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Aaron J. W. Hsueh - One of the best experts on this subject based on the ideXlab platform.
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hormonal regulation of apoptosis in early antral follicles follicle stimulating hormone as a major survival factor
Endocrinology, 1996Co-Authors: Sangyoung Chun, Karen M Eisenhauer, Sawako Minami, Hakan Billig, Emerald Perlas, Aaron J. W. HsuehAbstract:Hormonal regulation of apoptosis has been studied in cultured preovulatory follicles. Because early antral follicles are most vulnerable to undergo atretic degeneration under physiological conditions in vivo, the present studies were designed to investigate the hormonal regulation of apoptosis using in vitro culture of early antral follicles. Rats were implanted with diethylstilbestrol at 24 days of age to stimulate the development of early antral follicles, and ovaries were collected at day 27 of age. Early antral follicles were dissected and cultured (four per vial) for 24 h with or without hormonal treatments. After culture, DNA was extracted from follicles, and the degree of Apoptotic DNA Fragmentation was determined using 3'-end labeling and gel electrophoresis. In situ analysis of Apoptotic DNA Fragmentation revealed that granulosa cells in these follicles are the main cell type undergoing apoptosis. Follicles cultured in the absence of hormones showed a 12-fold increase in the level of Apoptotic DN...
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hormonal regulation of apoptosis in early antral follicles follicle stimulating hormone as a major survival factor
Endocrinology, 1996Co-Authors: Sangyoung Chun, Karen M Eisenhauer, Sawako Minami, Hakan Billig, Emerald Perlas, Aaron J. W. HsuehAbstract:Hormonal regulation of apoptosis has been studied in cultured preovulatory follicles. Because early antral follicles are most vulnerable to undergo atretic degeneration under physiological conditions in vivo, the present studies were designed to investigate the hormonal regulation of apoptosis using in vitro culture of early antral follicles. Rats were implanted with diethylstilbestrol at 24 days of age to stimulate the development of early antral follicles, and ovaries were collected at day 27 of age. Early antral follicles were dissected and cultured (four per vial) for 24 h with or without hormonal treatments. After culture, DNA was extracted from follicles, and the degree of Apoptotic DNA Fragmentation was determined using 3'-end labeling and gel electrophoresis. In situ analysis of Apoptotic DNA Fragmentation revealed that granulosa cells in these follicles are the main cell type undergoing apoptosis. Follicles cultured in the absence of hormones showed a 12-fold increase in the level of Apoptotic DN...
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apoptosis in testis germ cells developmental changes in gonadotropin dependence and localization to selective tubule stages
Endocrinology, 1995Co-Authors: Hakan Billig, J. S. Tapanainen, I Furuta, C Rivier, M Parvinen, Aaron J. W. HsuehAbstract:Recent studies have demonstrated Apoptotic DNA Fragmentation in the testis of immature rats deprived of gonadotropins. However, the exact cell type undergoing apoptosis during testis development and the age differences of gonadotropin dependence of testis cell apoptosis are unclear. The present study used gel fractionation and in situ methods to quantitate developmental changes of testis cell DNA Fragmentation and to localize the specific cell type affected in developing rats with and without treatment with a GnRH antagonist. Apoptotic DNA Fragmentation in whole testis was measured in rats between 8-70 days of age. A gradual increase (1.8- to 2.0-fold) in testis Apoptotic DNA Fragmentation was seen in rats between 16-28 days of age, compared with 8-day-old animals, followed by a decrease in adult animals. To study gonadotropin dependence of testicular apoptosis, serum FSH and, to a lesser extent, LH were suppressed by treatment with a long-acting GnRH antagonist (azaline-B, 250 micrograms/kg body wt, two ...
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ovarian follicle atresia a hormonally controlled Apoptotic process
Endocrine Reviews, 1994Co-Authors: Aaron J. W. Hsueh, Hakan Billig, A TsafririAbstract:GREATER THAN 99% of ovarian follicles undergo a degenerative process called atresia during reproductive life. Extending earlier morphological analysis, recent studies have demonstrated that Apoptotic cell death is the molecular mechanism underlying follicle atresia. The use of DNA 3′-end-labeling methods allows quantitation and identification of internucleosomal degradation of DNA after gel fractionation as well as in situ analysis of specific cell types undergoing DNA Fragmentation in histological sections. Using rats as the experimental model, gonadotropins, epidermal growth factor (EGF)/transforming growth factor-α (TGFα), basic fibroblast growth factor (bFGF), insulin-like growth factor-I (IGF-I), and estrogens have been identified as follicle survival factors capable of suppressing Apoptotic DNA Fragmentation, whereas androgens, interleukin-6 (IL-6), and GnRH are potential atretogenic factors. This review summarizes the historical background of studies on follicle atresia and selection as well as rec...
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gonadotropin suppression of apoptosis in cultured preovulatory follicles mediatory role of endogenous insulin like growth factor i
Endocrinology, 1994Co-Authors: Sangyoung Chun, Hakan Billig, I Furuta, Jonathan L Tilly, A Tsafriri, Aaron J. W. HsuehAbstract:Although the majority of ovarian follicles undergo atresia through a mechanism involving Apoptotic cell death, in vivo studies concerning the hormonal regulation of atresia have been difficult due to the presence of heterogeneous population of follicles in the ovary. In the present study, the regulation of follicle apoptosis by gonadotropins, insulin-like growth factor I (IGF-I), and IGF-binding protein 3 (IGFBP-3) was examined using a serum-free culture of preovulatory follicles. Immature rats at 26 days of age received a single dose of PMSG. Two days later, the largest preovulatory follicles were collected for in vitro culture with or without hormones. After 24 h of culture, follicular Apoptotic DNA Fragmentation was analyzed by autoradiography of size-fractionated DNA labeled at 3'-ends by [32P]dideoxy-ATP. A spontaneous increase in Apoptotic DNA Fragmentation occurred after 24 h of culture in the absence of hormones, whereas treatment with human CG (hCG) or FSH suppressed follicular apoptosis in a dose-dependent manner, with 0.1 microgram/ml causing maximal suppression by 60-62%. Cotreatment with hCG and FSH had no additional effect. Like gonadotropins, treatment with IGF-I and insulin also suppressed the spontaneous onset of apoptosis, with IGF-I being more effective than insulin. Cotreatment with IGFBP-3 and hCG dose-dependently reversed the suppressive effect of hCG on apoptosis by 42%, suggesting a mediatory role of endogenously produced IGF-I. The addition of IGFBP-3 also blocked the suppressive action of IGF-I by 49%, whereas it did not affect the suppressive action of an IGF-I agonist or insulin. Treatment with IGFBP-3 alone had no effect on Apoptotic DNA Fragmentation. Estrogen and progesterone production by the cultured follicles were also analyzed by RIA. Gonadotropin treatment resulted in a marked stimulation of the production of both steroid productions. In contrast, treatment with IGF-I caused a small increase in estrogen but decreased progesterone production. Although treatment with IGFBP-3 alone decreased both estrogen and progesterone production, cotreatment with IGFBP-3 and hCG resulted in a slight decrease in estrogen production but an increase in progesterone production. Furthermore, IGFBP-3 did not affect IGF-I action on steroid production. To further substantiate the hypothesis that IGFBP-3 blocks the suppressive effect of hCG on apoptosis by neutralizing endogenously produced IGF-I, solution hybridization analysis was performed, and hCG treatment was shown to increase IGF-I messenger RNA levels in cultured follicles by 1.9-fold.(ABSTRACT TRUNCATED AT 400 WORDS)
Hakan Billig - One of the best experts on this subject based on the ideXlab platform.
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hormonal regulation of apoptosis in early antral follicles follicle stimulating hormone as a major survival factor
Endocrinology, 1996Co-Authors: Sangyoung Chun, Karen M Eisenhauer, Sawako Minami, Hakan Billig, Emerald Perlas, Aaron J. W. HsuehAbstract:Hormonal regulation of apoptosis has been studied in cultured preovulatory follicles. Because early antral follicles are most vulnerable to undergo atretic degeneration under physiological conditions in vivo, the present studies were designed to investigate the hormonal regulation of apoptosis using in vitro culture of early antral follicles. Rats were implanted with diethylstilbestrol at 24 days of age to stimulate the development of early antral follicles, and ovaries were collected at day 27 of age. Early antral follicles were dissected and cultured (four per vial) for 24 h with or without hormonal treatments. After culture, DNA was extracted from follicles, and the degree of Apoptotic DNA Fragmentation was determined using 3'-end labeling and gel electrophoresis. In situ analysis of Apoptotic DNA Fragmentation revealed that granulosa cells in these follicles are the main cell type undergoing apoptosis. Follicles cultured in the absence of hormones showed a 12-fold increase in the level of Apoptotic DN...
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hormonal regulation of apoptosis in early antral follicles follicle stimulating hormone as a major survival factor
Endocrinology, 1996Co-Authors: Sangyoung Chun, Karen M Eisenhauer, Sawako Minami, Hakan Billig, Emerald Perlas, Aaron J. W. HsuehAbstract:Hormonal regulation of apoptosis has been studied in cultured preovulatory follicles. Because early antral follicles are most vulnerable to undergo atretic degeneration under physiological conditions in vivo, the present studies were designed to investigate the hormonal regulation of apoptosis using in vitro culture of early antral follicles. Rats were implanted with diethylstilbestrol at 24 days of age to stimulate the development of early antral follicles, and ovaries were collected at day 27 of age. Early antral follicles were dissected and cultured (four per vial) for 24 h with or without hormonal treatments. After culture, DNA was extracted from follicles, and the degree of Apoptotic DNA Fragmentation was determined using 3'-end labeling and gel electrophoresis. In situ analysis of Apoptotic DNA Fragmentation revealed that granulosa cells in these follicles are the main cell type undergoing apoptosis. Follicles cultured in the absence of hormones showed a 12-fold increase in the level of Apoptotic DN...
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apoptosis in testis germ cells developmental changes in gonadotropin dependence and localization to selective tubule stages
Endocrinology, 1995Co-Authors: Hakan Billig, J. S. Tapanainen, I Furuta, C Rivier, M Parvinen, Aaron J. W. HsuehAbstract:Recent studies have demonstrated Apoptotic DNA Fragmentation in the testis of immature rats deprived of gonadotropins. However, the exact cell type undergoing apoptosis during testis development and the age differences of gonadotropin dependence of testis cell apoptosis are unclear. The present study used gel fractionation and in situ methods to quantitate developmental changes of testis cell DNA Fragmentation and to localize the specific cell type affected in developing rats with and without treatment with a GnRH antagonist. Apoptotic DNA Fragmentation in whole testis was measured in rats between 8-70 days of age. A gradual increase (1.8- to 2.0-fold) in testis Apoptotic DNA Fragmentation was seen in rats between 16-28 days of age, compared with 8-day-old animals, followed by a decrease in adult animals. To study gonadotropin dependence of testicular apoptosis, serum FSH and, to a lesser extent, LH were suppressed by treatment with a long-acting GnRH antagonist (azaline-B, 250 micrograms/kg body wt, two ...
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ovarian follicle atresia a hormonally controlled Apoptotic process
Endocrine Reviews, 1994Co-Authors: Aaron J. W. Hsueh, Hakan Billig, A TsafririAbstract:GREATER THAN 99% of ovarian follicles undergo a degenerative process called atresia during reproductive life. Extending earlier morphological analysis, recent studies have demonstrated that Apoptotic cell death is the molecular mechanism underlying follicle atresia. The use of DNA 3′-end-labeling methods allows quantitation and identification of internucleosomal degradation of DNA after gel fractionation as well as in situ analysis of specific cell types undergoing DNA Fragmentation in histological sections. Using rats as the experimental model, gonadotropins, epidermal growth factor (EGF)/transforming growth factor-α (TGFα), basic fibroblast growth factor (bFGF), insulin-like growth factor-I (IGF-I), and estrogens have been identified as follicle survival factors capable of suppressing Apoptotic DNA Fragmentation, whereas androgens, interleukin-6 (IL-6), and GnRH are potential atretogenic factors. This review summarizes the historical background of studies on follicle atresia and selection as well as rec...
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gonadotropin suppression of apoptosis in cultured preovulatory follicles mediatory role of endogenous insulin like growth factor i
Endocrinology, 1994Co-Authors: Sangyoung Chun, Hakan Billig, I Furuta, Jonathan L Tilly, A Tsafriri, Aaron J. W. HsuehAbstract:Although the majority of ovarian follicles undergo atresia through a mechanism involving Apoptotic cell death, in vivo studies concerning the hormonal regulation of atresia have been difficult due to the presence of heterogeneous population of follicles in the ovary. In the present study, the regulation of follicle apoptosis by gonadotropins, insulin-like growth factor I (IGF-I), and IGF-binding protein 3 (IGFBP-3) was examined using a serum-free culture of preovulatory follicles. Immature rats at 26 days of age received a single dose of PMSG. Two days later, the largest preovulatory follicles were collected for in vitro culture with or without hormones. After 24 h of culture, follicular Apoptotic DNA Fragmentation was analyzed by autoradiography of size-fractionated DNA labeled at 3'-ends by [32P]dideoxy-ATP. A spontaneous increase in Apoptotic DNA Fragmentation occurred after 24 h of culture in the absence of hormones, whereas treatment with human CG (hCG) or FSH suppressed follicular apoptosis in a dose-dependent manner, with 0.1 microgram/ml causing maximal suppression by 60-62%. Cotreatment with hCG and FSH had no additional effect. Like gonadotropins, treatment with IGF-I and insulin also suppressed the spontaneous onset of apoptosis, with IGF-I being more effective than insulin. Cotreatment with IGFBP-3 and hCG dose-dependently reversed the suppressive effect of hCG on apoptosis by 42%, suggesting a mediatory role of endogenously produced IGF-I. The addition of IGFBP-3 also blocked the suppressive action of IGF-I by 49%, whereas it did not affect the suppressive action of an IGF-I agonist or insulin. Treatment with IGFBP-3 alone had no effect on Apoptotic DNA Fragmentation. Estrogen and progesterone production by the cultured follicles were also analyzed by RIA. Gonadotropin treatment resulted in a marked stimulation of the production of both steroid productions. In contrast, treatment with IGF-I caused a small increase in estrogen but decreased progesterone production. Although treatment with IGFBP-3 alone decreased both estrogen and progesterone production, cotreatment with IGFBP-3 and hCG resulted in a slight decrease in estrogen production but an increase in progesterone production. Furthermore, IGFBP-3 did not affect IGF-I action on steroid production. To further substantiate the hypothesis that IGFBP-3 blocks the suppressive effect of hCG on apoptosis by neutralizing endogenously produced IGF-I, solution hybridization analysis was performed, and hCG treatment was shown to increase IGF-I messenger RNA levels in cultured follicles by 1.9-fold.(ABSTRACT TRUNCATED AT 400 WORDS)
Shivendra V Singh - One of the best experts on this subject based on the ideXlab platform.
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phenethyl isothiocyanate inhibits oxidative phosphorylation to trigger reactive oxygen species mediated death of human prostate cancer cells
Journal of Biological Chemistry, 2010Co-Authors: Dong Xiao, Ajay Bommareddy, Eunryeong Hahm, Anna A Powolny, Suhyeong Kim, Michelle Barbi De Moura, Eric E Kelley, Daniel P Normolle, Bennett Van Houten, Shivendra V SinghAbstract:Phenethyl isothiocyanate (PEITC), a constituent of edible cruciferous vegetables such as watercress, not only affords significant protection against chemically induced cancer in experimental rodents but also inhibits growth of human cancer cells by causing Apoptotic and autophagic cell death. However, the underlying mechanism of PEITC-induced cell death is not fully understood. Using LNCaP and PC-3 human prostate cancer cells as a model, we demonstrate that the PEITC-induced cell death is initiated by production of reactive oxygen species (ROS) resulting from inhibition of oxidative phosphorylation (OXPHOS). Exposure of LNCaP and PC-3 cells to pharmacologic concentrations of PEITC resulted in ROS production, which correlated with inhibition of complex III activity, suppression of OXPHOS, and ATP depletion. These effects were not observed in a representative normal human prostate epithelial cell line (PrEC). The ROS production by PEITC treatment was not influenced by cyclosporin A. The Rho-0 variants of LNCaP and PC-3 cells were more resistant to PEITC-mediated ROS generation, Apoptotic DNA Fragmentation, and collapse of mitochondrial membrane potential compared with respective wild-type cells. The PEITC treatment resulted in activation of Bax in wild-type LNCaP and PC-3 cells, but not in their respective Rho-0 variants. Furthermore, RNA interference of Bax and Bak conferred significant protection against PEITC-induced apoptosis. The Rho-0 variants of LNCaP and PC-3 cells also resisted PEITC-mediated autophagy. In conclusion, the present study provides novel insight into the molecular circuitry of PEITC-induced cell death involving ROS production due to inhibition of complex III and OXPHOS.
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abstract 3782 benzyl isothiocyanate induced apoptosis in human breast cancer cells is mediated by downregulation of x linked inhibitor of apoptosis protein
Cancer Research, 2010Co-Authors: Suhyeong Kim, Shivendra V SinghAbstract:Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Benzyl isothiocyanate (BITC) is a highly promising cancer chemopreventive constituent of edible cruciferous vegetables such as gardencress. We have shown previously that BITC treatment inhibits viability of MDA-MB-231 (an estrogen-independent cell line expressing mutant p53) and MCF-7 (an estrogen-responsive cell line with wild-type p53) by causing reactive oxygen species-mediated apoptosis. In addition, oral administration of BITC inhibits growth of MDA-MB-231 human breast cancer cells subcutaneously or orthotopically implanted in female athymic mice without causing weight loss or any other side effects. More recent studies from our laboratory have indicated that dietary feeding of BITC offers protection against mammary hyperplasia and carcinoma development in MMTV-neu transgenic mice. The present study extends these exciting observations and determines role of p53 and inhibitor of apoptosis (IAP) family proteins in regulation of BITC-induced apoptosis. Exposure of MCF-7 and MDA-MB-231 cells to apoptosis inducing concentrations of BITC (2.5 and 5.0 μM) resulted in up-regulation of p53 in MCF-7 cell line and down-regulation of X-linked inhibitor of apoptosis (XIAP) in both cell lines. The BITC-mediated down-regulation of XIAP was evident as early as 8 h post-treatment. BITC-treated cell also exhibited induction of survivin especially in the MCF-7 cell line. The level of cIAP2 protein was only modestly increased upon treatment of MCF-7 and MDA-MB-231 cells with BITC. The BITC-induced apoptosis was only marginally affected by knockdown of p53 protein level by RNA interference. On the otehr hand, forced expression of XIAP protein conferred significant protection against BITC-mediated cytoplasmic histone-associated Apoptotic DNA Fragmentation in both cell lines. The BITC-induced cytoplasmic histone-associated Apoptotic DNA Fragmentation was significantly increased by knockdown of survivin protein in the MCF-7 cell line but not in the MDA-MB-231. These results indicate that down-regulation of XIAP contributes to the BITC-induced apoptosis in breast cancer cells. In addition, induction of survivin confers resistance against BITC-mediated cell death at least in the MCF-7 cell line. This study was supported by the National Cancer Institute grant CA129347-03. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3782.
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p66shc is indispensable for phenethyl isothiocyanate induced apoptosis in human prostate cancer cells
Cancer Research, 2010Co-Authors: Dong Xiao, Shivendra V SinghAbstract:Naturally occurring phenethyl isothiocyanate (PEITC) selectively inhibits growth of cancer cells by causing apoptosis, but the mechanism of cell death induction is not fully understood. We now show, for the first time, that growth factor adapter protein p66(Shc) is indispensable for PEITC-induced apoptosis. Mouse embryonic fibroblasts derived from p66(Shc) knockout mice were significantly more resistant to PEITC-mediated growth inhibition, cytoplasmic histone-associated Apoptotic DNA Fragmentation, and caspase-3 activation compared with wild-type fibroblasts. The PEITC treatment resulted in induction as well as increased Ser(36) phosphorylation of p66(Shc) in PC-3 and LNCaP human prostate cancer cells. Knockdown of p66(Shc) protein conferred significant protection against PEITC-mediated cytoplasmic histone-associated DNA Fragmentation as well as production of reactive oxygen species in both PC-3 and LNCaP cells. The PEITC-treated PC-3 and LNCaP cells exhibited increased binding of p66(Shc) with prolyl isomerase Pin1, a protein implicated in translocation of p66(Shc) to mitochondria. Consistent with these results, treatment of PC-3 cells with PEITC resulted in translocation of p66(Shc) to the mitochondria as judged by immunoblotting using cytosolic and mitochondrial fractions and immunofluorescence microscopy. Growth suppression and apoptosis induction in tumor xenografts in vivo by oral administration of PEITC to the PC-3 tumor-bearing male athymic mice were accompanied by statistically significant increase in the level of Ser(36)-phosphorylated p66(Shc). Collectively, these results provide novel insight into the critical role of p66(Shc) in regulation of PEITC-induced Apoptotic cell death in human prostate cancer cells.
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atg5 regulates phenethyl isothiocyanate induced autophagic and Apoptotic cell death in human prostate cancer cells
Cancer Research, 2009Co-Authors: Ajay Bommareddy, Eunryeong Hahm, Dong Xiao, Anna A Powolny, Alfred L Fisher, Yu Jiang, Shivendra V SinghAbstract:Phenethyl isothiocyanate (PEITC) is a promising cancer chemopreventive agent but the mechanism of its anticancer effect is not fully understood. We now show, for the first time, that PEITC treatment triggers Atg5-dependent autophagic and Apoptotic cell death in human prostate cancer cells. Exposure of PC-3 (androgen independent, p53 null) and LNCaP (androgen responsive, wild-type p53) human prostate cancer cells to PEITC resulted in several specific features characteristic of autophagy, including appearance of membranous vacuoles, formation of acidic vesicular organelles, and cleavage and recruitment of microtubule-associated protein 1 light chain 3 (LC3) to autophagosomes. A normal human prostate epithelial cell line (PrEC) was markedly more resistant toward PEITC-mediated cleavage and recruitment of LC3 compared with prostate cancer cells. Although PEITC treatment suppressed activating phosphorylations of Akt and mammalian target of rapamycin (mTOR), which are implicated in regulation of autophagy by different stimuli, processing and recruitment of LC3 was only partially/marginally reversed by ectopic expression of constitutively active Akt or overexpression of mTOR-positive regulator Rheb. The PEITC-mediated Apoptotic DNA Fragmentation was significantly attenuated in the presence of a pharmacologic inhibitor of autophagy (3-methyl adenine). Transient transfection of LNCaP and PC-3 cells with Atg5-specific small interfering RNA conferred significant protection against PEITC-mediated autophagy as well as Apoptotic DNA Fragmentation. A xenograft model using PC-3 cells and Caenorhabditis elegans expressing a lgg-1:GFP fusion protein provided evidence for occurrence of PEITC-induced autophagy in vivo. In conclusion, the present study indicates that Atg5 plays an important role in regulation of PEITC-induced autophagic and Apoptotic cell death.
Sangyoung Chun - One of the best experts on this subject based on the ideXlab platform.
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hormonal regulation of apoptosis in early antral follicles follicle stimulating hormone as a major survival factor
Endocrinology, 1996Co-Authors: Sangyoung Chun, Karen M Eisenhauer, Sawako Minami, Hakan Billig, Emerald Perlas, Aaron J. W. HsuehAbstract:Hormonal regulation of apoptosis has been studied in cultured preovulatory follicles. Because early antral follicles are most vulnerable to undergo atretic degeneration under physiological conditions in vivo, the present studies were designed to investigate the hormonal regulation of apoptosis using in vitro culture of early antral follicles. Rats were implanted with diethylstilbestrol at 24 days of age to stimulate the development of early antral follicles, and ovaries were collected at day 27 of age. Early antral follicles were dissected and cultured (four per vial) for 24 h with or without hormonal treatments. After culture, DNA was extracted from follicles, and the degree of Apoptotic DNA Fragmentation was determined using 3'-end labeling and gel electrophoresis. In situ analysis of Apoptotic DNA Fragmentation revealed that granulosa cells in these follicles are the main cell type undergoing apoptosis. Follicles cultured in the absence of hormones showed a 12-fold increase in the level of Apoptotic DN...
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hormonal regulation of apoptosis in early antral follicles follicle stimulating hormone as a major survival factor
Endocrinology, 1996Co-Authors: Sangyoung Chun, Karen M Eisenhauer, Sawako Minami, Hakan Billig, Emerald Perlas, Aaron J. W. HsuehAbstract:Hormonal regulation of apoptosis has been studied in cultured preovulatory follicles. Because early antral follicles are most vulnerable to undergo atretic degeneration under physiological conditions in vivo, the present studies were designed to investigate the hormonal regulation of apoptosis using in vitro culture of early antral follicles. Rats were implanted with diethylstilbestrol at 24 days of age to stimulate the development of early antral follicles, and ovaries were collected at day 27 of age. Early antral follicles were dissected and cultured (four per vial) for 24 h with or without hormonal treatments. After culture, DNA was extracted from follicles, and the degree of Apoptotic DNA Fragmentation was determined using 3'-end labeling and gel electrophoresis. In situ analysis of Apoptotic DNA Fragmentation revealed that granulosa cells in these follicles are the main cell type undergoing apoptosis. Follicles cultured in the absence of hormones showed a 12-fold increase in the level of Apoptotic DN...
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gonadotropin suppression of apoptosis in cultured preovulatory follicles mediatory role of endogenous insulin like growth factor i
Endocrinology, 1994Co-Authors: Sangyoung Chun, Hakan Billig, I Furuta, Jonathan L Tilly, A Tsafriri, Aaron J. W. HsuehAbstract:Although the majority of ovarian follicles undergo atresia through a mechanism involving Apoptotic cell death, in vivo studies concerning the hormonal regulation of atresia have been difficult due to the presence of heterogeneous population of follicles in the ovary. In the present study, the regulation of follicle apoptosis by gonadotropins, insulin-like growth factor I (IGF-I), and IGF-binding protein 3 (IGFBP-3) was examined using a serum-free culture of preovulatory follicles. Immature rats at 26 days of age received a single dose of PMSG. Two days later, the largest preovulatory follicles were collected for in vitro culture with or without hormones. After 24 h of culture, follicular Apoptotic DNA Fragmentation was analyzed by autoradiography of size-fractionated DNA labeled at 3'-ends by [32P]dideoxy-ATP. A spontaneous increase in Apoptotic DNA Fragmentation occurred after 24 h of culture in the absence of hormones, whereas treatment with human CG (hCG) or FSH suppressed follicular apoptosis in a dose-dependent manner, with 0.1 microgram/ml causing maximal suppression by 60-62%. Cotreatment with hCG and FSH had no additional effect. Like gonadotropins, treatment with IGF-I and insulin also suppressed the spontaneous onset of apoptosis, with IGF-I being more effective than insulin. Cotreatment with IGFBP-3 and hCG dose-dependently reversed the suppressive effect of hCG on apoptosis by 42%, suggesting a mediatory role of endogenously produced IGF-I. The addition of IGFBP-3 also blocked the suppressive action of IGF-I by 49%, whereas it did not affect the suppressive action of an IGF-I agonist or insulin. Treatment with IGFBP-3 alone had no effect on Apoptotic DNA Fragmentation. Estrogen and progesterone production by the cultured follicles were also analyzed by RIA. Gonadotropin treatment resulted in a marked stimulation of the production of both steroid productions. In contrast, treatment with IGF-I caused a small increase in estrogen but decreased progesterone production. Although treatment with IGFBP-3 alone decreased both estrogen and progesterone production, cotreatment with IGFBP-3 and hCG resulted in a slight decrease in estrogen production but an increase in progesterone production. Furthermore, IGFBP-3 did not affect IGF-I action on steroid production. To further substantiate the hypothesis that IGFBP-3 blocks the suppressive effect of hCG on apoptosis by neutralizing endogenously produced IGF-I, solution hybridization analysis was performed, and hCG treatment was shown to increase IGF-I messenger RNA levels in cultured follicles by 1.9-fold.(ABSTRACT TRUNCATED AT 400 WORDS)
Steven Grant - One of the best experts on this subject based on the ideXlab platform.
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induction of Apoptotic DNA Fragmentation and c jun downregulation in human myeloid leukemia cells by the permeant ca2 chelator bapta am
Oncology Research, 1995Co-Authors: Steven Grant, A J Freemerman, P C Gregory, H A Martin, A J Turner, R Mikkelsen, Jeyananthan Chelliah, S Yanovich, W D JarvisAbstract:The permeant Ca 2+ chelator acetoxymethyl-1,2-bis(2-aminopheoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA/AM), an agent previously used to characterize drug-induced apoptosis in neoplastic cells, has been examined with respect to induction of DNA Fragmentation and cytotoxicity in the human leukemia cell lines HL-60 and U937. Exposure of cells to various concentrations of BAPTA/AM for 6 h resulted in a biphasic induction of internucleosomal DNA cleavage, with maximal damage occurring at 10-μM concentrations. Higher BAPTA/AM concentrations were associated with the loss of internucleosomal cleavage products, but with the appearance of larger (i.e., 50-kilobase) fragments on pulsed-field gel electrophoresis. Cells exposed to 10 μM BAPTA/AM exhibited classic Apoptotic morphology, whereas cells exposed to 50-μM concentrations displayed atypical features (e.g., cell swelling, chromatin clumping) ; in each case, substantial cytotoxicity was noted. The actions of BAPTA/AM did not depend upon the presence of extracellular Ca 2+ , nor were they affected by impermeant Ca 2+ chelators. Measurement of cytosolic Ca 2+ by Fura-2/AM or Indo-1 revealed late but not early increases in intracellular Ca 2+ in BAPTA/AM-treated cells. Finally, BAPTA/AM-induced apoptosis was accompanied by the concentration-dependent downregulation of the immediate early response gene c-jun. These findings suggest a complex role for Ca 2+ chelators such as BAPTA/AM in the regulation of human myeloid leukemic cell apoptosis, and indicate that this agent may selectively antagonize internucleosomal DNA Fragmentation without interfering with other aspects of the Apoptotic response and/or cell lethality.
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induction of Apoptotic DNA Fragmentation and cell death in hl 60 human promyelocytic leukemia cells by pharmacological inhibitors of protein kinase c
Cancer Research, 1994Co-Authors: W D Jarvis, A J Turner, L F Povirk, R S Traylor, Steven GrantAbstract:The present studies were undertaken to characterize further the potential role of protein kinase C (PKC) in the regulation of apoptosis in HL-60 promyelocytic leukemia cells. The capacity of acute exposure to specific and nonspecific pharmacological inhibitors of PKC to promote Apoptotic DNA Fragmentation was examined both quantitatively and qualitatively and correlated with effects on cellular differentiation and proliferation. Incubation of HL-60 cells for 6 h with chelerythrine and calphostin C (highly specific inhibitors that act at the regulatory domain) or H7 and gossypol (nonspecific inhibitors that act at the PKC catalytic domain) produced concentration-dependent increases in DNA Fragmentation. Induction of DNA Fragmentation by chelerythrine, calphostin C, and gossypol was biphasic, resulting in a sharp decline in effect at concentrations above 5 microM, 0.1 microM, and 100 microM, respectively, whereas maximal and more stable effects were observed in response to H7 (100 microM). A 6-h exposure to staurosporine, a nonspecific but potent PKC inhibitor, failed to induce DNA Fragmentation at concentrations generally used to achieve maximal inhibition of enzyme activity (e.g., 50 nM) but promoted Fragmentation at considerably higher concentrations (e.g., > or = 200 nM). In contrast, 6-h exposures to the nonspecific protein kinase inhibitor hypericin (0.1 to 100 microM) or to the nonspecific inhibitor of protein kinase A, HA1004 (50 microM), were without effect on DNA Fragmentation. DNA obtained from cells exposed to chelerythrine (5 microM), calphostin C (100 nM), H7 (50 microM), gossypol (50 microM), and staurosporine (200 nM)--but not hypericin (25 microM)--exhibited clear evidence of internucleosomal DNA cleavage on agarose gel electrophoresis; moreover, these cells exhibited the classical morphological features of apoptosis (cell shrinkage, nuclear condensation, and the formation of Apoptotic bodies). All of the PKC inhibitors that induced apoptosis, and one of the inhibitors that did not (hypericin), substantially inhibited HL-60 cell clonogenicity at the concentrations evaluated. None of the agents tested induced cellular maturation as assessed by nonspecific esterase and nitro-blue tetrazolium positivity. DNA fragments obtained from cells exposed to specific and nonspecific PKC inhibitors possessed predominantly 5'-phosphate termini, consistent with the action of a Ca(2+)-/Mg(2+)-dependent endonuclease. Finally, Northern blot analysis revealed that exposure to calphostin C at a concentration that induced apoptosis (100 nM) failed to alter expression of bcl-2, an oncogene known to block apoptosis in both lymphoid and myeloid leukemia cells.(ABSTRACT TRUNCATED AT 400 WORDS)
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induction of Apoptotic DNA Fragmentation and cell death in hl 60 human promyelocytic leukemia cells by pharmacological inhibitors of protein kinase c
Cancer Research, 1994Co-Authors: W D Jarvis, A J Turner, L F Povirk, R S Traylor, Steven GrantAbstract:Abstract The present studies were undertaken to characterize further the potential role of protein kinase C (PKC) in the regulation of apoptosis in HL-60 promyelocytic leukemia cells. The capacity of acute exposure to specific and nonspecific pharmacological inhibitors of PKC to promote Apoptotic DNA Fragmentation was examined both quantitatively and qualitatively and correlated with effects on cellular differentiation and proliferation. Incubation of HL-60 cells for 6 h with chelerythrine and calphostin C (highly specific inhibitors that act at the regulatory domain) or H7 and gossypol (nonspecific inhibitors that act at the PKC catalytic domain) produced concentration-dependent increases in DNA Fragmentation. Induction of DNA Fragmentation by chelerythrine, calphostin C, and gossypol was biphasic, resulting in a sharp decline in effect at concentrations above 5 µm, 0.1 µm, and 100 µm, respectively, whereas maximal and more stable effects were observed in response to H7 (100 µm). A 6-h exposure to staurosporine, a nonspecific but potent PKC inhibitor, failed to induce DNA Fragmentation at concentrations generally used to achieve maximal inhibition of enzyme activity (e.g., 50 nm) but promoted Fragmentation at considerably higher concentrations (e.g., ≥200 nm). In contrast, 6-h exposures to the nonspecific protein kinase inhibitor hypericin (0.1 to 100 µm) or to the nonspecific inhibitor of protein kinase A, HA1004 (50 µm), were without effect on DNA Fragmentation. DNA obtained from cells exposed to chelerythrine (5 µm), calphostin C (100 nm), H7 (50 µm), gossypol (50 µm), and staurosporine (200 nm)—but not hypericin (25 µm)—exhibited clear evidence of internucleosomal DNA cleavage on agarose gel electrophoresis; moreover, these cells exhibited the classical morphological features of apoptosis (cell shrinkage, nuclear condensation, and the formation of Apoptotic bodies). All of the PKC inhibitors that induced apoptosis, and one of the inhibitors that did not (hypericin), substantially inhibited HL-60 cell clonogenicity at the concentrations evaluated. None of the agents tested induced cellular maturation as assessed by nonspecific esterase and nitro-blue tetrazolium positivity. DNA fragments obtained from cells exposed to specific and nonspecific PKC inhibitors possessed predominantly 5′-phosphate termini, consistent with the action of a Ca2+-/Mg2+-dependent endonuclease. Finally, Northern blot analysis revealed that exposure to calphostin C at a concentration that induced apoptosis (100 nm) failed to alter expression of bcl-2, an oncogene known to block apoptosis in both lymphoid and myeloid leukemia cells. Together, these observations suggest that certain inhibitors of PKC, administered alone and on a transient basis, are capable of inducing Apoptotic DNA Fragmentation and cell death in HL-60 promyelocytic leukemia cells in a highly concentration-dependent manner. These findings also suggest a protective involvement of basal PKC activity in the regulation of programmed cell death in myeloid leukemia cells.