Tsukasa Ohno

Gonadotropin-Releasing Hormone Receptor in Gynecologic Tumors Frequent Expression in Adenocarcinoma Histologic Types

Background. Gonadotropin‐releasing hormone (GnRH) analogs have been used in the therapy of the endocrine‐dependent cancers. The authors attempted to determine the frequency with which Gn‐RH receptor (Gn‐RHR) is present in gynecological cancers.

Presence of gonadotropin-releasing hormone and its messenger ribonucleic acid in human ovarian epithelial carcinoma

Objective: The purpose of this study was to investigate the expression of gonadotropin-releasing hormone messenger ribonucleic acid and the presence of gonadotropin-releasing hormone in human ovarian carcinoma known to have gonadotropin-releasing hormone binding sites and to be affected by gonadotropin-releasing hormone analog. Study Design: Human ovarian carcinomas surgically removed and human ovarian carcinoma cell lines were examined. Gonadotropin-releasing hormone was determined by a radioimmunoassay and a bioassy. Gonadotropin-releasing hormone messenger ribonucleic acid was determined by reverse transcription polymerase chain reaction using oligonucleotide primers synthesized according to the published human gonadotropin-releasing hormone sequence. Results: Gonadotropin-releasing hormone was shown to be present in extracts of ovarian mucinous cystadenocarcinoma sample (0.8 ± 0.12 pg/mg of protein) and ovarian adenocarcinoma cell line SK-OV3 (0.92 ± 0.17 pg/mg of protein) but not in the normal ovary and placenta. Two of two extract samples from individual cases evoked dose-dependent phosphoinositide breakdown in rat granulosa cells similar to that caused by authentic gonadotropin-releasing hormone. Gonadotropin-releasing hormone messenger ribonucleic acid was detected in two of two mucinous cystadenocarcinoma specimens, one of one serous cystadenocarcinoma, and SK-OV3 cells but not in the dysgerminoma, mucinous cystadenoma, and normal ovary and placenta. Conclusion: The demonstration of gonadotropin-releasing hormone and its messenger ribonucleic acid raises the possibility that gonadotropin-releasing hormone may play an autocrine regulatory role in the growth of ovarian carcinoma.

Direct protection by a gonadotropin-releasing hormone analog from doxorubicin-induced granulosa cell damage.

Background/Aims: Recent clinical applications suggest a beneficial effect of gonadotropin-releasing hormone analog (GnRHa) as a gonadal protector from chemotherapy-induced premature ovarian failure. This study aimed to determine cellular mechanisms involved in the protective action of GnRHa against granulosa cell damage caused by doxorubicin. Methods: Granulosa cells were obtained by ultrasound-guided follicular aspiration from patients undergoing in vitro fertilization, and screened for GnRH receptor expression prior to analyses. The cellular function was assessed by measuring the conversion of exogenously supplied androstenedione to estradiol-β (E2) in response to follicle-stimulating hormone (FSH) (1 µM). Results: Exposing to doxorubicin for 12 h before FSH stimulation caused a concentration-dependent inhibition of the E2 secretion to a minimum level of 20% of control. When the cells were incubated with a GnRHa for 12 h before and during exposure to doxorubicin, granulosa cells produced an equal level of E2 to that of control cells. The protective action of GnRHa was dose-dependent; a half-maximal effect occurred at 10 nM. Preincubation with GnRHa alone had no effect on FSH-induced E2 production. Conclusion: These findings demonstrate that a GnRHa may retard doxorubicin-induced granulosa cell damage, suggesting an additional GnRH activity to protect the gonads during chemotherapy through GnRH receptor-mediated mechanism(s).

Human ovary and placenta express messenger RNA for multiple activin receptors

In the present study, we examined the expression of activin receptor (ActR) mRNAs in human ovary and placenta. Primers specific for two type I and two type II activin receptors (ActR-I, ActR-IB, ActR-II, and ActR-IIB) were used in polymerase chain reaction (PCR) to amplify cDNAs prepared from granulosa-luteal cells, placental tissues and isolated trophoblast cells. PCR products with the expected sizes for ActR-I, ActR-IB, ActR-II, and ActR-IIB mRNAs were detected in freshly dissociated and 5-day cultured granulosa-luteal cells; and in trophoblast cells from both first trimester and term placentas. The identity of these PCR products were confirmed by Southern blot hybridization, as well as cloning and sequencing. These results suggest that multiple activin receptors are present in human ovary and placenta and may mediate activin function in these tissues. The demonstration of activin receptor mRNAs in granulosa-luteal and trophoblast cells further supports the notion that activin is an important local regulator in the human ovary and placenta.

Frequent expression of Fas in gonadotropin-releasing hormone receptor-bearing tumors

OBJECTIVE Fas, a cell surface receptor, mediates cell death by means of apoptosis in a variety of cell types. Gonadotropin-releasing hormone (GnRH) receptor-bearing tumors undergo the apoptosis with GnRH analogs. The authors attempted to determine the frequency with which Fas is present in the GnRH receptor-bearing tumors. STUDY DESIGN Surgically removed gynecological tumors were screened for GnRH receptor expression prior to analyses. Fas was characterized by immunoblotting of membrane proteins with the specific antibodies. Fas messenger ribonucleic acid (mRNA) was determined by reverse transcription-polymerase chain reaction using oligonucleotide primers synthesized according to the published Fas sequence. RESULTS Immunoreactive Fas and Fas mRNA were detected in a high proportion (94.4%) of the specimens from endometrial carcinomas (8 of 9), ovarian carcinomas (7 of 7), and uterine leiomyosarcomas (2 of 2); all these expressed GnRH receptor. There was neither substantial Fas nor GnRH receptor in 9 cervical carcinomas. Cloned cell lines gave identical results to those obtained in their respective mother tumors. CONCLUSION These data might suggest the frequent expression of Fas in the GnRH receptor-bearing tumors, but not in the GnRH receptor-negative tumors. Despite a poorly understood processes of apoptosis at present, there may be at least some similarity in signal transduction pathway utilized by GnRH analogs and Fas ligands.

Expression of Gonadotropin-Releasing Hormone Receptor in Human Epithelial Ovarian Carcinoma

We have previously demonstrated the presence of gonadotropin-releasing hormone (Gn-RH) messenger ribonucleic acid (mRNA) in epithelial ovarian carcinoma. In this study, the expression of Gn-RH receptor (Gn-RHR) was investigated in human ovarian carcinoma and human ovarian carcinoma cell line. Gn-RHR was determined by [3H] Gn-RH binding assay. Gn-RHR mRNA was determined by reverse transcription-polymerase chain reaction using oligonucleotide primers synthesized based on published human Gn-RHR sequence. Specific Gn-RH binding sites were shown to be present in plasma membrane isolated from five ovarian mucinous cystadenocarcinoma samples and one serous cystadenocarcinoma (Kd = 15·3 ± 8·08 nmol/L). Gn-RHR mRNA was detected in four mucinous cystadenocarcinoma specimens, one serous cystadenocarcinoma, and SK-OV-3 cells, but not in white blood cells. These results suggest that Gn-RH may play an autocrine regulatory role in the growth of ovarian carcinoma.

Gonadotropin-releasing hormone stimulates phospholipase C but not protein phosphorylation/dephosphorylation in plasma membrane from human epithelial ovarian cancer.

In view of advances in treatment of certain hormone-dependent cancers with analogues of gonadotropin-releasing hormone (Gn-RH), this study was undertaken to establish the signal transduction events interacting with Gn-RH receptor in a cell-free system prepared from human ovarian mucinous cystadenocarcinoma samples. A high affinity specific binding (Kd=8 × 10−9 M) of [3H] Gn-RH was demonstrated in two from two plasma membrane preparations. Gn-RH showed no effects on the rate of protein phosphorylation from [γ-32P] adenosine triphosphate in the plasma membrane preparations. On the other hand, incubation of plasma membrane isolated form [3H]inositol-labeled specimens with Gn-RH in the presence of guanosine thiotriphosphate resulted in the rapid production of inositol phosphates. The Gn-RH effects was concentration-dependent, and half-maximal activation occurred with 1–3 nm Gn-RH. The Gn-RH-stimulated membrane event was observed in all plasma membrane isolations tested, but not in those from uterine endometrial carcinoma of a given case. These results provide the first direct evidence that Gn-RH receptor is coupled to phosphoinositide hydrolysis but not to certain membrane protein phosphorylation/dephosphorylation in ovarian carcinoma plasma membrane. Though the functional role of this event in human ovarian cancer is still obscure, it might be part of a possible point of attack for therapeutic approaches using Gn-RH analogues in this malignancy.

Enhancement of Growth-Promoting Activity in Extract from Uterine Cancers by Protein Kinase C in Human Endometrial Fibroblasts

Uterine cervical and corpus cancers have been reported to synthesize and secrete a putative peptide mitogen, which elicits a potent proliferative response in fibroblasts by a mechanism independent of phosphoinositide turnover. The extract from human uterine cervical cancer stimulated [3H]thymidine incorporation into human endometrial fibroblasts in a dose-dependent manner. Concomitant exposure of the fibroblasts to thrombin or fibroblast growth factor (FGF) led to synergistic enhancement of the extract-stimulated [3H]thymidine incorporation into fibroblasts. An apparent maximal activity of the extract in the presence of thrombin or FGF was 2-fold higher than that in the absence of them, implying that thrombin or FGF acted at a stage after the interaction of the mitogen in the extract with its specific receptor. Insulin or epidermal growth factor failed to augment the growth-promoting activity in the extract. The stimulatory action of thrombin or FGF was mimicked by protein kinase C activators, phorbol-12-myristate-13-acetate (PMA) or 1-oleoyl-2-acetyl glycerol, but not by Ca2+ ionophore A23187. When the fibroblasts were first exposed to the extract for 4 h and then to PMA, PMA succeeded to augment the mitogenic activity in the same manner. The identical effects of protein kinase C activators with thrombin or FGF suggested that both types of ligand share a similar signaling cascade of action, activation of protein kinase C. These results demonstrated that the growth-promoting activity in uterine cancer extract could be enhanced by the agents which promote phosphoinositide metabolism through activation of protein kinase C. These findings could give a new insight into pathophysiology of the interaction between malignant cells and their stromal cells, fibroblasts.

DEHYDROEPIANDROSTERONE SULFATE BINDING SITES IN PLASMA MEMBRANE FROM HUMAN UTERINE CERVICAL FIBROBLASTS

Dehydroepiandrosterone sulfate (DHA-S) plays a critical role in cervical dilation at labor. Incubation of cervical fibroblasts with [3H]DHA-S caused a rapid and saturable increase in cellular radioactivity: an apparent equilibrium was reached by 2 min. There was no detectable conversion of DHA-S into DHA or oestradiol. When the fibroblasts loaded with [3H]DHA-S were homogenized and fractionated, the specific radioactivity in the plasma membrane fraction was enriched approximately 8- to 9-fold compared with the whole homogenate; only low amounts of radioactivity were observed in the other subcellular fractions. The binding of DHA-S to plasma membrane preparations showed saturation kinetics with an apparent equilibrium dissociation constant (K d) of 12 nM, and the binding capacity (B max) was calculated to be 1.25 fmol/mg protein. Neither DHA nor oestrone sulfate affected [3H]DHA-S binding to the plasma membrane. The plasma membranes of skin fibroblasts did not show specific binding sites for DHA-S. These findings demonstrate the presence of specific binding sites for DHA-S in the plasma membrane of cervical stroma cells. The fetal adrenal steroid may exert its action on cervical ripening at least in part through membrane-associated binding sites, or receptors.

Successful Outcomes of Pregnancy Complicated with Dermatomyositis

There are few reports in the literature of pregnancy in dermatomyositis (DM), in contrast with those of other connective tissue diseases. We describe 2 patients who had an established diagnosis of DM and then became pregnant. In these cases, both the fetal and maternal outcome were good, and differed from those in previously reported cases.