Induction of programmed cell death in Kaposi's sarcoma cells by preparations of human chorionic gonadotropin

Extensive genomic deletions involving chromosome 10 are the most common genetic alteration in glioblastoma multiforme (GBM). To localize and examine the potential roles of two chromosome arm 10q tumor suppressor regions, we used two independent strategies: mapping of allelic deletions, and functional analysis of phenotypic suppression after transfer of chromosome 10 fragments. By allelic deletion analysis, the region of 10q surrounding the MMAC/PTEN locus was shown to be frequently lost in GBMs but maintained in most low-grade astrocytic tumors. An additional region at 10q25 containing the DMBT1 locus was lost in all grades of gliomas examined. The potential biological significance of these two regions was further assessed by examining microcell hybrids that contained various fragments of 10q. Somatic cell hybrid clones that retained the MMAC/PTEN locus have a less transformed phenotype with clones exhibiting an inability to grow in soft agarose. However, presence or absence of DMBT1 did not correlate with any in vitro phenotype assessed in our model system. These results support a model of molecular progression in gliomas in which the frequent deletion of 10q25-26 is an early event and is followed by the deletion of the MMAC/PTEN during the progression to high-grade GBMs.     

Beta-core fragment of human chorionic gonadotropin in cervical intraepithelial neoplasia (CIN)

The effect of inhalation of paprine dust on the bronchopulmonary tract and antibody-forming function of the spleen was studied experimentally on two lines of mice: CBA and CC57W, differing from each other by haplotype H-2 and antioxidant status of the organisms under study. During comparative histomorphological examination of lungs and bronchi of the experimental animals a more intensive reaction was found in mice of the CBA line, whereas in mice of the CC57W line vascular changes prevailed without any reactions in the bronchioles. Reactions of the spleen to paprine dust in animals of different lines were also different: the quality of antibody-forming cells in the spleen of CBA line mice with comparatively higher antioxidant status increased under experimental conditions, while it decreased in those of the CC57W line. The data obtained showed dependance of the sensitivity of the organism to action of operational dust on individual genetic properties, demonstrating thus the necessity of applying principles of medical genetics in order to prevent effectively the development of occupational diseases in exposed teams of workers.     

Direct modulation of tumor suppressor connexin 26 gene by human chorionic gonadotropin in rat mammary glands

Human chorionic gonadotropin (hCG) has been shown to reduce the incidence of carcinogen-induced rat mammary tumors. Because connexin 26 (Cx26), a tumor suppressor gene candidate, can be up-regulated in mammary epithelial cells during lactation, we examined the in vivo and ex vivo effects of hCG on Cx26 expression in rat mammary tissues and used its effect on the expressions of beta-casein and Cx43 as controls. The Cx26 mRNA and protein expressions were up-regulated by daily administrations of 100 units of hCG, starting on day 5 and reaching a 14-fold maximum increment on days 16 through 21. It remained elevated above the basal level even 20 days after hCG withdrawal. The changes in beta-casein expression ran parallel to that of Cx26, whereas the expression of Cx43 was down-regulated. There was no correlation between steroidal hormone levels and Cx26 expression, except for the first 5 days of hCG treatment. In the ex vivo organ culture system, exposure of mammary glands to 10 units/ml hCG for 5 days up-regulated Cx26 but had no effect on beta-casein expression. These results imply a direct induction of the tumor suppressor Cx26 gene by hCG in mammary epithelial cells, a mechanism unrelated to lactation.     

The effect of human chorionic gonadotropin on methotrexate concentration in the rat testes

PURPOSE: We analyzed the effect of human chorionic gonadotropin (hCG) on drug concentrations in testicular interstitial fluid and whole testis tissue samples in rats receiving hCG prior to methotrexate (MTX) administration and in animals that did not receive hCG. MATERIALS AND METHODS: Adult male Sprague-Dawley rats were injected subcutaneously with 200 i.u. hCG (Goldline Laboratories, Ft. Lauderdale, FL.). Controls were injected subcutaneously with normal saline (0.2 cc). Sixteen hours after injection, each rat was given methotrexate (Methotrexate LPF, Immunex Corp. Seattle WA.) via a carotid artery cannula in a dose of 30 mg./kg. Methotrexate (MTX) levels were collected 60 minutes post infusion time in 27 rats and 90 minutes post infusion in 27 rats. MTX levels were measured in serum, testicular interstitial fluid and testicular tissue. MTX levels were measured using high performance liquid chromatography (HPLC). RESULTS: A significantly higher concentration of MTX was found in testicular interstitial fluid (TIF) in rats injected with hCG when specimens were collected 60 minutes post infusion. MTX levels in TIF had reversed 90 minutes post infusion with higher levels found in control rats. Tissue levels of MTX demonstrated no significant difference at either 60 or 90 minutes in the hCG treated animals or controls. CONCLUSION: Our results suggest that hCG effects the tissue distribution of MTX within the testis. Human chorionic gonadotropin may have this effect on the testicular microvasculature by 1) selectively increasing capillary permeability, 2) increasing lymphatic flow within the testes or 3) increasing testicular blood flow.     

Free alpha-subunits of human chorionic gonadotropin in preeclampsia

OBJECTIVE: To investigate free alpha-human chorionic gonadotropin (hCG) as a marker of preeclampsia. METHODS: Four groups of patients were studied: normal pregnancies, preeclampsia, eclampsia and normal pregnant women <20 weeks' gestation. Patients were further divided according to parity and gestational age (< or =20, 21-30, 31-40 weeks). An immunoradiometric assay employing monoclonal antibodies specific for free alpha-hCG was used. RESULTS: A total of 313 patients were analyzed. Thirty-four patients < or =20 weeks' gestation were followed until delivery: five (14.7%) developed preeclampsia; none had abnormal alpha-hCG levels before onset of preeclampsia. Patients with preeclampsia (21-30 weeks' gestation) demonstrated a mean alpha-hCG level greater than that of normotensive controls but this was not statistically significant. Between 31 and 40 weeks' gestation, mean alpha-hCG levels in the hypertensive and control groups were 210.8 ng/ml and 115.8 ng/ml, respectively (P < 0.001). A stronger association was observed between alpha-hCG and preeclampsia with increasing gestational age (relative risk [RR] 2.07, 21-30 weeks; RR 3.02, 31-40 weeks) and severity (RR 4.51, mild; RR 12.15, severe; RR 16.88, eclampsia). CONCLUSION: There is a strong association between alpha-hCG and preeclampsia, nevertheless this test is unsuitable for predicting preeclampsia.     

Polypeptide pattern of human breast epithelial cells following human chorionic gonadotropin (hCG) treatment

Numerous attempts have made to describe the particular protein pattern of malignant cells by using high resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The placental hormone human chorionic gonadotropin (hCG) inhibits tumor initiation and progression in experimental animals and has an inhibitory effect on the proliferation of human breast epithelial cells (HBEC) in vitro. The inhibitory effect on the immortalized HBEC MCF-10F is accompanied by the immunocytochemical expression of inhibin alpha and beta subunits by treated cells. With the purpose of clarifying the molecular mechanisms involved in this effect, the pattern of protein synthesis and mRNA were studied by 2-D PAGE in the immortalized HBEC MCF-10F cells treated in vitro 1001U for 24 h. The effect of hCG treatment on the synthesis of MCF-10F cells was monitored by labeling both control and treated cells with [S35]methionine and separation by 2-D PAGE. At least 11 proteins were preferentially synthesized and five specific polypeptides were decreased in hCG treated cells in comparison with controls. The hCG induced at least four new mRNAs which encoded protein in the molecular mass range of 24-72 kDa. It also increased the expression of at least six mRNAs and reduced the expression of least four mRNAs in comparison with control cells. The hCG-treated cells actively synthesized a 33-kDa polypeptide which was not present in control cells. The nature of this hCG-inducible 33 kDa protein elucidated by immunoprecipating [S35]methionine-labeled proteins with antisera directed against rat inhibin subunit alpha and beta b.(ABSTRACT TRUNCATED AT 250 WORDS)     

Time study of effects of pregnant mare's serum gonadotropin on surface characteristics of granulosa cells in rat ovaries

A single injection of pregnant mare's serum gonadotropin (PMSG) into immature female rats was used to stimulate graafian follicle growth. The surface of granulosa cells in the growing follicles was examined at 12-hour intervals for 72 hours by scanning electron microscopy. Between 24 and 36 hours after injection of PMSG, microvillous processes appeared on the surface of the cells. These villous processes became fully developed at 48 hours and remained for at least 72 hours after PMSG injection. The significance of this PMSG-induced microvilli formation on granulosa cells is discussed.     

Suppression of luteinizing hormone in castrated women by the administration of human chorionic gonadotropin

The suppressive effect of human chorionic gonadotropin (hCG) on luteinizing hormone (LH) and/or LH-beta was studied by specific LH-beta radioimmunoassay following hCG administration. Eight castrated women were each administered 10,000 IU of hCG in a single intramuscular injection and five women in the control group were injected with saline. The serum level of hCG increased after the injection, reaching 217.6 mIU/ml after 8 h. There was a significant suppression of LH levels as compared to those of the control group and the pre-injection levels: 68.2% 1 h after injection, 64.7% after 2h, 65.5% after 4 h, 77.0% after 8 h, 78.6% after 12 h, and 78.2% after 24 h. There was no significant suppression of the follicle-stimulating hormone (FSH) as compared to the preinjection and control values. Serum concentration of estradiol1 was not detectable either before or after the hCG injection. We conclude that hCG has a suppressive effect on LH and/or LH-beta secretion not mediated by estradiol.     

Site-directed mutagenesis of recombinant equine chorionic gonadotropin/luteinizing hormone: differential role of oligosaccharides in luteinizing hormone- and follicle-stimulating hormone-like activities

Equine chorionic gonadotropin (eCG) consists of highly glycosylated alpha- and beta-subunits and belongs to the glycoprotein hormone family that includes LH and FSH. eCG is a unique member of the gonadotropin family because it elicits response characteristics of both FSH and LH in other species than the horse. To determine the biological role of the N-linked oligosaccharide at Asn 56 of the alpha-subunit and O-linked oligosaccharides at the carboxyl-terminal peptide (CTP) of the beta-subunit, two mutant eCGs, in which Asn 56 of the alpha-subunit was replaced with Gln (eCG alpha 56/beta) or CTP was deleted (eCG alpha/ beta-CTP), were produced by site-directed mutagenesis and transfecting chinese hamster ovary (CHO-K1) cells. LH- and FSH-like activities were assayed in terms of testosterone production and aromatase activity in primary cultured rat Leydig cells and granulosa cells, respectively. The wild type eCG showed similar LH- and FSH-like activities to native eCG in the in vitro bioassays. The LH-like activity of eCG alpha 56/beta was greatly reduced, whereas that of eCG alpha/beta-CTP was unaffected, demonstrating that the oligosaccharide at Asn 56 of the alpha-subunit of eCG plays an indispensable role in LH-like activity. Interestingly, the FSH-like activity of eCG alpha 56/beta was increased markedly in comparison with the wild type, and that of eCG alpha/beta-CTP was also considerably increased. These data indicate that the dual activities of eCG, LH- and FSH-like activities, could be separated by removal of the N-linked oligosaccharide on the alpha-subunit Asn 56 or CTP-associated O-linked oligosaccharides.     

Superovulation in llamas (Lama glama) with pFSH and equine chorionic gonadotrophin used individually or in combination

The purpose of this experiment was to evaluate the efficacy of pFSH and/or equine chorionic gonadotrophin (eCG) for inducing superovulation in llamas. Sixteen adult llamas weighing on average 130 kg (range 110-140) and which had been showing signs of oestrus for 5 days were randomly allocated to three treatment groups and one control group (n = 4). Llamas in Group A received eCG (500 IU, i.m.) once daily for 3 days, those in Group B received pFSH i.m., in decreasing doses every 12 h for 5 days for a total of 220 mg, while those in Group C received eCG (500 IU, i.m.) once, and pFSH (total of 156 mg, i.m.) in decreasing doses for the next 4 days. Llamas in Group D (control) received saline (5 ml, i.m.) every 12 h for 5 days. All llamas were allowed to be mated on the evening of Day 5 and were given hCG (750 IU, i.m.) at that time; a second mating was carried out 12 h later. A non-surgical ova/embryo collection technique was performed 7 days after the first mating and then the ovarian response was evaluated by way of laparoscopy. All 16 llamas were mated successfully. The mean (SEM) number of ovulations (7.3 +/- 3.1) in Group B was significantly higher (P < 0.05) than in the other groups (1.5 +/- 0.5, 2.0 +/- 0.7, and 0.3 +/- 0.3 for groups A, C and D, respectively). The number of follicles > 10 mm at the time of ova/embryo collection was significantly higher (P < 0.05) in the groups treated with eCG. A total of 21 ova/embryos was recovered from the all flushed llamas, corresponding to 47.7% of corpora lutea observed. Of the 21 ova, 15 were fertilised; 13 of those were classified as excellent blastocysts and the remaining 2 were classified as dead or degenerating. Results demonstrate that llamas can be successfully ovarian superstimulated while expressing behavioural oestrus and suggest that pFSH is more effective than eCG to induce superovulation.     

Human chorionic gonadotropin in the treatment of HIV-related Kaposi's sarcoma

To evaluate the antineoplastic activity of human chorionic gonadotropin (hCG) in the treatment of HIV-related Kaposi's sarcoma (KS), two clinical trials focusing on two different schedules of administration and types of hCG preparation were conducted. In the low-dose group, hCG (Profasi-HP) was administered three times a week intramuscularly at a dose ranging from 4000 to 32,000 IU for 4 months and no objective response was observed among 5 evaluable patients. In the high-dose group, hCG (Gonadotrafon) was given intramuscularly three times a week at a dose ranging from 100,000 to 300,000 IU for 3 months with 1 partial response among 8 evaluable patients. In 6 patients evaluated for HIV viral load, no significant reduction in HIV viraemia was observed. In conclusion, hCG showed very limited activity against KS and no influence on HIV viral load, along with emerging dose-limiting toxicity and high cost of the therapy.     

Negative influence of O-linked oligosaccharides of high molecular weight equine chorionic gonadotropin on its luteinizing hormone and follicle-stimulating hormone receptor-binding activities

Three equine CG (eCG) forms with identical amino acid sequences but different mol wt and monosaccharide compositions were isolated from a crude eCG preparation and designated eCG-L (low mol wt), eCG-M (medium mol wt), and eCG-H (high mol wt). No differences in primary structure between each form and the known sequence of eCG were observed. SDS-PAGE of these preparations under reducing conditions revealed that the mol wt differences between them were due only to the different sizes of their beta-subunits. Carbohydrate compositions suggested an increase in O-glycosylation in the higher mol wt forms. N-Linked glycopeptide fragments obtained from eCG beta-subunits by endoproteinase Lys-C digestion had identical electrophoretic mobilities. Thus, the different molecular sizes of the beta-subunits were associated only with disparities in O-glycosylation of their C-terminal extension. When tested in a LH and several FSH radioligand assay systems, eCG-H proved to have significantly lower receptor-binding activities than eCG-L and eCG-M. Endo-beta-galactosidase digestion increased the FSH receptor-binding activity of all eCG forms; however, partially deglycosylated eCG-H remained the least active form. Thus, the O-linked oligosaccharides of eCG-H exert a negative influence on its receptor-binding activity.     

Induction of fibroblast apoptosis by anti-CD44 antibody: implications for the treatment of fibroproliferative lung disease

Fibroblast migration and proliferation within the alveolar wall and airspace after lung injury can lead to the development of fibrosis. Fibroblast cell surface CD44 is an adhesion receptor for provisional matrix proteins and mediates fibroblast invasion into fibrin matrices. Here we show that incubation of cultured fibroblasts with an anti-CD44 monoclonal antibody induces fibroblast detachment from the substratum and morphological changes compatible with apoptosis. In addition, we show that anti-CD44 monoclonal antibody rapidly induces fibroblast apoptosis within fibrin matrices. The effect of anti-CD44 antibody on induction of fibroblast apoptosis occurred within 8 hours and was dose dependent. Anti-CD44 antibody also induced fibroblast apoptosis in suspension. Furthermore, fibroblasts plated on anti-CD44-antibody-coated surfaces initially attached and spread on the antibody; however, after an 8-hour incubation time, many of the cells developed characteristic morphological features of apoptosis. Collectively, these data indicate that apoptosis did not result solely due to detachment from the substratum. Our results identify a new function for fibroblast cell surface CD44 related to the control of cell viability. We suggest this function may be important in fibroblast population control and could potentially be exploited in designing anti-fibrotic therapies.     

Induction of apoptosis in HL-60 human promyelocytic leukemia cells by adenosine A(3) receptor agonists

The effects of adenosine (ADO) analogs on cells of the human promyelocytic HL-60 line were examined. ADO A(3) receptor agonists, N(6)-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide (IB-MECA, 30-60 microM) and 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (CI-IB-MECA, 10-30 microM) induced apoptotic cell death. In contrast, neither an A(1)/A(2) antagonist (XAC) nor other selective ADO receptor agonists (CPA, NECA and CGS21680) induced apoptosis at concentrations of <30 microM. Both IB-MECA and CI-IB-MECA significantly induced Ca(2+) release from intracellular Ca(2+) pools followed by Ca(2+) influx, suggesting the presence of phospholipase C-coupled ADO A(3) receptors on HL-60 cells. This was further supported by the presence of mRNA of ADO A3 receptor in the cells. These results suggest that activation of ADO A(3) receptors is responsible for the ADO-induced apoptosis in HL-60 cells and could be of potential therapeutic value in the treatment of leukemia.