suggesting the putative role of hormones in H19 transcription. To determine the mode of steroid action, we have detected levels of H19 RNA synthesis during mammary gland development by in situ hybridization (ISH): two peaks of H19 transcription occur during puberty and pregnancy. Furthermore, we demonstrated by ISH that in the uterus H19 RNA synthesis is high during estrus and metestrus phases. To test steroid control of H19 transcription, ovariectomized and adrenalectomized mice were supplemented, 1 week after surgery, with 17-beta-estradiol (E2, 20 microg/kg/day), progesterone (P, 1 mg/kg/day) or corticosterone (B, 0.3 mg/ kg/day) for 2 weeks. According to ISH data, E2 and to a lesser extent B stimulated H19 transcription in the uterus, whereas P inhibited it. To confirm the in vivo results, in vitro experiments were performed using cultures of MCF-7 cells (a hormone-sensitive mammary cell line). E2 stimulated the endogenous H19 gene of this cell line and tamoxifen inhibited this effect. Furthermore, we performed transient cotransfections in MCF-7, in HBL-100 (another hormone-sensitive mammary cell line) and in BT-20 (a hormone-insensitive mammary cell line) with various constructs of ERalpha (WT or mutated) and PR-A, in presence or absence of steroid hormones. We demonstrated that ERalpha up-regulated the H19 promoter in MCF-7 and in HBL-100, whereas PR-A did not have any effect per se. Moreover, in MCF-7, PR-A antagonized clearly the ERalpha-mediated promoter enhancement, but in HBL-100 this counteracting effect on the ERalpha up-regulation was not found. Interestingly, the same experiments performed in BT-20 cell line provided very similar results as those obtained in MCF-7 cells, with a clear down-regulation mediated by PR-A on the H19 promoter. All these in vitro data are in agreement with in vivo results. In addition, data obtained with ERalpha mutants indicate that H19 promoter activation is both ligand-dependent and ligand-independent. We have thus demonstrated that H19 gene expression is controlled by steroid hormones; furthermore, this gene is highly expressed in hormone-sensitive organs when the hormonal stimulation is accompanied with a morphological repair.

H19 overexpression in breast adenocarcinoma stromal cells is associated with tumor values and steroid receptor status but independent of p53 and Ki-67 expression.

In a previous study we described the expression of the H19 gene by in situ hybridization (ISH) in normal breast and in benign or malignant breast tumors (Dugimont T, Curgy JJ, Wernert N, Delobelle A, Raes MB, Joubel A, Stehelin D, Coll J: Biol Cell 1995, 85:117-124). In the present work, 1) we extend the previous one to a statistically useful number of adenocarcinomas, including 10 subclasses, 2) we provide information on the precise ISH localization of the H19 RNA by using, on serial tissue sections, antibodies delineating specifically the stromal or the epithelial component of the breast, and 3) we consider relationships between the H19 gene expression and various clinicopathological information as tumor values (T0 to T4), grades, steroid receptors, lymph node status, and molecular features as the p53 gene product and the Ki-67/MIB1 protein, which is specific to proliferating cells. Data indicate that 1) in 72.5% of studied breast adenocarcinomas an overall H19 gene expression is increased when compared with healthy tissues, 2) the H19 gene is generally overexpressed in stromal cells (92.2%) and rarely in epithelial cells (2.9% only), 3) an up-regulation of the H19 gene is significantly correlated with the tumor values and the presence of both estrogen and progesterone receptors, and 4) at the cellular level, the H19 gene demonstrates an independent expression versus accumulation of both the p53 protein and the Ki-67/MIB-1 cell-cycle marker.

The H19 TATA-less promoter is efficiently repressed by wild-type tumor suppressor gene product p53.

The developmentally regulated H19 gene displays several remarkable properties: expression of an apparently non-translated mRNA, genomic imprinting (maternal allele only expressed), relaxation of the imprinting and/or epigenetic lesions demonstrated in some tumors. Despite several observations after relaxation of imprinting status of the gene, data on trans and cis-acting factors required for the human H19 gene expression are still missing. As a first approach to address identification of factors involved in the regulation of the gene, we found that cells from a p53 antisense-transfected HeLa clone displayed increased amounts of H19 transcripts when compared to the non-transfected cells. Moreover, a HeLa clone stably transfected with a temperature sensitive (ts) 143 Ala p53 mutant exhibited temperature-dependent regulation of H19 expression. This preliminary indication of the repressing effect of the p53 protein on H19 expression has been confirmed by transient cotransfection experiments in HeLa cells, using luciferase surrogate constructs under the control of the 823 bp sequence immediately upstream of the transcription start point of the H19 gene, and different constructs containing sense, antisense or a ts 143 Ala mutant p53 cDNA. We observed an increase of H19 promoter-driven activity in transient cotransfections with the antisense p53 cDNA and the temperature sensitive mutant p53 at the non-permissive temperature, but a decrease with sense wild-type p53 cDNA. Furthermore, the cotransfection experiments were repeated in a cell line lacking endogenous p53. (Calu 6 cells) and the results provided additional evidence for a down regulation of the expression of the H19 gene by the p53 protein.

The H19 gene is expressed within both epithelial and stromal components of human invasive adenocarcinomas.

In a previous work, we have isolated the human H19 gene and shown accumulation of transcripts in various human tumors including breast carcinomas (Douc-Rasy et al (1993) Int J Oncol 2, 753-758). Questions arose, after Northern blot results, about the precise H19 mRNA location, specially in normal breast tissues and benign or malign primary breast tumors. Then we performed molecular in situ hybridization to get insight into tissue expression of the H19 gene. Examined resections included one normal tissue, one fibroadenoma and 13 cancers. Results obtained with the H19 probe can be summarized as follows: 1) in normal breast tissues signals were focally observed in epithelial cells, but more predominantly in the palleal tissue which is sensitive to hormones; 2) in the fibroadenoma, fibroblastic cells were extensively labeled at the stroma-epithelium boundary, but epithelial cells were negative; and 3) in primary cancers, eight specimens exhibited signals on stromal cells, one specimen on epithelial cells and four on both epithelial and stromal cells. Data provide the following evidence: 1) usually labeled cells are clustered, either within normal or pathological tissues; 2) the labeling pattern highly differs from one tumor to another; and 3) H19 probe displays very different signals from one cell to another in given compartment of a given tissue section. In conclusion, it seems that a high H19 expression matches the tumor invasion. Our results suggest that the expression of this gene is concerned by the relationships between epithelial and stromal cells, and can reflect peculiar physiological states of the cells. Furthermore, we discuss results showing an abundant expression of H19 gene in some adenocarcinomas of bad prognosis, in the context of the otherwise established tumor-suppressor role of this gene, or the strictly controlled gene dosage, which could be overridden in these particular cases.

Some polypeptides in the nervous system of the marine worm, Nereis diversicolor, are related to the sodium influx stimulating peptide of the pulmonate freshwater snail, Lymnaea stagnalis.

Total mRNA, extracted from brain of the marine worm, Nereis diversicolor (Annelida, Polychaeta), was translated either in vitro using a rabbit reticulocyte lysate or in ovo (Xenopus laevis oocyte). The synthesized polypeptides were analyzed by electrophoresis and Western blotting techniques using polyclonal antisera raised against three peptides: sodium influx stimulating peptide (SISP) sequences 10-19 and 67-76 and a monoclonal antibody raised against purified native SISP (1-77) of Lymnaea stagnalis. Among the products translated in vitro, three polypeptides of 80, 72, and 64 kDa were recognized by the anti-SISP (10-19) polyclonal antiserum and by the monoclonal antiserum, but not by anti-SISP (67-76). Some of the in ovo translated products showed almost identical immunoreactivity to both the anti-SISP (10-19) and the monoclonal antibody. These polypeptides have molecular masses of 80, 72, and 43 kDa. No polypeptides were recognized by anti-SISP (67-76). Western blotting analysis of brain extracts revealed a number of proteins that reacted with the antiserum raised against SISP (10-19) and the monoclonal antiserum. Several perikarya of brain ganglionic nuclei and ventral nerve cord were immunoreactive to anti-SISP (10-19). The monoclonal antiserum gave similar results, although with a less intense immunoreaction. The infracerebral region was also stained, suggesting that the immunoreactive material is released as a true neurohormone into the hemolymph. The largest polypeptides, in particular those translated from brain mRNA, could be neuropeptide precursors containing a SISP-related sequence.

Adaptability of the coccidian Coelotropha to parasitism.

The coccidian, Coelotropha durchoni, manages to develop in its host, the polycheate annelid, Nereis diversicolor, because of its ability to circumvent the host's internal defence system. First, it avoids phagocytes by penetrating other cells, principally eleocytes and muscular cells, where it undergoes a phase of intracellular development. After becoming extracellular, a thick coat protects it from being attacked by granulocytes. This coat then breaks and is shed from its surface to permit fertilization. Parasites that have lost their coats and remain unfertilized are surrounded with granulocytes and destroyed by encapsulation. A strict hormonal correlation exists between the biological cycles of the parasite and its host. Thus, the mature spores of the coccidian parasite are disseminated when the worm lays its gametes by rupture of teguments. C. durchoni and N. diversicolor have established a biological equilibrium that permits mutual survival of both partners and constitutes a simple model for the study of the host-parasite relationship.

Natural killer cells in a lower invertebrate, Nereis diversicolor.

Globular, non-adherent coelomocytes, called "G3 granulocytes", of the polychaetous annelid Nereis diversicolor display spontaneous cytotoxicity. These cells were found capable of killing invertebrate as well as vertebrate target cells by a contact-dependent cytolytic process. Cytotoxic activity of G3 granulocytes against foreign cells develops in three steps. At first, the cells become motile and form lamellipodia. In a second step, short, pointed pseudopodia arise from the edge of the lamellipodia and are making contact with the stimulating foreign object. In a third step, the G3 granulocytes release dense granules by exocytosis onto the foreign substrate or cell which finally will undergo lysis. Within few minutes after activation, the G3 granulocyte will alter its polarity, realigning both Golgi apparatus and centrosome towards the target cell. A pore-forming protein may be involved in the cytotoxic activity of the G3 granulocytes. These cells were observed to burst after contact with and release of granules onto an abiotic solid substrate, indicating that under certain circumstances the G3 granulocytes may be sensitive to their own cytotoxic activity. These data support the postulate of Franceschi et al. (Eur. J. Immunol. 21, 489-493 (1991) that a primitive natural killer cell-like activity had been developed early in phylogenesis. A simple method for preparing invertebrate coelomocytes for scanning electron microscopy is described.

Precursors of molecules related to mammalian opioid peptides in brain of a marine worm.

Total mRNA were extracted from brain of Nereis diversicolor (Annelida, Polychaeta) and were translated in vitro or in ovo. The newly synthesized polypeptides were analyzed through electrophoresis of immunoprecipitated products or the Western blotting technique using polyclonal antibodies raised against mammalian dynorphin 1-17 and mammalian alpha-neo-endorphin. Among the products translated in vitro, only one class of polypeptide of 70 kDa was recognized by anti-dynorphin 1-17 antibodies. Furthermore, some in ovo translated products as well as proteins extracted from brain of worms showed identical immunoreactivity. These polypeptides, 60-70 kDa, reacted with anti-dynorphin 1-17 and anti alpha-neo-endorphin antibodies. These results suggest the existence of epitopes common to in ovo and in vitro translated products, to polypeptides extracted from the brain and to some mammalian opioid peptides of the prodynorphin family. We postulate the presence, in the brain of N. diversicolor, of precursors of peptides related to mammalian dynorphin 1-17 and alpha-neo-endorphin. Data reported in this investigation do not allow us to propose or even postulate the presence, in the brain of the worm, of one precursor molecule common to polypeptides related to mammalian dynorphin 1-17 and alpha-neo-endorphin. Furthermore, the Nereis precursor molecules exhibit a clear-cut difference in molecular mass with the mammalian prodynorphin: 70 kDa versus 30 kDa.