RESUMEN

LH and prostaglandin E(2) (PGE(2)) share many similar effects on the pre-ovulatory follicle. They can induce independently cumulus expansion, the resumption of meiosis and progesterone production. However, cyclooxygenase-2 (COX-2) inhibitors were found to hinder most of the LH-induced effects. Recently, EGF-like growth factors amphiregulin (Ar) and epiregulin (Ep) were found to be produced in response to LH stimulation and to induce cumulus expansion and oocyte maturation. We aimed at evaluating whether PGE(2) induces Ar and Ep syntheses in human granulosa cells and whether the inhibition of PGE(2) production by selective COX-2 inhibitor, nimesulide, affects LH-induced Ar and Ep biosynthesis. Ar and Ep mRNA levels increased following PGE(2) stimulation, in a dose- and time-dependent manner, which resembled those of LH. The blockade of protein kinase A (PKA) (by H89) and mitogen-activated protein kinase (MAPK) (by UO126) reduced the expression of PGE(2)-induced Ar and Ep biosynthesis. Although the stimulation of the cells with LH in the presence of nimesulide did not change the progesterone levels, it resulted in a significant reduction of Ar and Ep biosynthesis. In conclusion, PGE(2) may mimic LH action, at least in part, by the induction of Ar and Ep biosynthesis, which involves cAMP/PKA and MAPK pathways. The negative effect of nimesulide on the ovulatory process may be due to the reduction of Ar and Ep biosynthesis, which implies a possible collaborative role between PGE(2) and LH on their induction.

Asunto(s)

Dinoprostona/metabolismo , Factor de Crecimiento Epidérmico/biosíntesis , Glicoproteínas/biosíntesis , Células de la Granulosa/metabolismo , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Hormona Luteinizante/metabolismo , Ovulación/metabolismo , Adulto , Anfirregulina , Animales , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Inhibidores de la Ciclooxigenasa 2/farmacología , Dinoprostona/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Familia de Proteínas EGF , Factor de Crecimiento Epidérmico/genética , Epirregulina , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Glicoproteínas/genética , Células de la Granulosa/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Ratones , Ratones Endogámicos C57BL , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Ovulación/efectos de los fármacos , Progesterona/metabolismo , ARN Mensajero/biosíntesis , Transducción de Señal/efectos de los fármacos , Sulfonamidas/farmacología , Factores de Tiempo

RESUMEN

Single chain variants of the heterodimeric gonadotropins were engineered by tethering the genes of the individual subunits into one polypeptide. In tethered human (h) gonadotropins, the carboxyl terminal peptide (CTP) of the choriogonadotropin (CG) beta subunit serves as an effective linker to enhance the secretion of the analogs compared to variants lacking the CTP. The gonadotropin subunits of non-primate, non-equid species lack a CTP domain that precludes the use of a homologous CTP in tethered analogs in many species. Here we used the bovine LH as a model to examine the impact of the CTP domain of the hCGbeta subunit (denoted as huCTP) and of a previously untranslated CTP-like sequence decoded from the bovine LHbeta gene on the secretion and bioactivity of tethered analogs. This cryptic CTP (designated boCTP) was incorporated into the bovine LHbeta reading frame by deletion frame-shift mutations analogous to these that presumably occurred in primates and equids. We genetically engineered single chain variants in which the beta and alpha subunit domains were linked directly or via the heterologous huCTP or the homologous boCTP sequences and expressed them in CHO cells. The data suggest that the tethered analogs were expressed and N-glycosylated, but unlike the huCTP, the boCTP appears as devoid of mucin O-glycans. The incorporation of the boCTP or huCTP linkers enhanced by about 3fold the rate and efficiency of secretion from the transfected cells. The tether variants were bioactive, as estimated by induction of steroid production in immortalized granulosa cells expressing the rat LH receptor. Furthermore, the variants were about equally potent, as judged by their EC50s (0.7-0.9 ng/ml). Thus, the hCGbeta CTP maintains pro-secretory determinants without inhibiting receptor activation when applied as a linker in tethered bovine LH, implying that these CTP features are preserved when the domain is incorporated into non-primate single chain analogs. The study suggests that the boCTP and huCTP domains are advantageous for the secretion of tethered bovine gonadotropins, and also demonstrates strategies for the design of bioactive LH analogs in ruminant species.

Asunto(s)

Gonadotropina Coriónica/genética , Gonadotropina Coriónica/metabolismo , Hormona Luteinizante/genética , Hormona Luteinizante/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células CHO , Bovinos , Cricetinae , Femenino , Variación Genética , Glicosilación , Células de la Granulosa/metabolismo , Humanos , Datos de Secuencia Molecular , Ingeniería de Proteínas , Procesamiento Proteico-Postraduccional , Ratas , Transfección

RESUMEN

Fertility preservation is of major importance for women with cancer in whom ovarian function may be disturbed by the use of potentially sterilizing chemotherapeutic drugs and/or pelvic irradiation. Cryopreservation of ovarian cortical tissue is one of the potential options for preserving fertility among these women. Cryopreserved thawed human ovarian tissue can be autografted either orthotopically or heterotopically, but may also be transplanted first into an animal host with subsequent maturation and collection of oocytes. The objective of this study was to investigate the prevalence of ovarian follicular apoptosis in fresh and frozen/ thawed human ovarian tissue as a measure of follicular viability. The study group included 6 women with cancer who underwent ovarian tissue cryopreservation (OTCP). Ovarian tissue samples (n = 2) were obtained from each woman with one sample undergoing evaluation for apoptosis immediately following removal (control, group A) and the other evaluated for apoptosis following freezing/thawing (group B). Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) and 4'6' diamido-2-phenylindole hydrochloride (DAPI) staining methods were used to investigate follicular apoptosis. Morphological changes in the same samples were evaluated in hematoxylin and eosin (H&E)-stained sections. In each slide, only primordial and primary follicles were evaluated for abnormal morphology and apoptosis. Abnormal morphology was demonstrated in 23.8+/-8.7% of group A follicles compared to 48.3+/-11.2% of group B follicles (p < 0.05). Apoptosis was demonstrated in 25.4+/-8.4% of group A follicles compared to 60.9+/-6.0% of group B follicles (p < 0.05). We have shown that the ovarian follicles in group B demonstrated a higher incidence of apoptosis compared to those of group A. Therefore, the data suggest that follicular apoptosis might be a consequence of the freezing and thawing procedure. This may be used as a method for evaluating and comparing the outcome of different freezing/thawing protocols.

Asunto(s)

Apoptosis , Criopreservación/normas , Ovario/fisiología , Adulto , Criopreservación/métodos , Eosina Amarillenta-(YS) , Femenino , Hematoxilina , Histocitoquímica/métodos , Humanos , Etiquetado Corte-Fin in Situ , Oocitos/citología , Oocitos/fisiología , Folículo Ovárico/citología , Folículo Ovárico/fisiología , Ovario/citología , Reproducibilidad de los Resultados , Fijación del Tejido/métodos

RESUMEN

Gemcitabine and cisplatin are commonly used in chemotherapy, however, these drugs may cause severe cytotoxic side effects. Theophylline and aminophylline are commonly used as anti-asthma drugs and can block anti-phosphodiesterase activity. We examined whether these methylxanthins could effect lung cancer cell survival and synergise with gemcitabine and cisplatin to induce apoptosis. We found that theophylline induced apoptosis in the cultured H1299 cell line already at concentrations of 30 microg/ml, reaching an ED50% at 100 microg/ml. In contrast, aminophylline induced apoptosis at concentrations of 300 microg/ml and 17% apoptosis was evident at concentrations as high as 900 microg/ml, which is a lethal dose for in vivo treatment. Cisplatin induced apoptosis with ED50% of 0.8 microg/ml, while gemcitabine induced apoptosis with ED50% of 20 ng/ml. Using a combination of 20 microg/ml of theophylline (calculated as an effective but not toxic anti-asthma drug) with 10 ng/ml gemcitabine or with 0.3 microg/ml cisplatin significantly elevated incidence of apoptosis compared to gemcitabine or cisplatin alone at similar concentrations. In contrast, an observed synergistic effect between aminophylline and gemcitabine was evident only at concentrations of 80 microg/ml and 10 ng/ml respectively. However, no effect was apparent in combination doses of aminophylline (80 microg/ml) with cisplatin (0.3 microg/ml). The combined treatments involved reduction in the intracellular level of the anti-apoptotic Bcl-2 gene product. This corresponded with the extent of apoptosis induced by the various drug combinations. Thus, theophylline is significantly more effective than aminophylline in increasing the sensitivity of the H1299 lung cancer cells to the induction of cell death by gemcitabine and cisplatin. Thus, combination of theophylline with these drugs may permit a reduction in the effective dose needed in chemotherapy treatment of lung cancer patients.

Asunto(s)

Antiasmáticos/administración & dosificación , Apoptosis , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Cisplatino/administración & dosificación , Desoxicitidina/análogos & derivados , Desoxicitidina/administración & dosificación , Quimioterapia Combinada , Neoplasias Pulmonares/tratamiento farmacológico , Teofilina/administración & dosificación , Western Blotting , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Separación Celular , Cisplatino/farmacología , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Fase G1 , Humanos , Neoplasias Pulmonares/patología , Propidio/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Fase de Descanso del Ciclo Celular , Teofilina/farmacología , Gemcitabina

RESUMEN

Epiregulin and amphiregulin are growth factors involved in cancer development, but their potential role in signaling in the gonads is still obscure. We report here that basal expression of these growth factors is evident in human granulosa cells obtained from women treated for in vitro fertilization, when examined by RT-PCR using RNA isolated from primary cultures of ovarian granulosa cells. Expression of these factors was elevated concomitantly with elevation of progesterone production in these cells upon stimulation with luteinizing hormone (LH), and to a lesser extent with follicle stimulating hormone (FSH), both essential stimulants for ovulation and luteinization. Epiregulin and amphiregulin gene expression was dose- and time-dependent when measured subsequent to LH stimulation. Moreover, forskolin, which activates adenylate cyclase, was as efficient as LH in stimulating expression of these growth factors. It is suggested that upregulation of the epiregulin and amphiregulin expression is part of the signal transduction pathway which leads to ovulation and luteinization in the human ovary.

Asunto(s)

AMP Cíclico/metabolismo , Factor de Crecimiento Epidérmico/biosíntesis , Factor de Crecimiento Epidérmico/química , Glicoproteínas/biosíntesis , Gonadotropinas/química , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Folículo Ovárico/metabolismo , Anfirregulina , Western Blotting , Células Cultivadas , Colforsina/metabolismo , Relación Dosis-Respuesta a Droga , Familia de Proteínas EGF , Factor de Crecimiento Epidérmico/metabolismo , Epirregulina , Femenino , Hormona Folículo Estimulante/metabolismo , Glicoproteínas/química , Células de la Granulosa/citología , Humanos , Péptidos y Proteínas de Señalización Intercelular/química , Hormona Luteinizante/metabolismo , Ovario/metabolismo , Progesterona/química , Progesterona/metabolismo , ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Regulación hacia Arriba

RESUMEN

Gonadotropins play a crucial role in ovarian homeostasis and fertilization through the activation of the cAMP cascade. However, gonadotropin hyper-stimulation may be associated with higher risk for ovarian cancer development. It has been suggested, that high gonadotropin levels in peritoneal and ovarian cystic fluids of patients suffering from benign ovarian cysts, may lead to malignancy. Moreover, we have recently discovered that gonadotropin stimulation can activate the MAPK cascade in target cells. Using DNA microarray technology and RNA from human granulosa cells, we discovered that stimulation with saturating doses of gonadotropins dramatically elevates activity of genes coding for epiregulin and amphiregulin. These gene products can bind and activate the EGF receptor and ERBB4, which are associated with the development of various cancers such as ovarian, breast endometrial and other non-gynecological malignancies. Gonadotropin receptors are expressed not only in the gonads, but also in non-gonadal tissues and in cancer cells. The discovery that gonadotropins activate certain mitogenic signal transduction pathways, may serve as a guide for novel anti-cancer therapy by (1) specific interference at the receptor level to block the gonadotropic response, or arresting the receptor expression and (2) blocking downstream mitogenic signals generated by these hormones, like attenuation of the expression of epiregulin and amphiregulin that belong to the EGF family, using anti-sense and/or SiRNA techniques targeted to suppress their expression. Moreover, since amphiregulin and epiregulin act as mediators of luteinizing hormone (LH) action in the mammalian ovulatory follicles, regulation of the expression of these factors may open new possibilities in treatment of ovarian malfunction implicated with ovarian hyper-stimulation.

Asunto(s)

Antineoplásicos/farmacología , Factor de Crecimiento Epidérmico/biosíntesis , Glicoproteínas/biosíntesis , Gonadotropinas/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Transducción de Señal/efectos de los fármacos , Anfirregulina , Antineoplásicos/uso terapéutico , Transformación Celular Neoplásica , Diseño de Fármacos , Familia de Proteínas EGF , Epirregulina , Femenino , Expresión Génica , Humanos , Hormona Luteinizante/fisiología , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patología , Factor de Crecimiento Transformador alfa/fisiología

RESUMEN

Polycystic ovarian syndrome is seen in 5% of fertile aged women. However, there is no satisfactory PCOS model in experimental animals. To induce polycystic ovary phenotype in immature female rats, Wistar rats 21 days of age were injected daily with testosterone propionate 1 mg/100 g body weight dissolved in propylene glycol or propylene glycol for up to 35 days. Seven days of injection with testosterone (T) resulted in the appearance of large cystic follicles and a dramatic accumulation of multi-layer preantral follicles. At 42 days of age puberty in control animals was evident by the appearance of corpora lutea. In contrast in T treated animals no corpora lutea formation was seen even at the age of 56 days. Progesterone in the control animals was elevated at the age of 42 days in contrast with the T treated animals in which progesterone remained low (20% of control). While during 14 days of T injection most of the follicles did not have progressive apoptosis, at 21-35 days of injection (42-56 days of age) the vast majority of follicles became apoptotic. Progressive degeneration of oocytes was evident in T treated animals reaching 70-85% of total oocytes at 21-35 days of T injection compared to 30-40% in control animals. Western blot analysis of ovarian homogenates revealed gradual decrease in Bcl-2 content, evident at 28 and 35 days of T injection compared to control animals. Interestingly, the fasting glucose/insulin ratio was dramatically reduced in T treated animals following 14 days of testosterone treatment compared to controls. Our data suggest that T injection to immature female rats can induce polycystic ovaries, block ovulation and attenuate progesterone production. Moreover, normal/low glucose and high insulin blood levels in the testosterone treated rats raises the possibility that elevated androgens can lead to insulin resistance in this experimental PCOS model.

Asunto(s)

Glucosa/metabolismo , Insulina/metabolismo , Ovario/patología , Síndrome del Ovario Poliquístico/inducido químicamente , Síndrome del Ovario Poliquístico/patología , Testosterona/farmacología , Animales , Apoptosis , Western Blotting , Cuerpo Lúteo/metabolismo , Fragmentación del ADN , Modelos Animales de Enfermedad , Femenino , Etiquetado Corte-Fin in Situ , Resistencia a la Insulina , Oocitos/metabolismo , Fenotipo , Progesterona/metabolismo , Propilenglicol/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Ratas Wistar , Esteroides/metabolismo , Factores de Tiempo

RESUMEN

Gonadotropins play a crucial role in ovarian homeostasis and fertilization. However, hypergonadotropin stimulation has been thought to increase the risk for ovarian cancer. Moreover, some correlation between high levels of gonadotropins in the circulation and Alzheimer's disease has been implicated, with no clear evidence on the molecular mechanism involved. Using DNA microarray technology and RNA from gonadotropin-stimulated human granulosa cells, which comprise the main bulk of the ovarian follicular somatic cells, we discovered that stimulation of cells with saturating doses of gonadotropins gives rise to the expression of genes coding for presenilin 1 and 2, along with the up-regulation of genes involved in steroidogenesis such as StAR, cytochrome P450scc enzyme system and aromatase. Moreover, gonadotropin stimulation in these cells dramatically elevates activity of genes coding for epiregulin and amphiregulin, which can bind and activate the EGF receptor and ERB4. These gene products may elevate the risk for ovarian, breast, endometrial and other non-gynecological cancers. Gene transcripts for oncogenes and tumor markers such as pleiomorphic adenoma gene-like 1 (Plagl1) tumor antigen (L6) and claudin 3 were markedly elevated following LH and FSH stimulation. In parallel, downregulation in ovarian cancer 1 (DOC1) and suppression of tumorigenicity (ST5) genes was observed, suggesting a potential increase for cancer development. In contrast, increase in tumor rejection antigen (gp96) 1 and decrease in connective tissue growth factor (CTGF), transforming growth factor-beta 1 induced transcript 1 (TGFB1Il), pim-1 oncogene (PIM1), v-maf musculoaponeurotic fibrosarcoma oncogene homologue (MAF) and CD24 antigen may be associated with a decreased risk for specific cancers. In conclusion, gonadotropin stimulation may modulate specific sets of gene transcripts that may either elevate or reduce the risk for specific diseases.

Asunto(s)

Fertilización In Vitro , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Gonadotropinas/farmacología , Células de la Granulosa/efectos de los fármacos , Células de la Granulosa/metabolismo , Enfermedad , Femenino , Sustancias de Crecimiento/metabolismo , Humanos , Neoplasias , Análisis de Secuencia por Matrices de Oligonucleótidos , Embarazo , Receptores de Factores de Crecimiento/metabolismo

RESUMEN

Gonadotrophins exert a major effect on ovarian development and on the control of fertilization. By stimulating cells with forskolin (FK), it is possible to study which genes are activated by gonadotrophins via the cAMP cascade, and which by alternative pathways. Using RNA isolated from stimulated cells, we found that 59% of the total genes modulated by LH were also modulated by FK, while 69% of the genes modulated exclusively by FSH were also modulated by FK. Gene transcripts involved in steroidogenesis/progesterone production were highly elevated, while 17beta-hydroxysteroid dehydrogenase was down-regulated. This suggests that a decrease in the conversion of androstenedione to testosterone and estrone to estradiol occurs during luteinization. Down-regulation of genes coding for actin cytoskeleton proteins and cytokeratin 18 was observed in response to gonadotrophin and cAMP stimulation. Several of the genes coding for the microtubule network were also modulated, implying that rearrangement of the cytoskeletal proteins permits better coupling between organelles involved in steroidogenesis. A dramatic change in gene transcripts coding for signalling enzymes was observed following LH stimulation. This includes the down-regulation of adenylyl cyclase 7 and 9, elevation of cAMP-dependent phosphodiesterase, and the up-regulation of a negative regulator of G-protein signalling (RGS16) that may negate gonadotrophin signalling via guanine nucleotide binding proteins. Thus luteinized cells, despite increased gene transcripts to LH/chorionic gonadotrophin (CG) receptors, respond inefficiently to gonadotrophin stimulation, due to attenuation of signal transduction in the cAMP cascade at multiple steps. Novel genes involved in the regulation of apoptosis were found for the first time to be up-regulated by gonadotrophin stimulation, including: BAX inhibitor-1, granulysin and apoptosis repressor with caspase recruitment domain (ARC). These proteins may be involved in a unique alternative pathway of ovarian cell death. Such a pathway could temporarily preserve the mitochondria and progesterone production during the initial stages of granulosa cell apoptosis.

Asunto(s)

Apoptosis/genética , Proteínas del Citoesqueleto/genética , Fertilización In Vitro , Regulación de la Expresión Génica , Gonadotropinas/metabolismo , Células de la Granulosa/fisiología , Proteínas Quinasas/genética , Animales , Apoptosis/fisiología , Células Cultivadas , Femenino , Perfilación de la Expresión Génica , Células de la Granulosa/ultraestructura , Humanos , Datos de Secuencia Molecular , Proteínas Quinasas/metabolismo , Transducción de Señal/fisiología

RESUMEN

We have recently demonstrated that glucocorticoids protect against serum-deprivation, cAMP-, TNFalpha-, and p53-induced apoptosis in ovarian follicular cells involved in up-regulation of Bcl-2. We demonstrated that dexamethasone, which enhances steroidogenesis by up-regulation of the p450scc enzyme system, stimulates the MAPK cascade by phosphorylation of ERK1, ERK2 as well as by Akt phosphorylation within 1-5min with no effect on p38 MAPK phosphorylation. Moreover, glucocorticoids enhance expression of connexin 43, formation of gap junctions, expression of cadherins, and formation of adherence junctions within 24h of hormone stimulation of ovarian granulosa cells. It is suggested that the protective effects of glucocorticoids against apoptosis are mediated by both genomic and non-genomic mechanisms. Moreover, for the first time we show that protein phosphorylation, cell-cell contact, and intracellular communication are important mediators in glucocorticoid protection against apoptosis in ovarian follicular cells.

Asunto(s)

Dexametasona/farmacología , Células de la Granulosa/efectos de los fármacos , Células de la Granulosa/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Comunicación Celular/efectos de los fármacos , Comunicación Celular/fisiología , Línea Celular , Colforsina/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Glucocorticoides/farmacología , Células de la Granulosa/citología , Folículo Ovárico/citología , Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/fisiología , Progesterona/biosíntesis , Ratas , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/fisiología

RESUMEN

Ovarian cell death is an essential process for the homeostasis of ovarian function in human and other mammalian species. It ensures the selection of the dominant follicle and the demise of excess follicles. In turn, this process minimizes the possibility of multiple embryo development during pregnancy and assures the development of few, but healthy embryos. Degeneration of the old corpora lutea in each estrus/menstrual cycle by programmed cell death is essential for maintaining the normal cyclicity of ovarian steroidogenesis. Although there are multiple pathways that can determine cell death or survival, crosstalk among endocrine, paracrine and autocrine factors, as well as among protooncogenes, tumor suppressor genes, survival genes and death genes, play an important role in determining the fate of ovarian somatic and germ cells. The establishment of immortalized rat and human steroidogenic granulosa cell lines and the investigation of pure populations of primary granulosa cells allows for systematic studies of the mechanisms that control steroidogenesis and apoptosis of granulosa cells. We have discovered that during initial stages of granulosa cell apoptosis progesterone production does not decrease. In contrast, we found that it is elevated for up to 24hr following the onset of the apoptotic stimuli exerted by starvation, cAMP, p53 or tumor necrosis factor alpha stimulation, before total cell collapse. These observations raise the possibility for an alternative unique apoptotic pathway, one that does not involve mitochondrial cytochrome C release associated with the destruction of mitochondrial structure and steroidogenic function. Using mRNA from apoptotic cells and Affymetrix DNA microarray we discovered that Granzyme B, a protease that normally resides in T cytotoxic lymphocytes and natural killer cells of the immune system is expressed and activated in granulosa cells, thereby allowing the apoptotic signals to bypass mitochondrial signals for apoptosis, which can preserve their steroidogenic activity until complete cell destruction. This unique apoptotic pathway assures the cyclicity of estradiol and progesterone release in the estrus/menstrus cycle even during the initial stage of apoptosis.

Asunto(s)

Apoptosis/fisiología , Células de la Granulosa/fisiología , Ovario/citología , Animales , Femenino , Células de la Granulosa/citología , Granzimas , Humanos , Serina Endopeptidasas/fisiología

RESUMEN

Human granulosa cells were immortalized by transfection of the primary cells with a mutated p53 gene in combination with the Harvey-ras oncogene, yielding established cell lines designated HGP53. Here we report that forskolin, 8-Br-cAMP and FSH modulate cell growth and steroidogenesis in HGP53 cells. Low concentrations of 8-Br-cAMP or FSH stimulated cell proliferation, while higher doses attenuated cell proliferation. Progesterone production was already evident at an FSH concentration of 0.3 mIU/ml and was maximally stimulated (50-135-fold) at 50 mIU/ml of FSH. Expression levels of steroidogenic acute regulatory protein (StAR), adrenodoxin and cytochrome P450scc were enhanced 64-, 48- and 3.1-fold respectively by FSH stimulation. Dexamethasone enhanced FSH/cAMP-induced steroidogenesis and this effect involved a marked elevation in the intracellular level of adrenodoxin and P450scc, concomitantly with a marked decrease in StAR. Conversely, basic fibroblast growth factor attenuated FSH-stimulated progesterone production, and this effect involved reductions in adrenodoxin, P450scc and StAR levels. These data suggest that the rate of steroidogenesis may be determined by the ratio of StAR and P450scc, rather than by the level of each protein alone. Whereas FSH at a low dose slightly reduced apoptosis induced by serum withdrawal from HGP53 cells, higher doses enhanced it. Dexamethasone dramatically attenuated FSH- or forskolin-enhanced apoptosis. In conclusion, FSH-dependent mechanisms of differentiation, luteinization and apoptosis can be preserved in human granulosa cells immortalized by mutated p53. Moreover, this system lends itself to studies on cross-talk between the endocrine and paracrine factors that control these processes.

Asunto(s)

Línea Celular , Hormona Folículo Estimulante/farmacología , Genes p53 , Genes ras , Células de la Granulosa/efectos de los fármacos , Proteínas Nucleares , Transfección , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Adrenodoxina/metabolismo , Apoptosis/fisiología , División Celular/efectos de los fármacos , Tamaño de la Célula , Colforsina/farmacología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/genética , Ciclinas/metabolismo , Dexametasona/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Glucocorticoides/farmacología , Células de la Granulosa/citología , Células de la Granulosa/fisiología , Humanos , Progesterona/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2

RESUMEN

Human and rat granulosa cells express receptors to leptin which synergies with glucocorticoid hormones in stimulation of ovarian steroidogenesis. To examine whether leptin affects follicular development and maturation, we injected recombinant ovine leptin (300 ng-10 microg/animal) daily to immature 21 day-old female rats. Non-treated rats reached puberty at 44.5+/-1.6 (n=9) days. In contrast, in leptin treated animals, puberty was reached at 34.5+/-1.6 (n=9) days. Ovarian sections revealed hypertrophy of granulosa cells in leptin treated animals. Moreover, the number of ovulations was 2-fold higher in the treated animals compared to controls (3-4 ovulations versus 7-8 on the first three estrous cycles, P<0.001). Leptin dramatically reduced incidence of follicular apoptosis measured by TUNEL, and was already evident after 7 days of leptin injection (12% of apoptosis in leptin treated group compared to 52% in controls, P<0.001). Maximal protection against apoptosis was achieved at 1-3 microg leptin/animal. The levels of FSH, LH, progesterone and the steroidogenic factors ADX and STAR were elevated earlier in development in the leptin treated animals compared to control animals which is in line with the achievement of early puberty in the leptin treated animals compared to non treated ones. To reveal whether modulation of death and survival genes is involved in leptin attenuation of follicular apoptosis, we examined the expression of the survival gene Bcl-2 and the death gene Bax in Western blots of ovarian homogenates. There was a pronounced elevation in Bcl-2 expression during 7-14 days of leptin injections up to 16.3-fold (P<0.001) compared to Bcl-2 expression in controls. Bax expression was elevated only 3.4 fold (P<0.001), leading to an increase in the Bcl-2/Bax ratio of 4.7 fold (P<0.001). Expression of the tumor suppressor gene p 53 and the oncogene Mdm2 did not change significantly. Our data suggests that leptin may be involved in accelerating follicular maturation by attenuating follicular atresia and increasing the ratio of Bcl-2/Bax.

Asunto(s)

Apoptosis/efectos de los fármacos , Leptina/farmacología , Folículo Ovárico/fisiología , Ovario/efectos de los fármacos , Maduración Sexual/efectos de los fármacos , Adrenodoxina/genética , Adrenodoxina/metabolismo , Animales , Femenino , Hormona Folículo Estimulante/sangre , Etiquetado Corte-Fin in Situ , Hormona Luteinizante/sangre , Masculino , Tamaño de los Órganos , Ovario/fisiología , Ovulación/fisiología , Fosfoproteínas/metabolismo , Progesterona/sangre , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Ratas Wistar , Proteínas Recombinantes/farmacología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteína X Asociada a bcl-2

RESUMEN

Fibroblast growth factors play an important role in the control of ovarian folliculogenesis, but the complete repertoire of ovarian receptors which can transduce the fibroblast growth factor signals and their precise localization in the ovary have not yet been characterized. The most common form of inherited human dwarfism results from a point mutation in the transmembrane region of fibroblast growth factor receptor 3. A mouse model for achondroplasia was generated by introducing the human mutation (glycine 380-arginine) into the mouse fibroblast growth factor receptor 3 (G374R) by a "knock-in" approach using gene targeting leading to a constitutively active receptor. This resulted in the development of dwarf mice that share many features with human achondroplasia. Here we report that female (fibroblast growth factor receptor 3 G374R) dwarf mice become infertile. While no significant changes were observed in the anatomical and histological appearance of ovaries of 3-wk-old dwarf mice, a dramatic difference was observed in ovaries of 3-month-old mice. The normal ovary consists mainly of healthy corpora lutea and follicles at different stages of development, whereas the ovaries of the dwarf mice remain small and contain mainly follicles with a progressive apoptosis in the granulosa cells, and no corpora lutea could be observed. The levels of LH, FSH, and progesterone were lower by 72.3%, 38.0%, and 40.0%, respectively, in the blood of the dwarf mice compared with normal mice, and the total bioactivity of pituitary FSH and LH was lower by 65.6% and 79.6%, respectively, in the dwarf mice compared with normal mice. However treatment with PMSG and human CG of the dwarf mice led to rapid follicular development and formation of corpora lutea. Interestingly, the expression of the tumor suppressor gene p53 was increased dramatically in ovaries of the dwarf mice. The presence of the fibroblast growth factor receptor 3 cellular receptors in both normal and dwarf animals was demonstrated by Western blot and immunostaining. However, the distribution of the fibroblast growth factor receptors in the two strains shows significant differences. In the normal ovaries fibroblast growth factor receptor 3 was homogeneously distributed on the cell membrane of the granulosa cells and was absent in theca as well as corpora lutea cells, whereas in dwarf mice ovaries it was highly clustered on granulosa cells and very often appears in endocytic vesicles. Aged oocytes were more frequently observed in preantral follicles of ovaries of the dwarf mice. Nevertheless, oocytes isolated from antral follicles resume their meiotic division at a high percentage, similar to oocytes obtained from normal ovaries. The results imply fibroblast growth factor receptor 3 involvement in the control of follicular development through regulation of granulosa cell growth and differentiation, and that unovulation in the dwarf mice could be overcome in part by administration of exogenous gonadotropins. Moreover, it is suggested that the infertile phenotype is partially due to defects in the pituitary-gonadal axis.

Asunto(s)

Acondroplasia/fisiopatología , Apoptosis/fisiología , Células de la Granulosa/fisiología , Receptores de Factores de Crecimiento de Fibroblastos/genética , Acondroplasia/genética , Acondroplasia/patología , Animales , Peso Corporal , Femenino , Hormona Folículo Estimulante/sangre , Hormona Folículo Estimulante/metabolismo , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Hormona Luteinizante/sangre , Hormona Luteinizante/metabolismo , Ratones , Oocitos/fisiología , Tamaño de los Órganos , Ovario/patología , Ovario/fisiopatología , Hipófisis/fisiopatología , Progesterona/sangre , Progesterona/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo

RESUMEN

Ovarian cancer is the most lethal of gynecological malignancies. Yet early diagnosis and prognosis are far from being satisfactory. Degradation of heparan sulfate proteoglycans by heparanase appears to play an important role in the invasiveness of tumor cells through the basement membrane and into the extracellular matrix. Recent cloning of the heparanase gene and generation of monoclonal antibodies against the enzyme permit to examine tumor cell expression of the enzyme. The aim of the present study was to assess heparanase activity and localization in various subtypes of epithelial ovarian cancer in correlation with oncogene expression. Histologically confirmed malignant ovarian tissue from ten women and tissue from 2 benign ovarian tumors and 4 normal ovaries were assessed for heparanase presence, activity and localization, incidence of apoptosis and expression of the oncogenes erbB2 and Mdm2. Heparanase immunohistostaining and activity were present in mucinous carcinomas and were more intense than in endometrioid and in serous carcinomas. The lowest activity was observed in benign ovarian tumors and normal ovaries. In ovarian carcinomas the enzyme was intensely concentrated in the cytoplasm of the cancerous cells. In contrast, in normal ovaries and benign tumors the enzyme was predominantly localized in endothelial cells lining blood capillaries. The rate of apoptosis was considerably higher in mucinous and endometrioid carcinomas, and was lower in serous and primary peritoneal carcinomas. Extremely high concentration of heparanase was often demonstrated in apoptotic cells. Endometrioid and serous carcinomas showed high expression of Mdm2 and erbB2 while mucinous carcinomas showed low expression. In benign ovarian tumors and normal ovaries the expression of both oncoproteins was extremely low. In conclusion ovarian carcinomas demonstrate higher levels of heparanase than benign tumors and normal ovaries suggesting that the enzyme may play an important role in metastatic spread of the cancerous cells. Apoptosis may be a significant part of the mechanism of the enzyme release into the extracellular space. Although heparanase activity seems to play an essential role in tumor progression, expression of oncogenes, such as erbB2 and Mdm2 seems to play the dominant role in the development of ovarian cancer.

Asunto(s)

Adenocarcinoma Mucinoso/enzimología , Carcinoma Endometrioide/enzimología , Cistadenocarcinoma Seroso/enzimología , Glucuronidasa/metabolismo , Proteínas Nucleares , Neoplasias Ováricas/enzimología , Proteínas Proto-Oncogénicas/metabolismo , Receptor ErbB-2/metabolismo , Adenocarcinoma Mucinoso/patología , Adulto , Apoptosis , Western Blotting , Carcinoma Endometrioide/patología , Cistadenocarcinoma Seroso/patología , Femenino , Amplificación de Genes , Expresión Génica , Glucuronidasa/genética , Humanos , Técnicas para Inmunoenzimas , Hibridación in Situ , Persona de Mediana Edad , Estadificación de Neoplasias , Neoplasias Ováricas/patología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-mdm2 , Sondas ARN , Receptor ErbB-2/genética

RESUMEN

Ovarian cancer is among the most lethal cancers in women because of its high metastatic potential and lack of response to therapy. An experimental model to study this disease was developed using a transformed granulosa cell line expressing a mutant p53 and Ha-ras. When injected into the ovary of nude mice in the presence of laminin-1, tumors develop in the ovary and peritoneum and metastasize to various organs, leading to death within 21 days. In contrast, when cells were injected in the presence of gelatin, development of tumors was slower and no metastases were observed by day 21. Here we investigated the possible mechanism by which laminin-1 exerts its promotion of tumorigenesis and metastasis. Cells were co-injected with laminin-1 and active laminin peptides from the alpha1; (A13: RQVFQVAYIIIKA, A12: WVTVTLDL RQVFQ, AG73: LQVQLSIR, IKVAV) and beta1 (YIGSR) chains. Ovarian tumor growth and metastasis were increased in the presence of laminin-1 plus either AG73 peptide, IKVAV, or A13, and were significantly reduced in the presence of A12 or YIGSR. Expression of Bcl-2 and Mdm2 was higher by 3.5- and about 100-fold, respectively, in ovarian tumors grown in the presence of laminin compared to tumors grown in the presence of gelatin. Moreover, peptides A13 and AG73 further elevated Bcl-2 expression by 6- and 7-fold respectively, while IKVAV yielded expression similar to laminin-1. YIGSR and A12 reduced the expression of Bcl-2 by 7- and 3-fold, respectively, compared to treatment with laminin-1. A13 and AG73 increased Mdm2 expression by 1.8- and 1.3-fold, respectively, while IKVAV, A12, and YIGSR were without effect. Thus, laminin-1 exerts its proliferative effect on the development of ovarian tumors via upregulation of survival genes such as Bcl-2 and Mdm2. Peptides A13 and AG73 (which increased tumor growth and spread) enhance the expression of these genes and A12 and YIGSR (which decrease tumor growth and spread) attenuate their expression. IKVAV probably enhances tumor growth and metastasis by another mechanism.

Asunto(s)

Laminina/fisiología , Proteínas Nucleares , Neoplasias Ováricas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Apoptosis , Western Blotting , División Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Ratones Desnudos , Microscopía Electrónica de Rastreo , Metástasis de la Neoplasia , Trasplante de Neoplasias , Neoplasias Ováricas/patología , Fragmentos de Péptidos/fisiología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-mdm2 , Células Tumorales Cultivadas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia Arriba , Proteína X Asociada a bcl-2

RESUMEN

The response of granulosa cells to luteinizing hormone (LH) and follicle-stimulating hormone (FSH) is mediated mainly by cAMP/protein kinase A (PKA) signaling. Notably, the activity of the extracellular signal-regulated kinase (ERK) signaling cascade is elevated in response to these stimuli as well. We studied the involvement of the ERK cascade in LH- and FSH-induced steroidogenesis in two granulosa-derived cell lines, rLHR-4 and rFSHR-17, respectively. We found that stimulation of these cells with the appropriate gonadotropin induced ERK activation as well as progesterone production downstream of PKA. Inhibition of ERK activity enhanced gonadotropin-stimulated progesterone production, which was correlated with increased expression of the steroidogenic acute regulatory protein (StAR), a key regulator of progesterone synthesis. Therefore, it is likely that gonadotropin-stimulated progesterone formation is regulated by a pathway that includes PKA and StAR, and this process is down-regulated by ERK, due to attenuation of StAR expression. Our results suggest that activation of PKA signaling by gonadotropins not only induces steroidogenesis but also activates down-regulation machinery involving the ERK cascade. The activation of ERK by gonadotropins as well as by other agents may be a key mechanism for the modulation of gonadotropin-induced steroidogenesis.

Asunto(s)

Gonadotropinas/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Transducción de Señal , Esteroides/biosíntesis , Animales , Línea Celular , Gonadotropina Coriónica/metabolismo , Colforsina/farmacología , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Femenino , Flavonoides/farmacología , Hormona Folículo Estimulante/metabolismo , Células de la Granulosa/metabolismo , Humanos , Hormona Luteinizante/metabolismo , Sistema de Señalización de MAP Quinasas , Microscopía Fluorescente , Modelos Biológicos , Fosfoproteínas/biosíntesis , Fosforilación , Plásmidos/metabolismo , Progesterona/biosíntesis , Progesterona/metabolismo , Unión Proteica , Ratas , Factores de Tiempo , Transfección

RESUMEN

StAR, a protein synthesized in the cytoplasm and subsequently imported into mitochondria, regulates the rate-determining step in steroidogenesis, the transport of cholesterol from the outer to the inner mitochondrial membrane. The active form of StAR is the 37 kDa pre-protein, which has a short half-life. To determine whether proteasomes participate in the turnover of StAR, we incubated primary cultures of preovulatory rat granulosa cells and immortalized human granulosa cells in the presence of MG132, a specific inhibitor to proteasome catalysis. This treatment caused accumulation of StAR in unstimulated cells. Moreover, incubation of the cells with MG132 in the presence of forskolin (FK), luteinizing hormone/chorionic gonadotropin or follicular stimulating hormone augmented the accumulation of both the 37 kDa cytoplasmic protein and the 30 kDa mature mitochondrial protein, compared to cells incubated with FK or the gonadotropic hormones alone. Concomitantly, progesterone production was enhanced. In contrast no elevation in the 37 kDa StAR intracellular levels or progesterone production was observed following incubation of the cells with the cysteine protease inhibitor E-64. The increase of the 37 kDa StAR protein was evident after 15 min and 30 min of incubation with MG132 (143% and 187% of control values, respectively) with no significant elevation of the 30 kDa protein. Accumulation of the intermediate mitochondrial 32 kDa protein was evident after 1-2 h and the accumulation of the 30 kDa protein was evident only after 4 h of incubation with MG132. In contrast, no elevation in adrenodoxin, a component of the cytochrome P450scc enzyme system, was found. These data suggest that StAR protein is either directly or indirectly degraded by the proteasome which may explain, in part, its short half-life. Moreover, it seems that the cytosolic 37 kDa protein, which is responsible for the steroidogenic activity of StAR, is the primary proteasomal substrate and that the inhibition of its degradation by MG132 causes the up-regulation of progesterone production.

Asunto(s)

Leupeptinas/farmacología , Complejos Multienzimáticos/antagonistas & inhibidores , Fosfoproteínas/metabolismo , Adrenodoxina/metabolismo , Animales , Western Blotting , Línea Celular , Células Cultivadas , Colforsina/farmacología , Cisteína Endopeptidasas , Inhibidores de Cisteína Proteinasa/metabolismo , Citoplasma/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Células de la Granulosa/metabolismo , Humanos , Inmunohistoquímica , Microscopía Fluorescente , Microscopía de Contraste de Fase , Progesterona/biosíntesis , Complejo de la Endopetidasa Proteasomal , Ratas , Esteroides/biosíntesis , Factores de Tiempo , Regulación hacia Arriba

RESUMEN

Primary granulosa cells obtained from PMSG primed immature rats were triple transfected with SV40 DNA, Ha-ras oncogene and an expression vector containing human beta(2)-adrenergic receptors resulting in granulosa cell lines constitutively expressing the beta(2)-adrenergic receptors. Isoproterenol, a potent adrenergic agent, stimulated both cAMP accumulation and progesterone production in these cells in a dose dependent manner. Responsiveness of these cells was specific only to isoproterenol, while hCG (2.4 nM) and hFSH (2.4 nM) had no effect on steroid production. ED(50) for stimulation of cAMP and progesterone in these cells by isoproterenol was 2x10(-6) M and 7x10(-6) M, respectively. Forskolin also showed a dose dependent stimulation of cAMP and progesterone with ED(50) of 1.5 and 0.35 microg/ml, respectively. Epinephrine at a dose of 10(-5) M elicited maximum response to produce cAMP and progesterone. Isoproterenol induced accumulation of cAMP and progesterone in these cells were inhibited by beta(2)-adrenergic blocker, propranolol with an ED(50) of 6x10(-8) and 7x10(-9) M, respectively, whereas the beta(1)-adrenergic blocker, metoprolol was effective only at a very high concentration (ED(50)>10(-4) and 1.9x10(-5) M for inhibiting isoproterenol induced cAMP and progesterone production, respectively). Induction of steroidogenesis by isoproterenol or forskolin involved de novo synthesis of the cytochrome P450 side chain cleavage (SCC) enzyme complex, as assessed by indirect immunofluorescence staining for adrenodoxin. Western analysis indicate that expression of adrenodoxin is upregulated by forskolin, isoproterenol and adrenalin by 7.8-, 6.9- and 10.8-fold, respectively. The presence of StAR protein was identified by Western blotting. StAR expression was elevated by 8.3-, 2.5- and 4.7-fold upon stimulation with forskolin, isoproterenol and adrenalin, respectively. Thus, this cell line could serve as a good model system to study catecholamine mediated regulation of growth and differentiation of granulosa cells and the role of oncogenes in this process.

Asunto(s)

Adrenodoxina/metabolismo , Células de la Granulosa/metabolismo , Isoproterenol/farmacología , Fosfoproteínas/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Transfección , 1-Metil-3-Isobutilxantina/farmacología , Agonistas de Receptores Adrenérgicos beta 2 , Antagonistas de Receptores Adrenérgicos beta 2 , Adrenodoxina/inmunología , Animales , Western Blotting , Línea Celular Transformada , Gonadotropina Coriónica/farmacología , Colforsina/farmacología , AMP Cíclico/biosíntesis , AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Epinefrina/farmacología , Femenino , Hormona Folículo Estimulante/farmacología , Genes ras/genética , Células de la Granulosa/efectos de los fármacos , Inmunohistoquímica , Metoprolol/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Fosfoproteínas/inmunología , Plásmidos/genética , Plásmidos/metabolismo , Progesterona/metabolismo , Propranolol/farmacología , Ratas , Receptores Adrenérgicos beta 2/genética , Virus 40 de los Simios/genética , Virus 40 de los Simios/metabolismo

RESUMEN

Cisplatin is in common use in ovarian cancer therapy, although it is also implicated in cytotoxicity in normal tissue. We have examined the effect of cisplatin alone and in combination with theophylline, a phoshodiesterase inhibitor, on modulation of Bcl-2/Bax expression and induction of apoptosis in human granulosa cells transformed by stable transfection with mutant p53 plus Ha-ras. Theophylline elicited cell death only at relatively high concentrations with an EC50 of 200 microg/ml. Cisplatin exerted its lethal effect with an EC50 of 7 microM. In the presence of 15 or 50 microg/ml of theophylline (in the range used against asthma in humans), the EC50 for cisplatin was reduced to 2 microM or 1.2 microM, respectively. Using fluorescence-activated cell sorting analysis of DNA stained cells and the terminal deoxy-nucleotide tranferase-mediated dUTP nick end-labeling method, we found that even at concentrations of 0. 3 and 1 microM cisplatin, theophylline at 15 and 50 microg/ml increased the incidence of apoptosis in these cells by 3-5-fold, while theophylline alone induced extremely low apoptosis. Neither drug had any measurable effect on Bax protein expression. In contrast Bcl-2 protein expression levels were markedly reduced by theophylline and cisplatin in a dose-dependent manner. The combination of theophylline and cisplatin resulted in a further dramatic reduction in Bcl-2, under-scoring the pronounced synergy of these two drugs. These observations suggest that suppression of Bcl-2 expression may play an important role in mediating the synergistic effect of cisplatin and theophylline on induction of apoptosis in ovarian cancer cells.

Asunto(s)

Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Cisplatino/farmacología , Proteínas de Neoplasias/efectos de los fármacos , Inhibidores de Fosfodiesterasa/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/efectos de los fármacos , Teofilina/farmacología , Antineoplásicos/uso terapéutico , Apoptosis/fisiología , Cisplatino/uso terapéutico , Quimioterapia Combinada , Femenino , Genes ras/efectos de los fármacos , Genes ras/fisiología , Células de la Granulosa/efectos de los fármacos , Células de la Granulosa/metabolismo , Humanos , Proteínas de Neoplasias/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Inhibidores de Fosfodiesterasa/uso terapéutico , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Teofilina/uso terapéutico , Células Tumorales Cultivadas/efectos de los fármacos , Proteína p53 Supresora de Tumor/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo