Oncogene-transformed granulosa cells as a model system for the study of steroidogenic processes

Amsterdam, Abraham; Hanukoglu, Israel; Suh, Byung Sun; Keren-Tal, Iris; Plehn-Dujowich, Debora; Sprengel, Rolf; Rennert, Hanna; Strauss, Jerome F.

doi:10.1016/0960-0760(92)90315-a

Cited by 16 publications

(7 citation statements)

References 68 publications

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“…The fact that cAMP only moderately elevates the expression of this factor in HO-23 cells is in line with the observation that Ad4BP/SF-1 appears at an early stage of gonadal development before the acquisition of steroidogenic activity (19,20). In contrast, cAMP dramatically elevates both StAR and the electron carrier ADX, which is an intrinsic part of the P450scc enzyme system (15,16,43). This observation suggests that these cells preserve cAMP-induced steroidogenesis similar to primary granulosa cells.…”

Section: Discussionsupporting

confidence: 84%

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Induction of Ad4BP/SF-1, Steroidogenic Acute Regulatory Protein, and Cytochrome P450scc Enzyme System Expression in Newly Established Human Granulosa Cell Lines

Hosokawa¹

1998

Endocrinology

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We have established immortalized human granulosa cells by triple transfection of primary cells obtained from in vitro fertilization patients with SV40 DNA, Ha-ras oncogene, and a temperature sensitive (ts) mutant of the tumor suppressor gene p53 (p53val135). Forty-one clones were isolated, and their steroidogenic responses were analyzed. While all the cell lines proliferate rapidly and show only traces of progesterone production, upon stimulation with 50 M of forskolin (FK), which elevates intracellular cAMP, they become steroidogenic as evidenced by progesterone production. The steroidogenic response of the cell lines was stable even after 20 generations and several cycles of freezing and thawing. A highly responsive cell line (HO-23) was further examined for characteristics of the steroidogenic response. Cells stimulated with FK and 8-Br-cAMP produced high levels of pregnenolone, progesterone, and 20␣-hydroxy-4-pregnen-3-one (20␣-OH-progesterone) comparable with amounts produced by highly differentiated primary human granulosa-luteal cells. Hydrocortisone and dexamethasone highly augment the cAMP-stimulated progesterone production, whereas testosterone and PRL enhanced cAMPinduced progesterone synthesis only moderately. Estradiol, insulinlike growth factor I, and insulin showed no significant effect on cAMPinduced steroidogenesis. The phorbol ester TPA, and basic fibroblast growth factor, dramatically suppress cAMP-induced production of progesterone, whereas bovine corneal endothelial cell ECM (BCE/ ECM) enhanced cAMP-induced progesterone and antagonized basic fibroblast growth factor suppression of cAMP-induced steroidogenesis. Steroidogenic factor 1 (Ad4BP/SF-1) was expressed in control cells, and its expression was augmented by FK, whereas the steroidogenic acute regulatory protein showed low expression in the nonstimulated cells but was clearly elevated upon cAMP stimulation and was slightly decreased by TPA in cAMP-stimulated cells. Expression of the electron carrier adrenodoxin (ADX), which is a part of the cytochrome P450scc enzyme system, was very low in nonstimulated cells but was dramatically elevated in FK-and 8-Br-cAMP-stimulated cells, whereas no reduction of ADX was evident in cells costimulated with FK and TPA. Immunocytochemical studies revealed a weak staining of ADX in mitochrondria of nonstimulated cells and intensive staining in highly clustered mitochondria of FK-or 8-Br-cAMP-stimulated cells. Only moderate reduction in ADX staining was evident in cells costimulated with FK and TPA. These unique cell lines can provide a useful model for the investigation of induced steroidogenesis in human granulosa cells. (Endocrinology 139: 4679 -4687, 1998)

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Section: Discussionsupporting

confidence: 84%

“…This result is in line with the preservation of response to glucocorticoid hormones in SV40-ras transfected rat granulosa cells (4,42,43). Glucocorticoid hormones also enhance gonadotropin-/cAMP-stimulated progesterone production in primary granulosa cells (1,2,44).…”

Section: Discussionsupporting

confidence: 81%

Induction of Ad4BP/SF-1, Steroidogenic Acute Regulatory Protein, and Cytochrome P450scc Enzyme System Expression in Newly Established Human Granulosa Cell Lines

Hosokawa¹

1998

Endocrinology

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“…The CHO cell mutant CT60 and its variant CT60 neo R (resistant to neomycin) HAT S (hypoxanthine, aminopterin, and thymidine sensitive) were provided by T. Y. Chang (Dartmouth College of Medicine, Hanover, NH) and grown in CT60 medium: Ham's F-12 medium (F-12) (Biofluids, Rockville, MD) supplemented with 10% fetal bovine serum (FBS, HyClone), 2 mM glutamine, 100 units͞ml penicillin, and 100 g͞ml streptomycin. The mouse ovarian granulosa cell lines, ELN and ELC, obtained from normal and NP-C BALB͞c mice, respectively, by simian virus 40 transformation (18) were grown in a 1:1 mixture of F-12 and Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% FBS and the above reagents. Yeast spheroplast fusion clones derived from CT60 cells were selected and maintained in CT60 medium as described above, plus 400 g͞ml G-418 (GIBCO͞BRL).…”

Section: Methodsmentioning

confidence: 99%

“…Restoration of the normal intracellular distribution and esterification of exogenous cholesterol was achieved when human chromosome 18, a chromosome partly syntenic to mouse chromosome 18 (15), was transferred into an immortalized cell line derived from this strain (SPM-3T3 cells) (16). Genetic cross breeding studies suggested that the gene responsible for NP-C in the C56BL͞KsJ sphingomyelinosis mice was the same gene mutated in the other strain (NP-C BALB͞c) (17), whereas cell fusion studies with a simian virus 40-transformed ovarian granulosa cell line (18) derived from the NP-C BALB͞c mice suggested that these mice were genotypically allelic to human NP-C linked to chromosome 18 (data not shown). These data were consistent with the linkage of human NP-C to chromosome 18q11-12 (4,5) and strongly suggested that murine and human NP-C are caused by mutations in the same gene.…”

mentioning

confidence: 99%

Substantial narrowing of the Niemann–Pick C candidate interval by yeast artificial chromosome complementation

Carstea²,

Cummings³

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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Niemann-Pick disease type C (NP-C) is an autosomal recessive lipidosis linked to chromosome 18q11-12, characterized by lysosomal accumulation of unesterified cholesterol and delayed induction of cholesterol-mediated homeostatic responses. This cellular phenotype is identifiable cytologically by filipin staining and biochemically by measurement of low-density lipoprotein-derived cholesterol esterification. The mutant Chinese hamster ovary cell line (CT60), which displays the NP-C cellular phenotype, was used as the recipient for a complementation assay after somatic cell fusions with normal and NP-C murine cells suggested that this Chinese hamster ovary cell line carries an alteration(s) in the hamster homolog(s) of NP-C. To narrow rapidly the candidate interval for NP-C, three overlapping yeast artificial chromosomes (YACs) spanning the 1 centimorgan human NP-C interval were introduced stably into CT60 cells and analyzed for correction of the cellular phenotype. Only YAC 911D5 complemented the NP-C phenotype, as evidenced by cytological and biochemical analyses, whereas no complementation was obtained from the other two YACs within the interval or from a YAC derived from chromosome 7. Fluorescent in situ hybridization indicated that YAC 911D5 was integrated at a single site per CT60 genome. These data substantially narrow the NP-C critical interval and should greatly simplify the identification of the gene responsible in mouse and man. This is the first demonstration of YAC complementation as a valuable adjunct strategy for positional cloning of a human gene.Niemann-Pick disease type C (NP-C) is an autosomal recessive neurodegenerative disorder with heterogeneous manifestations. The classic clinical profile includes variable hepatosplenomegaly, vertical supra-nuclear ophthalmoplegia, progressive ataxia, dystonia, and dementia (1). The metabolic lesion is characterized by lysosomal sequestration of low-density lipoprotein (LDL)-derived cholesterol leading to delayed cholesterol-mediated homeostatic responses (2, 3). Lysosomal cholesterol accumulation can be depicted cytochemically by the cholesterol-specific fluorescent dye, filipin. Normally, endocytosed LDL-derived cholesterol is mobilized from lysosomes to the endoplasmic reticulum for esterification. As a result, there is little free cholesterol accumulation in lysosomes detectable by filipin staining in normal cells. In contrast, in NP-C cells the lysosomal accumulation of the endocytosed LDL-derived free cholesterol results in a specific perinuclear filipin-staining pattern. Biochemically, the NP-C phenotype can most conveniently be monitored by LDL-induced cholesterol ester synthesis. Cholesterol ester synthesis is markedly stimulated by LDL in normal cells, but not in NP-C cells.

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“…The screen for these compounds required an immortalized cell culture model for NPC disease and a biochemical assay that could be adapted for high throughput screening. The cell culture models that we used were simian virus 40-transformed mouse ovarian granulosa cell lines obtained from wildtype and npc nih BALB/c mice (24,25). The NPC1 gene in npc nih BALB/c mice has a retrotransposon-like insertion that leads to prematurely truncated NPC1 protein of approximately 500 amino acids (15).…”

Section: Development Of the Drug Screenmentioning

confidence: 99%

Identification of a pharmaceutical compound that partially corrects the Niemann-Pick C phenotype in cultured cells

Liscum

Arnio

Anthony

et al. 2002

Journal of Lipid Research

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Oncogene-transformed granulosa cells as a model system for the study of steroidogenic processes

Cited by 16 publications

References 68 publications

Induction of Ad4BP/SF-1, Steroidogenic Acute Regulatory Protein, and Cytochrome P450scc Enzyme System Expression in Newly Established Human Granulosa Cell Lines

Induction of Ad4BP/SF-1, Steroidogenic Acute Regulatory Protein, and Cytochrome P450scc Enzyme System Expression in Newly Established Human Granulosa Cell Lines

Substantial narrowing of the Niemann–Pick C candidate interval by yeast artificial chromosome complementation

Identification of a pharmaceutical compound that partially corrects the Niemann-Pick C phenotype in cultured cells

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