Developmental changes in the inhibition of cultured rat uterine cell proliferation by opioid peptides.

Opioid peptides are negative regulators of cell proliferation in several organs including the uterus. In the present study, the ontogeny of the direct inhibitory action of opioid peptides on the proliferation of cultured rat uterine cells was investigated. Uteri of 7, 14, 21, 28, 35 and 60-day-old rats were removed in a sterile way. Tissue blocks were dispersed by limited digestions with trypsin and collagenase. Cells were cultured in enriched Dulbecco's modified Eagle's medium (DMEM). Treatments were present during the entire culture period. Cell densities of the monolayers were determined by counting the cells following trypsinization and trypan blue exclusion. Rat uterine mixed cell cultures grew to confluence within 10 days. The average population doubling time gradually increased with the age of animals. Epidermal growth factor (EGF) increased cell densities of cultures from all age groups. The oestradiol (E2)-responsiveness appeared at 21 days of age. The effect of [D-Met2-Pro5]-enkephalinamide (ENK) was biphasic. ENK and [Met5]-enkephalin (OGF) decreased cell densities of both unstimulated and EGF-stimulated cultures from 7-day-old rats to the same extent. ENK failed to act in 14-day-old animals. From 21 days of age on, the E2- or EGF-stimulated proliferation was inhibited only by ENK and DAMGO, while 30 nm DPDPE, Dynorhin-A, OGF, [Leu5]-enkephalin, beta-endorphin, and morphiceptin were ineffective. The half-inhibitory concentration of ENK was 0.3 nm. The effects of ENK were prevented by concomitant treatment with naloxone. Our novel data demonstrate two different phases of the inhibitory action of opioid peptides on rat uterine cell proliferation during ontogeny with an insensitive interval in between.

Comparative analysis of cyclin D1 and oestrogen receptor (alpha and beta) levels in human leiomyoma and adjacent myometrium.

The aim of these experiments was to investigate the expression of cyclin D1 and of oestradiol receptors as well as the level of [(3)H]oestradiol binding in leiomyoma and adjacent myometrium from human uteri at different menstrual phases and at an early stage of menopause. [(3)H]oestradiol binding was determined by saturation analysis, while the oestradiol receptor (ER) alpha and beta and cyclin D1 levels were determined by Western blot analysis of 16 samples of human leiomyomas and corresponding myometria at different hormonal stages. In leiomyomas during all phases of the menstrual cycle, ERalpha expression, high affinity oestradiol binding and cyclin D1 expression were all elevated in comparison with adjacent myometrium. ERbeta expression and low affinity oestradiol binding were enhanced in leiomyomas only during the proliferative phase. During menopause, ERbeta expression and low affinity binding were enhanced in leiomyomas, while the ERalpha expression was not significantly enhanced and cyclin D1 levels were similar to that in myometrium. Only the oestradiol binding exhibited any menstrual cycle-related changes. Our data suggest the involvement of cyclin D1 in the growth of leiomyomas during the menstrual cycle. In menopause, there appears to be a switch from ERalpha to ERbeta expression in leiomyomas, and the induction of cyclin D1 is decreased. The regression of tumour may ensue from these changes at menopause.

Anti-mitogenic action of opioid peptides on epidermal growth factor-stimulated uterine cells.

Endogenous opioid peptides are negative regulators of estradiol-induced uterine cell proliferation. To investigate the possible molecular target site(s) of their anti-mitogenic action, we examined the effect of opioid peptides on epidermal growth factor-induced cell proliferation both in uterine primary cell cultures prepared from adult rats and in human myometrial smooth muscle cell lines. Epidermal growth factor (EGF) significantly increased cell density in both types of cultured monolayers. This EGF-induced stimulation of cell proliferation was blocked by [D-Met(2)-Pro(5)]enkephalinamide in a time-dependent, receptor-mediated manner. The effective concentrations were within the physiological nanomolar range. Enkephalinamide did not have any effect on the basal rate of proliferation of the uterine cells. Our results on this novel physiological cross-talk suggest that shared step(s) of the mechanism of action of estradiol and EGF might be targeted by opioid peptides and not the general machinery of cell proliferation.

Cytotoxic effects of cigarette smoke alkaloids inhibit the progesterone production and cell growth of cultured MA-10 Leydig tumor cells.

Inhibition of corpus luteum progesterone synthesis by cigarette smoke alkaloids might, in part, explain the generally poorer outcome of pregnancy in smoking women. The present experiments evaluate the effects of cigarette smoke alkaloids on progesterone biosynthesis and cell growth. Studies were based using the MA-10 Leydig tumor cell line. The steroid pathway in MA-10 cells has only two specific enzymatic steps. The cholesterol passes to the cholesterol side-chain cleavage enzyme and then metabolizes the resulting pregnenolone to progesterone and partly to 20alpha-dihydroprogesterone. Incubation of MA-10 cells with nicotine, cotinine, anabasine, all of these alkaloids, or an aqueous extract of cigarette smoke resulted in dose-dependent inhibition of progesterone and 20alpha-dihydroprogesterone synthesis. The number of cells in the treated dishes seemed less than the control. This latter finding prompted experiments evaluating the short-term effects of the alkaloids on cell growth. Growth of MA-10 cells influenced with alkaloids or smoke extract was also inhibited. All of the inhibitory effects of nicotine, cotinine, anabasine and cigarette smoke extract on MA-10 cells were explained by cytotoxicity. The cytotoxic effect could reduce the fertilization, implantation, and early human development. This mechanism entails the consequence of impaired placental growth, disorder in the placental vascular architecture and placental circulation, and small-for-date babies.

Barbiturates inhibit progesterone synthesis in cultured Leydig tumor cells and human granulosa cells.

Screening drugs used in obstetrical practice for effects on steroid hormone synthesis revealed that phenobarbital inhibited progesterone synthesis in MA-10 Leydig tumor cells. The inhibition was apparently a drug class effect since it could be reproduced by other barbiturates. Barbiturate blockade was reversible and could be bypassed in the MA-10 cells by using 22-hydroxycholesterol. Human granulosa cell progesterone synthesis was also inhibited in a dose dependent fashion by phenobarbital, secobarbital and barbituric acid. Significant inhibition occurred in dose ranges that would be therapeutic for treating epilepsy. From these data we conclude that barbiturates block steroidogenesis by inhibiting cholesterol transport to the cholesterol side chain cleavage enzyme.

Influence of nicotine, cotinine, anabasine and cigarette smoke extract on human granulosa cell progesterone and estradiol synthesis.

To reveal the well known effect of smoking on the incidence of early abortion, the possible effects of cigarette alkaloids on progesterone and estradiol synthesis were investigated. A suspected cause for early spontaneous abortion is corpus luteum insufficiency. The present experiments evaluate the effects of cigarette smoke alkaloids on progesterone and estradiol biosynthesis. Human granulosa cells were obtained from patients undergoing in vitro fertilization and embryo transfer treatment because of infertility. Incubation of the granulosa cells with cotinine, anabasine, with the combination of nicotine, cotinine and anabasine, or with an aqueous extract of cigarette smoke resulted in inhibition of progesterone synthesis. The alkaloids and smoke extract decreased the DNA content of the culture dish. These latter findings suggested a cytotoxic effect of the alkaloids. Both cotinine and anabasine slightly stimulated the synthesis of normalized estradiol. However, nicotine, combination of all three alkaloids, and cigarette smoke extract had no significant influence on estradiol production. Taken together, these data would suggest that cigarette alkaloids inhibit cellular progesterone synthesis both by inhibiting progesterone synthesis and by causing less specific toxic effects to the cell. In contrast, cigarette smoke alkaloids slightly stimulated or had no effect on estradiol production. These concomitant actions of cigarette alkaloids partly explain the higher incidence of early abortion in pregnant women who smoke.

Hormone synthesis and responsiveness of spontaneous granulosa cell tumors in (SWR x SWXJ-9) F1 mice.

Granulosa cell tumors spontaneously occur in approximately 10-25% of female (SWR x SWXJ-9) F1 mice. The present studies were designed to test whether tumor-bearing mice produce a distinct hormonal profile by which they could be identified and determine whether cultured tumor cells are responsive to hormones and growth factors that regulate normal granulosa cells. Samples of female mouse blood taken from age 3 to 10 weeks allowed estimation of serum FSH, 17beta-estradiol, and inhibin levels for normal mice and for mice destined to develop tumors. These studies indicated that FSH and 17beta-estradiol values differed between normal and tumor-bearing animals, but overlapped sufficiently that such values could not accurately predict the tumor-bearing state. Inhibin concentrations did differentiate normal from tumor-bearing animals in all cases. Increased levels of inhibin were observed coincident in time with visibly detectable tumors within the ovaries. Compared to inhibin synthesis in vivo, hormonal responsiveness in vitro was much more variable. Steroidogenesis was stimulated in all tumors by dibutyryl-cAMP and low-density lipoprotein (LDL). Some, but not all, tumors responded to IGF1, EGF, FSH, and hCG. In about one-half of the tumors tested, FSH could induce hCG or dibutyryl-cAMP responsiveness. IGF1 pretreatment consistently increased the responsiveness of tumor cells stimulated by dibutyryl-cAMP and LDL. Production of inhibin by isolated tumor cells was detectable and decreased by EGF or dibutyryl-cAMP treatments. We conclude that granulosa tumor cell secretion of inhibin may be under different control than secretion from normal granulosa cells and acts as an excellent marker for these tumors.

Effect of alkaloids in cigarette smoke on human granulosa cell progesterone synthesis and cell viability.

Inhibition of corpus lutea progesterone synthesis by alkaloids in cigarette smoke might, in part, explain the generally poorer outcome of pregnancy in women who smoke. The present experiments evaluated the effects of alkaloids in cigarette smoke on progesterone biosynthesis and cell viability. Studies were initiated using primary cultures of human granulosa cells. Incubation of the granulosa cells with nicotine, cotinine, anabasine, the combination of nicotine, cotinine and anabasine, or an aqueous extract of cigarette smoke resulted in inhibition of progesterone synthesis. The alkaloids and smoke extract decreased the DNA content of the culture dish. These findings suggested a cytotoxic effect of the alkaloids. Growth curves conducted using the gonadotropin-responsive, progesterone-synthesizing MA-10 cell line confirmed growth inhibition by the alkaloids and smoke extract. Together, these data suggest that cigarette alkaloids inhibit cellular progesterone synthesis both by inhibiting progesterone synthesis and by causing less-specific toxic effects to the cell.

Measurement of human chorionic gonadotropin-related immunoreactivity in serum, ascites and tumour cysts of patients with gynaecologic malignancies.

Human chorionic gonadotropin (hCG)-like molecules have been reported to be elevated in a substantial fraction of serum samples from patients with various gynaecologic tumours and have been discussed as possible markers in these malignancies. Employing highly sensitive and specific immunoradiometric assays, we determined total hCG-related immunoreactivity (hCG/hCG beta), as well as free alpha-subunit (alpha-SU), common to all glycoprotein hormones, in serum (n = 106) and malignant effusions (n = 26) of women with gynaecologic malignancies. For comparison, we also measured hCG/hCG beta in nonmalignant ascitic fluids (n = 21). HCG/hCG beta serum levels were elevated (> 5 IU L-1) in 39 of 106 patients (37%) with gynaecologic malignancies, whereas free alpha-SU was above normal range only in seven (6.6%). Frequencies of hCG/hCG beta elevations were similar in women with endometrial, (n = 39), cervical (n = 40) and ovarian (n = 27) cancer, being 30%, 35% and 41%, respectively. In malignant ascites (n = 15) and tumour cyst fluids (n = 11) of patients with ovarian cancer, hCG/hCG beta concentrations were significantly higher than in the corresponding serum samples and benign ascitic samples. Free alpha-SU, on the other hand, was increased in only one of 26 malignant effusions. In conclusion, hCG/hCG beta is frequently elevated in serum of patients with endometrial, cervical and ovarian cancer and may serve as a tumour marker in these malignancies, particularly in patients where other markers are negative. In this respect, analysis of ascitic or tumour cyst fluids may be of higher diagnostic value as serum measurements.

Immunohistochemical localization of pregnancy-related placental protein 4 in human placenta, umbilical cord and adult human female genital tissues.

Placental protein 4 (PP4) is a soluble placental tissue protein which was isolated from human placenta. The aim of the present study was to demonstrate the localization of cells containing PP4 in human placenta and in various female genital tissues under normal conditions. PP4 immunoreactive structures were demonstrated by using the peroxidase-antiperoxidase immunohistochemical technique. The samples were obtained from normal human placenta, umbilical cord, uterine cervix, endometrium, ovary and vulva. The most differentiated trophoblastic cells, the syncytiotrophoblasts, as well as the intermediate trophoblast cells contained PP4. PP4 immunoreactivity was present in umbilical cord as well. Occasionally PP4 was detected in normal ovarian, endometrial or vulvar tissue samples. Cervix and myometrium were free of PP4 immunoreactive material. PP4 staining was cytoplasmic. Our findings indicate that PP4 cannot be considered specific for the placenta since it is present in some human adult tissues as well.

Factors underlying the variability of lipid droplet fluorescence in MA-10 Leydig tumor cells.

Neutral lipids accumulate in cellular lipid droplets. These droplets vary remarkably in number and amount between cells. In the present studies, the variability in lipid content was quantified by comparing the coefficient of variation of fluorescence histograms of nile red lipid-stained cells to the variability of cell size or cell protein distributions. This measure of lipid droplet variability persisted through a wide range of cell lipid content and averaged 4.4-fold more variability than cell size and 2.6-fold more variability than cell protein content. While looking for possible explanations for this variability, it was determined that cell to cell variability could not be explained by multiple clonal populations of cells or the confluence of the cell monolayer. Analysis of lipid variability using a more droplet-specific fluorescent dye, bodipy, reduced variability by about 44%. Cell cycle analysis revealed that G2 + M cells contained more lipid than S-phase cells, which in turn contained more lipid than G0 + G1 cells, but that variability was equally large throughout the cell cycle. The cholesteryl ester hydrolase inhibitor, diethylumbelliferyl phosphate, inhibited hydrolysis of both cholesteryl esters and triglycerides. Lipid content of diethylumbelliferyl phosphate-treated cells increased while the variability in lipid staining decreased by an average of 72%. Thus, the excess lipid fluorescence variability compared to cell size or protein fluorescence could in part be explained by variability in cellular hydrolysis of triglyceride and cholesteryl ester. Excess lipid fluorescent variability could be reduced by an average of 44% when a more lipid droplet-specific stain was used instead of nile red.

Serum levels of squamous cell carcinoma antigen and ovarian carcinoma antigen (CA 125) in patients with benign and malignant diseases of the uterine cervix.

In the present study we evaluated the clinical usefulness of the tumor antigens, squamous cell carcinoma antigen (SCC) and ovarian carcinoma antigen (CA 125), in populations of patients with benign and malignant cervical disease. SCC and CA 125 levels were determined in the serum of 59 patients with invasive carcinoma of the uterine cervix and in 21 patients with benign cervical diseases. Before treatment of cervical cancer, SCC levels were elevated in 63% of the patients with squamous cell cancer while all 5 patients with adenocarcinoma had normal levels. CA 125 levels were elevated in 21% of the patients with cervical squamous cell cancer and in 3 of the 5 cases of adenocarcinoma of the cervix. In patients with benign cervical diseases, only 1 had a positive SCC level and none were positive for CA 125. No correlation was found between SCC levels and histological differentiation or clinical stage. In positive patients, serial SCC determinations correlated with the clinical course in 72.2%. Increasing levels were always associated with progression and increased on average 3 months before there was clinical evidence for disease progression. It is concluded from these studies that SCC levels are a useful marker for cervical cancer progression and recurrence. Levels of CA 125 were more likely to be elevated in patients with adenocarcinoma than squamous cell carcinoma, but when elevated in these latter patients, it also tended to predict tumor recurrence.

Immunohistochemical localization of pregnancy-related placental protein 4 in nontumorous tissues and in gynecological tumors.

The aim of the study was to demonstrate the localization of placental protein 4 (PP4) in different nontumorous and tumorous tissues originating from the female genital tract. PP4 immunoreactivity was demonstrated using the peroxidase-antiperoxidase immunohistochemical technique. Tissue samples were obtained from the cervix and body of the uterus, ovary, vulva and from gestational trophoblastic tumors. PP4-positive cells were present in nontumorous tissues with various pathologic findings, and in many but not all benign gynecological tumors. Similar numbers of PP4-positive cells were located in malignant and benign gynecological tumors; however, PP4 staining intensity was greater in the malignant lesions. PP4-positive cells were found in hydatidiform moles and in choriocarcinoma. PP4 was distributed mainly in the cytoplasm, but it was also bound to the cell membrane. We conclude from these studies that PP4 is located in a variety of benign and malignant cells of the female genital tract and that these cells may be the source of plasma PP4 found in patients with these conditions.

Plasma membrane cholesterol is utilized as steroidogenic substrate in Y-1 mouse adrenal tumor cells and normal sheep adrenal cells.

Previous studies from this laboratory indicate that plasma membrane cholesterol acts as an important source of steroidogenic substrate for MA-10 Leydig tumor cells. The present studies were designed to generalize these findings to other steroidogenic cells and to another species. Studies were performed using the Y-1 murine adrenal tumor cell line and primary cultures of sheep adrenocortical cells. Treating Y-1 cells with the acyl coenzyme A:cholesterol acyltransferase inhibitor, 58-035, caused cellular cholesteryl ester depletion and rendered more apparent the effect of dibutyryl-cAMP to cause cellular free cholesterol depletion. Radioactive 20 alpha-dihydroprogesterone was synthesized by Y-1 cells that had been plasma membrane-labeled with [3H]-cholesterol. Primary sheep adrenal cultures that had been cholesteryl ester-depleted also demonstrated cellular free cholesterol depletion after stimulation with dibutyryl cAMP. Plasma membrane label was converted to steroid hormones in these cells as well. Taken together, these data indicate that the use of plasma cholesterol is not restricted to the MA-10 cells. The present data indicate that both neoplastic mouse adrenal tumor cells and normal sheep adrenal cells utilize plasma membrane cholesterol.

Finasteride blocks progesterone synthesis in MA-10 Leydig tumor cells.

The 5 alpha-reductase inhibitor, finasteride, inhibits progesterone synthesis in the MA-10 Leydig tumor cells. Inhibition is dose-dependent with half maximal inhibition occurring at 10 ng/ml, a concentration significantly less than serum concentrations detected in finasteride-treated patients. Experiments to localize the site of inhibition by this compound revealed that the 3 beta-hydroxysteroid dehydrogenase delta 5-->delta 4 isomerase enzyme was not blocked by finasteride, but that cholesterol side-chain cleavage was inhibited. Thus, both dibutyryl-cAMP-stimulated and 22-hydroxycholesterol-stimulated steroidogenesis were inhibited by finasteride. This effect of finasteride to block cholesterol side-chain cleavage may be species-specific. Inhibition is readily detected in the mouse-derived MA-10 cells; however, human granulosa cell steroidogenesis is finasteride-insensitive while rat Leydig cell steroidogenesis is only minimally effected by finasteride.