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Endocrinology Vol. 139, No. 11 4688-4700
Copyright © 1998 by The Endocrine Society

ARTICLES

Modulation of Mdm2 Expression and p53-Induced Apoptosis in Immortalized Human Ovarian Granulosa Cells1

Kumiko Hosokawa, Dorit Aharoni, Ada Dantes, Eitan Shaulian, Carolina Schere-Levy, Ruth Atzmon, Fumikazu Kotsuji, Moshe Oren, Israel Vlodavsky and Abraham Amsterdam2

Department of Molecular Cell Biology, The Weizmann Institute of Science (K.H., D.A., A.D., E.S., C.S.-L., M.O., A.A.), Rehovot 76100, Israel; Department of Oncology, Hadassah-Hebrew University Hospital (R.A., I.V.), Jerusalem 91120, Israel; and Department of Obstetrics and Gynecology, Fukui Medical University (K.H., F.K.), Fukui 910–1193, Japan

Address all correspondence and requests for reprints to: A. Amsterdam, Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel. E-mail: lhamster{at}weizmann.weizmann.ac.il


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The activity of the tumor suppressor gene p53 is implicated in arrest of the cell cycle and the induction of apoptosis. The mdm2 oncogene is transcriptionally activated by p53, and the protein products of this gene can down-modulate biochemical activities and biological effects of p53 in a cell context-dependent manner. We have established highly steroidogenic human granulosa cell lines expressing the Ha-ras oncogene and a temperature sensitive (ts) mutant of p53 (p53val135) to test the involvement of p53-downstream genes in the modulation of apoptosis in these cells. We find that ras-transformed granulosa cells expressing p53val135 undergo apoptosis following a shift from 37 C to 32 C, a temperature at which p53val135 exerts its wild-type activity. Elevating the cellular content of cAMP at 32 C markedly enhances apoptosis. Basic fibroblast growth factor (bFGF) effectively blocks the p53/cAMP-induced apoptosis, but suppresses steroidogenesis. A naturally produced basement membrane-like extracellular matrix (ECM) containing immobilized bFGF exerts a similar antiapoptotic effect, but unlike soluble bFGF, it enhances steroidogenesis in these cells. While cAMP markedly suppresses the p53-induced Mdm2 expression, bFGF and ECM elevate Mdm2 expression 3–5-fold. These effects on Mdm2 expression are most pronounced 2–4 h after the shift to 32 C, before nuclear fragmentation is detected. Cells grown at 32 C in contact with ECM have a more developed actin cytoskeleton both in the absence and presence of cAMP stimulation, compared with cells grown on plastic dishes. We conclude that bFGF and components of the ECM can cross-talk with p53/cAMP-generated signals for apoptosis. These signals may, at least in part, be coordinated by the modulation of Mdm2 expression, which precedes the biochemical events characteristic of apoptosis. The multicomponent ECM also induced differentiation in these ras-transformed cells, while soluble bFGF inhibited differentiation, suggesting that ECM components other than bFGF stimulate differentiation. Organization of the actin cytoskeleton is likely to play an important role in the cross-talk between p53/cAMP- and bFGF/ECM-generated signals. Because the tumor supressor gene p53 is implicated with apoptosis of primary granulosa cells and the ECM is involved in the prevention of this process, the newly established cell lines can serve as a useful model for apoptosis in highly luteinized granulosa cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE SENSORY WORLD of eukaryotic cells includes receptors to hormones, growth factors, and receptors to extracellular matrix proteins such as integrins. These signals can lead to cell proliferation and transformation or to differentiation and programmed cell death. Coordination between various stimuli directs the cell to follow a specific developmental pathway, but the mechanism of such cross-talk is still obscure (reviewed in Refs. 1, 2, 3).

One of the main stimuli is exerted by the interaction of specific ligands with cell membrane receptors containing a seven transmembrane domain (4). A large family of these receptors is coupled to adenylate cyclase, the activation of which leads to an accumulation of cAMP (5, 6). As a second messenger, cAMP elicits a wide array of biological responses such as the induction of cell proliferation, cell differentiation, and apoptosis (7, 8, 9). The nature of the response depends on the amplitude and duration of the cAMP response (10), as well as on the cell type (7, 8, 9).

In ovarian granulosa cells, one of the main somatic components of the ovarian follicle, gonadotropin/cAMP-mediated signals, leads to activation of the steroidogenic factor (Ad4BP/SF-1) and up-regulation of both the steroidogenic acute regulatory protein (StAR) and the steroidogenic enzymes (11, 12, 13, 14). This up-regulation results in the production of estradiol and progesterone depending on the stage of the estrus or the menstrual cycle (13). Constant activation of adenylate cyclase with forskolin (FK) or stimulation of preovulatory granulosa cells with 8-Br-cAMP may lead to an excess of cAMP, causing the cells to undergo apoptosis (10). Transfection with SV40 DNA causes these cells to become resistant to cAMP-induced apoptosis (10). However, transfection with the temperature sensitive (ts) mutant of p53, p53val135, results in regaining the cAMP response in both the steroidogenic and apoptotic processes, suggesting that cAMP may cross-talk with p53-generated signals through as yet unknown mechanism (15).

Fibroblast growth factors (FGFs) are a family of polypeptides that stimulate proliferation, migration, and differentiation of cells of mesenchymal and neuroectodermal origin (16, 17). Basic FGF (bFGF), the most abundant member of the family, appears as a circulating molecule or as a complex with heparan sulfate sequestered in basement membranes and cell surfaces. Transfected NIH3T3 cells, expressing a chimeric bFGF modified by the addition of a signal peptide, undergo autocrine transformation accompanied by characteristic morphological and biochemical alterations (18, 19). The biological response of cells to FGF is mediated through high affinity cell surface receptors (FGFR), possessing intrinsic tyrosine kinase activity (20). FGF binding induces receptor dimerization (21), tyrosine kinase activation, and autophosphorylation (20). Basic FGF has also been implicated as a survival factor for a variety of cell types, including cAMP-stimulated rat ovarian granulosa cells (10). Moreover, bFGF can synergize with cAMP-generated signals leading to differentiation and steroidogenesis of granulosa cells through an as yet unknown mechanism (10, 22). Notably, bFGF can protect endothelial cells against radiation-induced apoptosis in vivo and in vitro (23) by largely unknown molecular mechanisms. Because p53 plays a key role in radiation-induced apoptosis (reviewed in Refs. 24, 25, 26), this suggests possible cross-talk between bFGF- and p53-generated signals. p53 is an important contributor to the regulation of apoptosis (reviewed in Ref. 27). In the mammalian ovary, where follicular apoptosis is essential for the selection of a dominant follicle and in eliminating excess developing follicles, elevation of p53 expression was associated with the appearance of atretic follicles. Atresia of these follicles is believed to occur via apoptosis (reviewed in Refs. 11, 28, 29), accompanied by up-regulation of the bax gene (30)—one of the key transcriptional targets of p53 (31). In recently established rat granulosa cell lines expressing the ts mutant p53val135, p53-induced apoptosis is dramatically enhanced by cAMP-generated signals, which are the principal signals of gonadotropin-stimulated differentiation in these cells (15).

One of the best studied p53 target genes is mdm2. p53 activates transcription of mdm2 gene (32, 33, 34). Full length Mdm2 protein, as well as several smaller forms thereof (35), bind to p53, which renders p53 biochemically inactive and targets it for rapid degradation within the cell (32, 34, 36, 37, 38, 39, 40, 41, 42). Activation of mdm2 gene expression by p53 thus drives a negative feedback loop, which ensures proper regulation and termination of the p53 signal (32, 34, 43, 44). In particular, Mdm2 can relieve p53-mediated growth arrest (36, 45), as well as protect cells from p53-dependent apoptosis (46, 47).

Recently, we found that bFGF can cause an elevation of cellular Mdm2 levels in fibroblasts (48), suggesting that such elevation may contribute to the antiapoptotic effects of bFGF through inactivation of endogenous p53. To study the possible mechanism of cross-talk between cAMP- and p53-generated signals on the one hand, and between p53- and bFGF/ECM-generated signals on the other hand, we established a unique system of steroidogenic human granulosa cell lines through triple-transfection with oncogenic Ha-ras and p53val135 as described in detail in the preceding paper (49).

Using these cell lines, we now demonstrate cross-talk between p53-generated signals and cAMP-generated signals in the induction of apoptosis. Such signals are markedly suppressed by bFGF/ECM. Moreover, this cross-talk involves modulation of Mdm2 expression that is inversely related to the synergistic effect of cAMP and p53 on apoptosis and is directly related to the survival activity exerted by bFGF/ECM. Finally, we show that development of the actin cytoskeleton may be involved in cell resistance to apoptotic signals.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies
Antibody against progesterone was a generous gift of Dr. F. Kohen (Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel). Monoclonal mouse antihuman Mdm2 and polyclonal rabbit antihuman p21 were purchased from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany). Both goat antimouse and antirabbit IgG coupled to horseradish peroxidase (HRP) were obtained from Biomakor (Rehovot, Israel). Phalloidin and antirabbit IgG both labeled with fluorescein isothiocyanate (FITC) were obtained from Sigma Chemical Co. (St. Louis, MO). The other antibodies were kindly provided by Dr. K. Morohashi (Okazaki National Research Institutes, Okazaki, Japan) (antibovine Ad4BP/SF-1, cross-reacted with human Ad4BP/SF-1), Dr. W. L. Miller (University of California, San Francisco, CA) (antihuman adrenodoxin), and Dr. D. P. Lane (University of Dundee, Dundee, UK) (p53-specific monoclonal antibody, PAb421).

Reagents
FK (a potent activator of adenylate cyclase), 8-Br-cAMP (a cAMP analog) and 4', 6-diamido-2-phenylindole hydrochloride (DAPI, for DNA staining) were purchased from Sigma Chemical Co. Highly purified bFGF was generously provided by Dr. A. Yayon (Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel).

Establishment of human granulosa cell lines
The human granulosa cell line HO–23 was established by triple transfection of primary human granulosa cells obtained from an in vitro fertilization (IVF) program, with SV40 DNA, Ha-ras oncogene, and a temperature sensitve (ts) mutant of p53 (p53val135) as described in the preceding paper (49). Cells were maintained in DMEM/F12 (1:1) containing 5% FCS, at 37 C on 5% CO2 in a humidified incubator.

Preparation of dishes coated with ECM
Dishes coated with ECM derived from bovine corneal endothelial cells (BCE) were prepared as described (22, 49, 50).

For preparation of HR9/ECM, PF-HR9 cells derived from a differentiated mouse endodermal carcinoma were seeded (1 x 105 cells/35 mm dish) into tissue culture dishes coated with fibronectin (50 µg/dish) to achieve firm adhesion of the ECM to the plastic. Ascorbic acid (50 µg/ml) was added on day 2 and 4 and the ECM denuded 6–7 days after seeding the cells (50). Major constituents of the HR-9/ECM are laminin, entactin, collagen type IV, and heparan sulfate proteoglycans (HSPGs) (50, 51). Wild-type PF-HR9 cells produce an FGF-free ECM (HR9/ECM), whereas PF-HR9 cells transfected with the gene encoding bFGF deposited an identical ECM (HR9-bFGF/ECM) except for the inclusion of bFGF as a complex with HSPGs.

Biochemical assays
Progesterone measurement. Progesterone accumulated in the culture medium was determined by RIA (10, 15, 52) at the end of cell stimulation.

Protein assay.Protein was quantified by the Bradford method (53).

Fluorescence-activated cell sorting (FACS) analysis
Floating cells and trypsinized attached cells from each treatment were collected and combined to ensure a complete representation of the cell population (22). Cells were washed with cold PBS and fixed in cold methanol (-20 C) for 1 h. Subsequently cells were centrifuged, resuspended in 0.5 ml cold PBS, and stained for at least 15 min with 50 µg/ml propidium iodide in the presence of RNase A (100 µg/ml). Cells were then analyzed in the fluorescence-activated cell sorter (FACSort; Becton Dickinson and Co.). Five thousand events from the gated subpopulation were recorded separately.

Western blot analysis
Western blot analysis was carried out as described (22, 47, 49). Samples containing equal amounts of protein (25 or 30 µg) were separated by 12% (to detect Mdm2, p53, Ad4BP/SF-1 and p21) or 15% [to detect adrenodoxin (ADX)] SDS-PAGE.

Phase contrast and immunofluorescent microscopy
Phase contrast and fluorescent microscopy of cells labeled with antiadrenodoxin antibodies and DAPI or with FITC-labeled phalloidin and DAPI was carried out as described (22, 49, 54). In some experiments, fixed and permeabilized cells were coincubated with 0.3 µg/ml FITC-labeled phalloidin and 0.5 µg/ml DAPI. Microscopic examination of the specimens was carried out using a Zeiss Axioskop microscope (Carl Zeiss, Oberkochen, Germany) in both phase and fluorescent modes.

Electron microscopy
Culture plates were fixed with 2.5% glutaraldehyde and 3% paraformaldehyde in PBS (pH 7.4). Cells were harvested using rubber policemen and centrifuged for 30 min at 12,000 x g (10, 15). The pellets were fixed in 1% osmium tetroxide, stained in block with 2% aqueous uranyl acetate, and embedded in polybed 812 embedding medium (Polyscience Inc., Washington, PA). Ultrathin sections were cut using an ultramicrotome and stained with uranyl acetate and lead acetate. Electron micrographs were taken at 80 kV on an EM 410 (Phillips Electronic Instruments, Eindthoven, The Netherlands).

Statistical analysis
Analysis of progesterone and densitometer tracing was performed using the t test for comparison of means (55). Differences between treatment groups were considered statistically significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Kinetics of inducing steroidogenesis in immortalized human granulosa cells
HO-23 cells grown in DMEM/F12 medium containing 5% FCS proliferate rapidly (generation time about 24 h) and produce traces of progesterone (<0.15 ng/2.5 x 105 cells per 24 h) (Fig. 1Go). When the cells were stimulated with 50 µM of FK, elevation of progesterone production was evident after a lag period of 3–6 h due to de novo synthesis of the steroidogenic cytochrome P450 side-chain cleavage enzyme system (P450scc), as evidenced by the de novo appearance of the electron carrier adrenodoxin (ADX), which is a specific and intrinsic component of this enzyme system (49). Maximum progesterone production was about 170 times above basal level (n = 3, P > 0.001) and was obtained 18–21 h following FK stimulation. Essentially the same pattern of progesterone production was evident following stimulation with 1 mM 8-Br-cAMP (not shown), reaching maximum progesterone production (x470 above basal level) at 24 h of stimulation. Shifting the temperature to 32 C, which leads to the manifestation of wild-type p53 activity (15, 56), further increased progesterone production, which reached maximum accumulation at 12 h subsequent to the temperature shift. Parallel cultures grown at 37 C essentially demonstrated a similar pattern of progesterone accumulation. When expression of steroidogenic proteins was analyzed by Western blot, it was found that nonstimulated cells expressed only traces of ADX, whereas cells stimulated with FK or 8-Br-cAMP at 37 C followed by 8 h incubation at either 32 C or 37 C (49) expressed a high amount of ADX (MW: 11 kDa).



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  		Figure 1. Induction of progesterone production in HO-23 human granulosa cell line. Cells were incubated for 24 h at 37 C in DMEM/F12 medium containing 5% FCS in the presence or absence of 50 µM FK and subsequently were incubated in serum-free medium with or without FK for an additional period of up to 24 h either at 37 C or 32 C. Data are mean ± SD of triplicate cultures.

 
Steroidogenesis and apoptosis
Because activation of wild-type p53 at 32 C was found to induce apoptosis in other cell types (56, 57), we monitored changes in nuclear morphology by DAPI staining. To correlate the apoptotic process with steroidogenic activity, the same cells were counter-stained with ADX specific antibodies. Nonstimulated cells showed a pronounced accumulation of lipid droplets and only weak staining of mitochondria with anti-ADX antibodies (Fig. 2Go, A and A'). In contrast, cells stimulated with 50 µM FK at 37 C showed pronounced accumulation of ADX located in mitochondria, both in the perinuclear region and cell processes. DAPI staining did not reveal any nuclear changes characteristic of apoptosis (Fig. 2Go, B and B').



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  		Figure 2. ADX appearance during the initial stage of apoptosis. HO-23 cells were incubated for 24 h at 37 C in 5% FCS in the presence or absence of 50 µM of FK and subsequently in serum-free medium for 5 h in the presence or absence of 50 µM FK either at 37 C or 32 C. Cultures were fixed with 3% paraformaldehyde and double stained with anti-ADX antiserum and DAPI. A and A', Nonstimulated cells visualized by phase contrast (A) and fluorescent microscopy for ADX (A'). B and B', Cells stimulated with 50 µM FK for 24 h at 37 C followed by 5 h at 37 C. ADX (B) and DNA (by DAPI staining) (B') were visualized by the fluorescent microscopy. C, C', and C'' cells stimulated with 50 µM FK for 24 h at 37 C and subsequently with FK for additional 5 h at 32 C. C, Phase contrast microscopy. C' and C'', visualization of ADX (C') and DNA (C'') by fluorescent microscopy. Arrows, Mitochondria. Arrowheads, Blebbing of the cell membrane. Asterisks, Nuclear fragmentation. Magnification: A and A', x1,000; B, B', C', C' and C'', x650.

 
Shifting the cells to 32 C for 5 h did not induce significant changes in cell morphology and nuclear DNA staining (not shown). In contrast, a similar temperature shift of cAMP-stimulated cells induced membrane blebbing characteristic of early stages of apoptosis (Fig. 2CGo). Mitochondria remained highly positive for ADX and were highly clustered in the perinuclear region.

Interestingly, nuclear staining in most of the cells at this stage remained normal even in cells showing intensive blebbing, suggesting that blebbing precedes extensive nuclear alterations. However, a significant fraction of the cells already showed clear nuclear fragmentation. Even in these cells, highly clustered mitochondria remained intensely stained with anti-ADX antibodies, suggesting that steroidogenesis and apoptosis can coexist, at least temporarily, in the same cell. When ultrathin sections of cells were analyzed by electron microscopy, extensive accumulation of large mitochondria was evident in the cytoplasm, and the cell surface was heavily loaded with microvilli. Intense blebbing was evident in apoptotic cells; these blebs were essentially free of mitochondria, which were found well preserved and intensely clustered in the perinuclear region (Fig. 3Go).



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  		Figure 3. Ultrathin section of control and apoptotic HO-23 cells. Cells were incubated with 50 µM FK for 24 h at 37 C and subsequently for 7 h at 37 C (A) or at 32 C (B and C). Cells were fixed with 2.5% glutaraldehyde, 3% paraformaldehyde and processed for electron microscopic examination. n, nucleus; m, mitochondria; v, microvilli; b, cytoplasmic blebs. Magnification: A and B, x5,400; C, x17,000.

 
Protective effect of bFGF and ECM on p53/cAMP- induced apoptosis
To follow the apoptotic process in HO-23 cells, FACS analysis was performed on propidium iodide stained cells following different treatments.

Cells cultured at 37 C displayed a rather complex cell cycle pattern (Fig. 4AGo). This pattern suggests that the HO-23 culture is composed of at least two subpopulations; diploid or near-diploid cells, and tetraploid or near-tetraploid cells. Hence, peak II probably contains the G1 fraction of the diploid subpopulation, whereas peak III is likely to represent a mixture of diploid cells in G2/M and tetraploid cells in G1. Peak IV is likely to represent tetraploid cells in G2/M. In addition, there was a small subG1 peak (marked as I in Fig. 4AGo), suggestive of a low rate of spontaneous apoptosis. Treatment with FK at 37 C resulted in two small increases in the subG1 population, coupled with a slightly reduced G1 population. A similar pattern, but with a further increase in the subG1 fraction was seen at 32 C. Most remarkably, exposure to FK at 32 C resulted in massive accumulation of cells with subG1 DNA content, at the expense of all other phases of the cell cycle. These data further confirm that the combination of cAMP-increase and p53-activation is a potent inducer of apoptosis in HO-23 cells.



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  		Figure 4. DNA breakdown in HO-23 cells during apoptosis. HO-23 cells were incubated for 24 h in DMEM/F12 medium supplemented with 5% FCS on uncoated or BCE/ECM-coated tissue culture dishes in the presence or absence of FK (50 µM), bFGF (10 ng/ml) or both. The culture medium was replaced with serum-free medium containing the same stimulants for 10 h either at 37 C or 32 C. Cells were fixed with methanol, stained with propidium iodide, and examined by FACS. CONT, nonstimulated cells; ECM, BCE/ECM. Data in panel B were calculated from panel A. The experiment was performed in duplicate culture plates and the deviation between different determinations did not exceed 5% of the mean.

 
Because bFGF and ECM exhibit a protective effect against apoptosis in primary cultured granulosa cells (22), we examined their effect on p53/cAMP-induced apoptosis in HO-23 cells. In cells cultured at 32 C without FK, both bFGF at 10 ng/ml and the ECM produced by bovine corneal endothelial cells (BCE/ECM) dramatically reduced the subG1 population from 26.4% to 6.1% and 8.3%, respectively (Fig. 4BGo). The protective effect of bFGF and ECM was clearly evident also in FK treated cells, reducing the subG1 population from 47.2% to 18.6% and 9.1%, respectively (Fig. 4BGo).

BCE/ECM contains sequestered bFGF (58, 59). To verify the possible contribution of the ECM-resident bFGF to the survival effect of the ECM in HO-23 cells, we compared the protective effect of ECMs produced by HR9 mouse endodermal cells containing or lacking bFGF (50). We found that the bFGF containing HR9-bFGF/ECM exerted a greater protective effect on p53-induced apoptosis than HR9/ECM lacking bFGF, suggesting that the protective effect of ECM can be attributed at least in part to bFGF (Fig. 5Go).



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  		Figure 5. Effect of different ECMs on p53-induced apoptosis in HO-23 cells. Cells were cultured on uncoated plastic, HR9/ECM, or HR9-bFGF/ECM for 24 h in medium containing 5% FCS. The medium was replaced by serum-free medium, and an additional incubation for 15 h was carried out either at 37 C or 32 C. Cultures were stained with propidium iodide and processed for FACS analysis as described in Materials and Methods and the legend to Fig. 4Go. Data are means of duplicate plates that did not deviate by more than 5% of the mean.

 
Expression of Mdm2 in HO-23 granulosa cells
mdm2 gene expression is induced by wild-type p53 (32, 34) and is believed to regulate p53-mediated processes (40, 45, 46, 47). We, therefore, decided to examine the expression of Mdm2 protein during p53/cAMP-induced apoptosis and to see whether it can be modulated by survival factors such as bFGF and BCE/ECM (Fig. 6Go). Cells grown at 37 C showed only traces of the 90 kDa Mdm2 protein. Incubation at 32 C for two h resulted in a clear elevation of Mdm2, which migrated as a doublet at ~90 kDa where the higher MW band was less pronounced compared with the lower band. bFGF and ECM enhanced the expression of Mdm2, which was less pronounced in FK-treated cells but still somewhat higher than in control-untreated cells maintained at 32 C. The same pattern was evident following 3 h of stimulation at 32 C. However, the expression of Mdm2 in FK-treated cells at 32 C was lower than in the control cells. Moreover, cells treated with FK on ECM showed a much lower Mdm2 expression compared with cells cultured on ECM alone. A similar ability of FK to reduce Mdm2 levels was also observed after 4 h, at which time the intensity of the higher MW band was more pronounced than at earlier time points in all treatments.



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  		Figure 6. Expression of p53, Mdm2, and p21 during early stages of apoptosis. HO-23 cells were cultured for 24 h in medium containing 5% serum and stimulants as indicated. The medium was changed to serum-free medium containing the same stimulants and incubation proceeded for 2–4 h at 32 C or 37 C. Cell lysates were then prepared, and Western blot analysis was performed using specific antibodies to p53, Mdm2, and p21. CONT, nonstimulated cultures; FK, 50 µM FK; bFGF, 10 ng/ml. ECM was deposited by bovine corneal endothelial cells (BCE/ECM). The experiment was repeated three times with essentially the same results.

 
The expression of p53 was the same in all treatments of 2 h of incubation. However, at 4 h there was a significant decrease in steady-state levels of p53 at 32 C compared with 37 C, with a further reduction evident in cAMP-stimulated cells. This suggests that enhanced degradation of p53 might have occurred at 32 C and was further enhanced by cAMP. Expression of p21, a target gene of p53 that mediates growth arrest (60), was clearly elevated at 32 C, but no changes in p21 expression were observed among the different treatments at 32 C. This indicates that the differential effects of bFGF, ECM, and FK on Mdm2 are unique to this gene product and are not a general feature of all p53 target genes.

Relationship between steroidogenesis and survival signals exerted by bFGF and ECM
The effect of bFGF, BCE/ECM, HR9/ECM, and HR9-bFGF/ECM on steroidogenesis was examined in cells stimulated with FK at 37 C and following 10 h of incubation at 32 C (Fig. 7Go). Treatment with bFGF almost completely abolished production of progesterone [5–8% compared with cells stimulated with FK, (P < 0.001)]. In contrast, cultivation of the cells on BCE/ECM enhanced progesterone production (400–500%) (P < 0.001) over cells treated with FK alone. Interestingly, HR9/ECM increased progesterone by only 150% (P < 0.01), whereas cultivation of the cells on HR9-bFGF/ECM decreased progesterone production at 32 C to 89% (P < 0.05) compared with cultures treated with FK alone. Thus, bFGF exerts survival signals concomitantly with inhibition of progesterone production, whereas other ECM components exert independent additional signals that stimulate progesterone production. The data demonstrate an uncoupling of survival effects from steroidogenic effects.



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  		Figure 7. Progesterone production by cells cultured on different ECMs. HO-23 cells cultured on BCE/ECM (ECM), HR9/ECM or HR9-bFGF/ECM were stimulated with 50 µM FK for 24 h. Cells were further incubated in serum-free medium containing 50 µM FK for additional 4 h either at 37 C or at 32 C. Data are means ± SD of triplicate culture plates. * and **, Represents difference (P > 0.001, P > 0.01) from cells cultured on plastic and stimulated by FK alone.

 
Because expression of Ad4BP/SF-1 was found to be moderately elevated by FK at 37 C in contrast to ADX, which was dramatically elevated in HO-23 cells following FK stimulation (49), we examined expression of key steroidogenic proteins during induction of apoptosis at 32 C. As shown in Fig. 8Go by Western blot quantitated by densitometric tracing (n = 3), expression of Ad4BP/SF-1 was not significantly changed during incubation at 32 C vs. 37 C. Moreover, no changes in Ad4BP/SF-1 expression were observed in cells treated with either BCE/ECM, bFGF or bFGF + BCE/ECM. However, there was an increase of 35% (P < 0.001) in the content of Ad4BP/SF-1 in cultures treated with FK alone and 69% increase (P < 0.001) in cells treated with FK on BCE/ECM (Fig. 8BGo). In contrast, very low expression of ADX was found in all treatments excluding cells treated with FK (Fig. 8BGo). Interestingly, the levels of ADX were significantly higher in cells costimulated with FK + bFGF (29%, P < 0.01), FK + BCE/ECM (71%, P < 0.001) and FK + bFGF + BCE/ECM (41%, P < 0.001) compared with cells stimulated with FK alone. No significant difference was found in the content of ADX in FK treated cells at 32 C compared with 37 C. These observations suggest that the content of Ad4BP/SF-1 and ADX do not change during initial steps of apoptosis when changes in expression of mdm2 were already clearly evident (Fig. 6Go).



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  		Figure 8. Expression of Ad4BP/SF1 and ADX following apoptotic stimuli in H0-23 cells. Cells were incubated in serum containing medium (5% FCS) in the absence or presence of 50 µM FK, BCE/ECM, 10 nM bFGF, bFGF + FK, BCE/ECM + FK, bFGF + BCE/ECM or bFGF + BCE/ECM + FK. After 24 h incubation, the medium in each treated culture was replaced by serum-free medium containing the same stimulant, either at 32 C or 37 C for 3 h as indicated in A. A, Western blot of Ad4BP/SF1 and ADX. On the left axis, position of molecular weight markers. On the right, position of rat adrenal Ad4BP/SF1 and pure bovine ADX. B, Densitometric tracing of ADX. Data are means ± SD of three independent measurements. *, Represents difference (P > 0.001) from nonstimulated cells (CONT).

 
Organization of the actin cytoskeleton and cell survival
To explore relationships between cell survival and the organization of the cytoskeleton, we double stained the cultures with fluorescein-conjugated phalloidin and DAPI (Fig. 9Go). Cells grown at 37 C showed a diffuse staining of actin in the cell bodies and aggregated masses of actin in the cell processes, often at contact sites with processes of neighboring cells (Fig. 9AGo). Cells treated for 10 h at 32 C underwent intensive apoptosis (Fig. 9BGo), which was further accelerated in the presence of 50 µM FK (not shown), as revealed by massive nuclear fragmentation (Fig. 9BGo'). In these cells, only remnants of aggregated actin, if any, could still be observed (Fig. 9BGo). Cells treated with bFGF showed patterns of actin organization and DAPI staining similar to control cultures (Fig. 9CGo and C'). However, some local increase in actin concentration was sometimes evident in the main cell bodies (Fig. 9CGo). Cells cultured on BCE/ECM showed a well developed network of actin filaments (Fig. 9DGo) and no signs of apoptosis (Fig. 9DGo') suggesting that actin cytoskeleton organization may play an important role in the induction of both cell survival and steroidogenic activity.



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  		Figure 9. Actin cytoskeleton organization in HO-23 cells. A and A', Cells were cultured on plastic dishes for 24 h in medium containing 5% serum and subsequently in serum-free medium for 10 h at 37 C. B and B', The same as in panel A but during the last 10 h the cells were incubated at 32 C. C and C', The same as in panel B but in the presence of 10 ng/ml bFGF. D and D', The same as in panel B but the cells were cultured on BCE/ECM. Cells were double stained with fluorescein-conjugated phalloidin and DAPI. A–D, Visualization of actin. A'–D', Visualization of DNA. Arrowheads, Aggregated actin. Arrows, Actin filaments. Asterisks, Nuclear fragmentation. Magnification, x1,000.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
p53, Mdm2, and apoptosis
Activation of p53 is able to induce apoptosis in numerous cell types including steroidogenic granulosa cells (15, 27). Moreover, we have demonstrated that cAMP can dramatically enhance apoptosis in preovulatory granulosa cells and that bFGF or a basement membrane-like extracellular matrix can protect cells from p53/cAMP-induced apoptosis (10, 15, 22). However, the mechanism of cross-talk between bFGF/ECM- and p53-generated signals on the one hand, and between p53- and cAMP-generated signals on the other hand, remains obscure. The present work suggests that cross-talk between these signals in controlling apoptosis may involve the modulation of Mdm2 expression and that the intensity of expression of this oncoprotein is inversely related to the apoptotic process. High expression of Mdm2 is characteristic of the survival activity of bFGF and ECM-FGF, whereas cAMP-induced apoptosis is associated with down-regulation of Mdm2 expression (summarized in Fig. 10Go).



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  		Figure 10. A simplified model illustrating possible cross-talk between p53-generated and bFGF-generated signals and between p53- and cAMP-generated signals in induction of growth arrest and apoptosis via modulation of Mdm2 expression. As suggested in this model, bFGF can independently modulate cell growth and Mdm2 expression. Similarly, cAMP may exert its effects on apoptosis and Mdm2 regulation through alternative pathways. TYR.PHOS., Tyrosine phosphorylation; MAPKK, mitogen-activated kinase kinase; MAPK, mitogen-activated kinase; p21, a product of the waf1/cip1 gene. ->, stimulatory. {dashv}, inhibitory.

 
The mechanism by which Mdm2 exerts its antiapoptotic activity is believed to involve protein-protein interactions with p53. Mdm2 binds directly to p53 and neutralizes its activity (37, 40, 41, 42). It was also recently demonstrated that Mdm2 can induce proteolysis of p53 and that this degradation is carried out by proteasomes, presumably following conjugation to ubiquitin (38, 39). The latter fact might account for the pronounced decrease in steady-state levels of p53 that we observed at 32 C, a temperature at which cellular Mdm2 levels become elevated. The modulation of Mdm2 expression, as revealed in our studies, was already evident within 2 h of incubation at 32 C, when no biochemical or morphological signs of apoptosis were evident yet. Along with related observations employing rodent fibroblasts (48), our data support the notion that bFGF and ECM may exert at least some of their antiapoptosis effect through increasing cellular Mdm2 levels. However, our observations were obtained using highly luteinized immortalized granulosa cells that apparently underwent partial transformation. Whether modulation of Mdm2 levels in primary granulosa cells plays an important role in their survival should be examined in future studies probably by RT-PCR because the levels of Mdm2 protein in normal granulosa cells are extremely low (Aharoni, D., and A. Amsterdam, unpublished results).

Apoptosis and differentiation in steroidogenic granulosa cells
One of the characteristic features of highly differentiated granulosa cells is a high expression of steroidogenic enzymes and other essential steroidogenic proteins such as Ad4BP/SF-1 and the StAR protein (11, 12, 14). HO-23 cells proliferate rapidly and can induce tumors in nude mice (Hosokawa et al., unpublished); however, when stimulated with FK and 8-Br-cAMP, they synthesize the P450scc enzyme system and produce high amounts of progesterone, characteristic of highly luteinized human granulosa cells (61). When these cells were stimulated for apoptosis there was no immediate down-regulation of steroidogenic activity, nor of the content of mitochondrial P450scc and Ad4BP/SF-1. These unique features of HO-23 cells permit us to address the question of how initial stages of apoptosis affect the differentiated functions of steroidogenic cells. Both the steroidogenic activity of the mitochondria as well as their ultrastructural features were not impaired during the initial step of apoptosis, in contrast to recent claims that deterioration in mitochondrial structure and function is a key event in apoptosis of human colorectal cancer cells induced by p53 (62).

As evident from this study, cAMP served not only as a powerful inducer of steroidogenesis in HO-23 cells but also synergized with p53-generated signals in the induction of apoptosis. This may explain the dramatic effect of cAMP on induction of apoptosis in highly differentiated normal preovulatory granulosa cells (10, 22). cAMP may also synergize with wild-type p53, the expression of which seems to be elevated during the apoptotic process in vivo (29, 30). The interesting observation that at initial stages of apoptosis there was no decrease in steroidogenic activity can be explained by the fact that the steroidogenic mitochondria were concentrated in the perinuclear region and thus were not eliminated by pinching off of the apoptotic blebs. These mitochondria were probably not exposed to degradation by proteasomes that are mainly concentrated in the apoptotic blebs (63). These observations are in line with our previous studies on apoptosis in rat granulosa cells (15, 63). Synchronization of the apoptotic process by p53 and cAMP revealed cells in which numerous apoptotic blebs were already formed but no nuclear alterations were evident. Hence, in this cell system, cytoplasmic apoptotic events can precede nuclear events.

Differential effect of bFGF, ECM-FGF, and other ECM components on granulosa cell differentiation and apoptosis
In this work we show that bFGF is able to block p53-induced apoptosis as well as p53/cAMP-induced apoptosis. This may suggest that bFGF exerts its effect on p53-induced apoptosis via elevation of Mdm2 expression, but bFGF may also modulate cAMP-generated signals for apoptosis through alternative pathways, such as activation of the MAP kinase cascade (Fig. 10Go) (64, 65). Although bFGF inhibited progesterone production, this inhibition could not be attributed to down-regulation of the P450scc enzyme system or Ad4BP/SF-1 because no reduction in expression of either ADX or Ad4BP/SF1 was evident following incubation with bFGF. Various basement membrane-like ECMs promote the survival of serum-deprived primary granulosa cells (22). In this study, we show that the ECM also provides a powerful survival signal against p53/cAMP-induced apoptosis in immortalized granulosa cells, as well as in primary granulosa cells (22). It should be noted that both BCE/ECM, which contained bFGF and soluble bFGF, enhanced Mdm2 expression to the same extent. However, whereas bFGF blocked steroidogenesis almost completely, BCE/ECM enhanced steroidogenesis by 3-fold (66, 67), suggesting that additional components of the BCE/ECM are able to stimulate steroidogenesis. Indeed, BCE/ECM was able to enhance adrenodoxin expression in FK-treated HO-23 cells, whereas bFGF showed only a marginal effect. By using ECMs lacking bFGF (HR9/ECM) or containing bFGF (HR9-bFGF/ECM), the net effect of bFGF on HO-23 cell survival could be demonstrated. Whether other components of the ECM such as laminin and fibronectin contribute to its survival activity needs to be further explored.

Possible role of actin organization in survival and differentiation of HO-23 cells
We have recently demonstrated that survival of primary and transformed rat granulosa cells involves the integrity of the actin cytoskeleton (22). On the other hand, degradation of fodrin, a multifunctional protein with spectrin-like properties, which is a major component of the cortical cytoskeleton in most eucaryotic cells, was implicated in apoptosis in a human T cell lymphoma line (68). Similarly, gelsolin, which functions as a cross-linker at actin filaments, was recently demonstrated to play an important role in controlling apoptosis in neutrophils and other mammalian cells (69). The present study suggests that the integrity of the actin cytoskeleton is essential for protection against p53/cAMP-induced apoptosis. Moreover, in cells maintained on BCE/ECM, an extensive network of actin filaments was created in contrast to the relatively poor organization of actin filaments characteristic of ras-transformed cells grown on plastic surfaces (55). Because components of the ECM and serum can interact with specific integrins that are linked to the actin cytoskeleton, the possible steroidogenic effect of the ECM may be a reflection of cytoskeleton reorganization, as demonstrated in primary granulosa cells (reviewed in Refs. 3, 70). Rearrangement of the actin cytoskeleton was found to play an important part in enhancing steroidogenesis in nonapoptotic granulosa cells (3, 70). Stabilization of the new organization of actin cytoskeleton in the highly steroidogenic cells may be important not only for the ongoing steroidogenic activity but may also prevent luteinized granulosa cells from undergoing apoptosis.


   Acknowledgments
 
We thank Dr. W. L. Miller for generous provision of anti-ADX antibodies, Dr. F. Kohen for antiprogesterone antibodies, Dr. K. Morohashi for anti Ad4BP/SF1 antibodies, Dr. D. P. Lane for anti p53 antibodies, Dr. A. Yayon for generous provision of bFGF, and Mrs. V. Laufer for excellent secretarial assistance.


   Footnotes
 
1 This work was supported by grants from the Israel Academy of Sciences (to I.V. and A.A.), by the Israeli Ministry of Science (to A.A.), by the Leo and Julia Forchheimer Center of Molecular Genetics at the Weizmann Institute of Science (to A.A.) and by a Grant-in-Aid 0704424 from the Ministry of Education, Science and Culture of Japan (to F.K., K.H., and A.A.). Back

2 Incumbent of the Joyce and Ben B. Eisenberg Chair of Molecular Endocrinology and Cancer Research. Back

Received February 26, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Amsterdam A, Plehn-Dujowich D, Suh BS 1992 Structure-function relationships during differentiation of normal and oncogene transformed granulosa cells. Biol Reprod 46:513–522[Abstract]
  2. Amsterdam A, Rotmensch S 1987 Structure-function relationships during granulosa cell differentiation. Endocr Rev 8:309–337[Medline]
  3. Amsterdam A, Rotmensch S, Ben-Ze’ev A 1989 Coordinated regulation of morphological and biochemical differentiation in a steroidogenic cell: the granulosa cell model. Trends Biochem Sci 14:377–382[CrossRef][Medline]
  4. Lefkowitz RJ 1993 G protein-coupled receptor kinases. Cell 74:409–412[CrossRef][Medline]
  5. Johnson GL, Dhanasedaron N 1989 The G protein family and their interaction with receptors. Endocr Rev 10:317–331[Medline]
  6. Hsueh AJW, LaPolt PS 1992 Molecular basis of gonadotropin receptor regulation. Trends Endocrinol Metab 3:164–170[Medline]
  7. Berridge MV, Tan AS, Hilton CJ 1993 Cyclic adenosine monophosphate promotes cell survival and retards apoptosis in a factor dependent bone marrow-derived cell line. Exp Hematol 21:267–276
  8. Duprez E, Gjersten BT, Bernard O, Lanotte M, Doskeland SO 1993 Antiapoptotic effect of heterozygously expressed mutant RI (Ala336-Asp) subunit of cAMP kinase I in a rat leukemia cell line. J Biol Chem 268:8332–8340[Abstract/Free Full Text]
  9. McConkey DJ, Orrenius S, Jondal M 1990 Agents that elevate cAMP stimulate DNA fragmentation in thymocytes. J Immunol 145:1227–1230[Abstract]
  10. Aharoni D, Dantes A, Oren M, Amsterdam A 1995 c-AMP mediated signals as determinants for apoptosis in primary granulosa cells. Exp Cell Res 218:271–282[CrossRef][Medline]
  11. Amsterdam A, Selvaraj N 1997 Control of differentiation, transformation, and apoptosis in granulosa cells by oncogenes, oncoviruses, and tumor suppressor genes. Endocr Rev 18:435–461[Abstract/Free Full Text]
  12. Lue X, Ikeda Y, Parker KL 1994 A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation. Cell 77:481–490[CrossRef][Medline]
  13. Richards JS 1994 Hormonal control of gene expression in the ovary. Endocr Rev 15:725–751[CrossRef][Medline]
  14. Stocco DM, Clark BJ 1996 Regulation of the acute production of steroids in steroidogenic cells. Endocr Rev 17:221–244[CrossRef][Medline]
  15. Keren-Tal I, Suh BS, Dantes A, Lindner S, Oren M, Amsterdam A 1995 Involvement of p53 expression in cAMP mediated apoptosis in immortalized granulosa cells. Exp Cell Res 218:283–295[CrossRef][Medline]
  16. Folkman J, Klagsburn M 1987 Angiogenic factors. Science 235:442–447[Abstract/Free Full Text]
  17. Rifkin DB, Moscatelli D 1989 Recent developments in the cell biology of basic fibroblast factor. J Cell Biol 109:1–6[Free Full Text]
  18. Rogelj S, Weinsberg RA, Fanning P, Klagsbrun M 1988 Basic fibroblast growth factor fused to a signal transforms cells. Nature 331:173–175[CrossRef][Medline]
  19. Yayon A, Klagsbrun M 1990 Autocrine transformation by chimeric signal peptide-basic fibroblast factor: reversal by suramin. Proc Natl Acad Sci USA 87:5346–5350[Abstract/Free Full Text]
  20. Coughlin SR, Barr PJ, Cousens LS, Fretto LJ, Williams LT 1988 Acidic and basic fibroblast growth factors stimulate tyrosine kinase activity in vivo. J Biol Chem 263:988–993[Abstract/Free Full Text]
  21. Bellot F, Crumley G, Kaplow JM, Schlessinger J, Jaye M, Dionne CA 1991 Ligand-induced transphosphorylation between different FGF receptors. EMBO J 10:2849–2954[Medline]
  22. Aharoni D, Meiri I, Atzmon R, Vlodavsky I, Amsterdam A 1997 Differential effect of components of the extracellular matrix on differentiation and apoptosis. Curr Biol 7:43–51[CrossRef][Medline]
  23. Fuks Z, Persaud RS, Alfieri A, McLoughlin M, Ehleiter D, Schwartz JL, Seddon AP, Cordon-Cardo C, Haimovitz FA 1994 Basic fibroblast growth factor protects endothelial cells against radiation-induced programmed cell death in vitro and in vivo. Cancer Res 54:2582–2590[Abstract/Free Full Text]
  24. Gottlieb T, Oren M 1996 p53 in growth control and neoplasia. Biochem Biophys Acta 1287:77–102[Medline]
  25. Ko LJ, Prives C 1996 p53:puzzle and paradigm. Genes Dev 10:1054–1072[Free Full Text]
  26. Levine AJ 1997 p53, the cellular gatekeeper for growth and division. Cell 88:323–331[CrossRef][Medline]
  27. Oren M 1994 Relationship of p53 to the control of apoptotic cell death. Semin Cancer Biol 5:221–227[Medline]
  28. Hsueh AJW, Billig H, Tsafriri A 1994 Ovarian follicle atresia: a hormonally controlled apoptotic process. Endocr Rev 15:707–724[CrossRef][Medline]
  29. Tilly JL 1996 Apoptosis and ovarian function. Rev Reprod 1:162–172[Abstract]
  30. Tilly JL, Tilly KI, Kenton LM, Johnson AL 1995 Expression of members of the Bcl-2 gene family in the immature rat ovary: equine chorionic gonadotropin-mediated inhibition of granulosa cell apoptosis is associated with decreased Bax and constitutive Bcl-2 and Bcl-xlong messenger ribonucleic acid levels. Endocrinology 136:232–241[Abstract]
  31. Miyashita T, Reed JC 1995 Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell 80:293–299[CrossRef][Medline]
  32. Barak Y, Juven T, Haffner R, Oren M 1993 mdm2 expression is induced by wild type-p53 activity. EMBO J 12:461–468[Medline]
  33. Juven T, Barak Y, Zauberman A, George DL, Oren M 1993 Wild type p53 can mediate sequence-specific transactivation of an internal promoter within the mdm2 gene. Oncogene 8:3411–3416[Medline]
  34. Wu X, Bayle JH, Olson D, Levine AJ 1993 The p53-mdm-2 autoregulatory feedback loop. Genes Dev 7:1126–1132[Abstract/Free Full Text]
  35. Olson DC, Marechal V, Momand J, Chen JD, Romocki C, Levine AJ 1993 Identification and characterization of multiple mdm-2 proteins and mdm-2-p53 protein complexes. Oncogene 8:2353–2360[Medline]
  36. Chen JD, Lin JY, Levine AJ 1995 Regulation of transcription functions of the p53 tumor suppressor by the mdm-2 oncogene. Mol Med 1:142–152[Medline]
  37. Haines DS, Landers JE, Engel LJ, George DL 1994 Physical and functional interaction between wild-type p53 and mdm2 proteins. Mol Cell Biol 14:1171–1178[Abstract/Free Full Text]
  38. Haupt Y, Maya R, Kazaz A, Oren M 1997 Mdm2 promotes the rapid degradation of p53. Nature 387:296–299[CrossRef][Medline]
  39. Kubbutat MHG, Jones SN, Vousden KH 1997 Regulation of p53 stability by Mdm2. Nature 387:299–303[CrossRef][Medline]
  40. Momand J, Zambetti GP, Olson DC, George DL, Levine AJ 1992 The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation. Cell 69:1237–1245[CrossRef][Medline]
  41. Oliner JD, Pietenpol JA, Thiagalingam S, Gvuris J, Kinzler KW, Vogelstein B 1993 Oncoprotein MDM2 conceals the activation domain of tumour suppressor-p53. Nature 362:857–860[CrossRef][Medline]
  42. Zauberman A, Barak Y, Ragimov N, Levy N, Oren M 1993 Sequence-specific DNA binding by p53: identification of target sites and lack of binding to p53-MDM2 complexes. EMBO J 12:2799–2808[Medline]
  43. Momand J, Zambetti GP 1997 Mdm-2: "big brother" of p53. J Cell Biochem 64:343–352[CrossRef][Medline]
  44. Picksley SM, Lane DP 1993 The p53-mdm2 autoregulatory feedback loop—a paradigm for the regulation of growth control by p53? BioEssays 15:689–690[CrossRef][Medline]
  45. Chen CY, Oliner JD, Zhan QM, Fornace AJ, Vogelstein B, Kastan MB 1994 Interactions between p53 and MDM2 in a mammalian cell cycle checkpoint pathway. Proc Natl Acad Sci USA 91:2684–2688[Abstract/Free Full Text]
  46. Chen JD, Wu X, Lin JY, Levine AJ 1996 mdm-2 inhibits the G1 arrest and apoptosis functions of the p53 tumor suppressor protein. Mol Cell Biol 16:2445–2452[Abstract]
  47. Haupt Y, Barak Y, Oren M 1996 Cell type-specific inhibition of p53-mediated apoptosis by mdm2. EMBO J 15:1596–1606[Medline]
  48. Shaulian E, Resnitzky D, Shifman O, Blandino G, Amsterdam A, Yayon A, and Oren M 1997 Induction of Mdm2 and enhancement of cell survival by bFGF. Oncogene 15:2717–2725[CrossRef][Medline]
  49. Hosokawa K, Dantes A, Schere-Levy C, Barash A, Kotsuji F, Vlodavsky I, and Amsterdam A 1998 Induction of Ad4BP/SF-1, StAR and cytochrome P450scc enzyme system expression in newly established human granulosa cell lines. 139:4679–4687
  50. Rogelj S, Klagsbrun M, Atzmon R, Kurokawa M, Haimovitz A, Fuks Z, Vlodavsky I 1989 Basic fibroblast growth factor is an extracellular matrix component required for supporting the proliferation of vascular endothelial cells and the differentiation of PC12 cells. J Cell Biol 109:823–831[Abstract/Free Full Text]
  51. Kramer HR, and Vogel KG 1984 Selective degradation of basement membrane macromolecules by metastatic cells. J Natl Cancer Inst 72:889–899
  52. Kohen F, Bauminger S, and Lindner HR 1975 Preparation of antigenic steroid-protein conjugate. In: Cameron EHD, Hillier SG, Griffiths K (eds) Steroid Immunoassay. Alpha Omega Publishing Ltd., Cardiff, Wales, 11–23
  53. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principles of protein-dye binding. Anal Biochem 72:248–254[CrossRef][Medline]
  54. Hanukoglu I, Suh BS, Himmelhoch S, Amsterdam A 1990 Induction and mitochondrial localization of cytochrome P450scc system enzymes in normal and transformed ovarian granulosa cells. J Cell Biol 111:1973–1981
  55. Suh BS, Amsterdam A 1990 Establishment of highly steroidogenic granulosa cell lines by cotransfection with SV40 and Ha-ras oncogene: induction of steroidogenesis by cyclic adenosine 3'-5'-monophosphate and its suppression by phorbol ester. Endocrinology 127:2489–2500[Abstract]
  56. Michalovitz D, Halevy O, Oren M 1990 Conditional inhibition of transformation and of cell proliferation by a temperature-sensitive mutant of p53. Cell 62:671–680[CrossRef][Medline]
  57. Yonish-Rouach E, Resnitzky D, Lotem J, Sachs L, Kimchi A, Oren M 1991 Wild-type p53 induces apoptosis of myeloid leukaemic cells that is inhibited by interleukin-6. Nature 352:345–347[CrossRef][Medline]
  58. Ruoslahti E, Yamaguchi Y 1991 Proteoglycans as modulators of growth factor activities. Cell 64:867–869[CrossRef][Medline]
  59. Vlodavsky I, Bar-Shavit R, Korner G, Fuks Z 1993 Extracellular matrix-bound growth factors, enzymes and plasma proteins. In: Rohrbach DH, Timpl R (eds) Basement Membranes: Cellular and Molecular Aspects. Academic Press Inc, Orlando, pp 327–343
  60. El-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, Lin D, Mercer WE, Kinzler KW, Vogelstein B 1993 WAF1, a potential mediator of p53 tumor suppression. Cell 75:817–825[CrossRef][Medline]
  61. Breckwoldt M, Selvaraj N, Aharoni D, Barash A, Segal I, Insler V, Amsterdam A 1996 Expression of Ad4BP/cytochrome P450 side chain cleavage enzyme and induction of cell death in long term cultures of human granulosa cells. Mol Hum Reprod 2:391–400[Abstract/Free Full Text]
  62. Polyak K, Xia Y, Zweier JL, Kinzler KW, Vogelstein B 1997 A model for p53-induced apoptosis. Nature 389:300–305[CrossRef][Medline]
  63. Pitzer F, Dantes A, Fuchs T, Baumeister W, Amsterdam A 1996 Removal of proteasomes from the nucleus and their accumulation in apoptotic blebs during programmed cell death. FEBS Lett 394:47–50[CrossRef][Medline]
  64. Bogoyevitch MA, Glennon PE, Andersson MB, Clerk A, Lazou A, Marshall CJ, Parker PJ, Sugden PH 1994 Endothelin-1 and fibroblast growth factors stimulate the mitogen-activated protein kinase signaling cascade in cardiac myocytes. The potential role of the cascade in the integration of two signaling pathways leading to myocyte hypertrophy. J Biol Chem 269:1110–1119[Abstract/Free Full Text]
  65. Creuzet C, Loeb J, Barbin G 1995 Fibroblast growth factors stimulate protein tyrosine phosphorylation and mitogen-activated protein kinase activity in primary cultures of hippocampal neurons. J Neurochem 64:1541–1547[Medline]
  66. Amsterdam A, Rotmensch S, Furman A, Venter EA, Vlodavsky I 1989 Synergistic effect of human chorionic gonadotropin and extracellular matrix on in vitro differentiation of human granulosa cells: progesterone production and gap junction formation. Endocrinology 124:1956–1964[Abstract]
  67. Furman A, Rotmensch S, Kohen F, Mashiach S, Amsterdam A 1986 Regulation of rat granulosa cell differentiation by extracellular matrix produced by bovine corneal endothelial cells. Endocrinology 118:1878–1885[Abstract]
  68. Martin SJ, O’Brien GA, Nishioka WK, McGahon AJ, Mahboubi A, Saido TC, Green DR 1995 Proteolysis of fodrin (non-erythroid spectrin) during apoptosis. J Biol Chem 270:6425–6428[Abstract/Free Full Text]
  69. Kothakota S, Azuma T, Reinhard C, Klippel A, Tang J, Chu K, McGarry TJ, Kirschner MW, Koths K, Kwiatkowski DJ, Williams LT 1997 Caspase-3-generated fragment of gelsolin: effector of morphological change in apoptosis. Science 278:294–298[Abstract/Free Full Text]
  70. Amsterdam A, Aharoni D 1994 Plasticity of cell organization during differentiation of normal and oncogene transformed granulosa cells. Micros Res Tech 27:108–124[CrossRef][Medline]



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