help button home button Endocrine Society JCEM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2004-0454
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
90/1/469    most recent
Author Manuscript (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Khan, S. M.
Right arrow Articles by Yeh, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Khan, S. M.
Right arrow Articles by Yeh, J.
Related Collections
Right arrow Female Endocrinology
The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 1 469-473
Copyright © 2005 by The Endocrine Society

Epidermal Growth Factor Receptor Inhibition by Tyrphostin 51 Induces Apoptosis in Luteinized Granulosa Cells

Shah M. Khan, Rush H. Oliver and John Yeh

Department of Obstetrics, Gynecology and Women’s Health (S.M.K., R.H.O.), University of Minnesota Medical School, Minneapolis, Minnesota 55455; and Department of Gynecology-Obstetrics (J.Y.), University at Buffalo, Buffalo, New York 14222

Address all correspondence and requests for reprints to: John Yeh, M.D., Department of Gynecology and Obstetrics, University at Buffalo, 219 Bryant Street, Buffalo, New York 14222. E-mail: jyeh{at}buffalo.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Growth factors may be involved in the control of ovarian cell fate and could contribute to regulation of ovarian cell apoptosis. Our objective is to test the hypothesis that, in human luteinized granulosa cells, epidermal growth factor (EGF) works through the MAPK signaling pathway and inhibition of EGF receptor by a specific tyrosine kinase inhibitor, tyrphostin 51, will inhibit the activation of MAPK and induce apoptosis. Luteinized granulosa cells from human in vitro fertilization aspirates were treated as follows: 1) vehicle (dimethylsulfoxide:ethanol), 2) EGF, 3) tyrphostin 51, and 4) tyrphostin 51 plus EGF. Blockage of EGF receptor by tyrphostin 51 reduced the MAPK activity and inhibited phosphorylation and nuclear translocation of activated MAPK. Blockage of EGF receptor also induced apoptosis as demonstrated by the activation of caspase-3, an executioner protease of the apoptotic pathway, and by an increased percentage of subdiploid apoptotic nuclei. These results support the hypothesis that in human luteinized granulosa cells, EGF works through the MAPK signaling pathway and that its inhibition by tyrphostin 51 inhibits MAPK phosphorylation and induces apoptotic nuclear changes. Our data thus provide additional information regarding regulation of apoptosis in luteinized granulosa cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
EPIDERMAL GROWTH FACTOR (EGF) is a polypeptide that stimulates cellular growth. EGF is one of the intraovarian signals that may contribute to regulation of cell fate during ovarian remodeling (1). EGF has been reported to be present or produced within the human ovary (2), and data show the presence of EGF receptor (EGF-R) in corpus luteum cells (3, 4). The process of luteinization has been suggested to be associated with the loss of sensitivity to the mitogenic actions of EGF (5).

MAPKs (p42MAPK and p44MAPK) are serine/threonine kinases that are activated rapidly in cells stimulated by various extracellular stimuli. An important signaling pathway that is activated by EGF-R is Ras to Raf to MAPK kinase (MEK) to ERK and the pathway implicated in proliferation, survival, and gene expression (6). MAPKs have been shown to play a pivotal role in modulation of cell growth and differentiation (7). In human carcinoma cell lines, programmed cell death can be induced by down-regulation of EGF-R activity by an inhibitor of the EGF-R tyrosine kinase (8). It has been reported that, in lung cancer cells, genistein, a tyrphostin kinase inhibitor, inhibits EGF-stimulated cell growth and results in programmed cell death (9). Gilmore et al. (10) recently described the ability of ZD1839, a small-molecule inhibitor of the EGF-R, to induce apoptosis of mammary cells that are dependent upon growth factors for survival.

In our previous studies, we demonstrated that PD98059, a specific inhibitor of MEK (11), and depletion of Raf-1 by geldanamycin (12) induced apoptosis in human luteinized granulosa cells. Others and we have shown the presence of proteolytic apoptotic machinery, apoptotic protease-activating factor-1, and caspase-3, in human granulosa cells (13, 14). In addition, the presence of caspase-3, the executioner protease, during apoptosis has been demonstrated in granulosa cells of atretic follicles (15). Recent studies have also demonstrated that apoptotic protease-activating factor-1 and caspase-9 play a crucial role in determining the fate of granulosa cells during atresia in pig ovaries (16). Tyrphostin, a receptor tyrosine kinase inhibitor, has been shown in different cell types to inhibit MAPK activation (17) and cell proliferation (18, 19). We therefore hypothesize that in human luteinized granulosa cells EGF works through the MAPK signaling pathway and that its inhibition by tyrphostin 51 will result in activation of caspase-3 and induction of apoptotic nuclear changes. This information will give us a better idea of the mechanism by which luteinized granulosa cell apoptosis is regulated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation of human luteinized granulosa cells

Luteinized granulosa cells were isolated from the follicular aspirates of patients undergoing in vitro fertilization, following the previously described method (13). The Human Subjects Committee of the University of Minnesota approved the use of these cells for our investigation. Briefly, red blood cells were removed from the follicular aspirates by centrifugation through Ficoll/Hypaque (density 1.07; Gallard-Schlesinger, Carle Place, NY). White blood cells were depleted by using anti-CD45-coated magnetic beads (Dynal, Miami, FL). Luteinized granulosa cells were dispersed by incubatin in trypsin (1x; Life Technologies, Inc.-BRL, Grand Island, NY). Trypsin digestion was stopped after 3 min by addition of six volumes of 10% fetal bovine serum (Intergen, Purchase, NY) supplemented with DMEM/Ham’s F12 culture medium, containing 100 IU/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin (Sigma, St. Louis, MO). Cells were plated at approximately 2 x 105 cells per 60-mm plastic culture dishes. The purity of the preparation was qualitatively high by visual inspection. The inspection of the purified cells under microscopic visualization confirmed single-cell populations.

Pooled granulosa cells from each patient were isolated, divided into treatment groups, and treated under the different conditions. This is called "n = 1" throughout the manuscript. Therefore, for example, in experiment 1 (Fig. 1Go), 14 different patients were studied. For every patient, all experimental conditions, including control, were used. Thus, control conditions were used in each of the 14 "n" cases.



View larger version (13K):
[in this window]
[in a new window]
  		FIG. 1. EGF-stimulated MAPK activity. MAPK activity in total cell lysates was determined by radiochemical assay of 32P incorporation into MBP. The mean for EGF differs significantly from the means of the other treatments (P < 0.05). Treatment groups were the following: control, vehicle; tyrphostin 51, 100 µM for 60 min; EGF, 10 ng/ml for 10 min; and tyrphostin 51 + EGF, 60 min preincubation with 100 µM tyrphostin 51 followed by a 10-min treatment with 10 ng/ml EGF. Granulosa cells from each patient (n = 14 patients) were isolated, aliquoted into wells, and treated with all four treatment conditions.

 
Treatment of luteinized granulosa cells

The luteinized granulosa cells were starved overnight in serum-free medium. The treatment groups were as follows: 1) vehicle (1:1 dimethylsulfoxide:ethanol), 2) EGF, 3) tyrphostin 51, and 4) tyrphostin 51 plus EGF. The cells were treated with 100 µM tyrphostin 51 (Sigma) for 1 h and/or 10 ng/ml EGF (Life Technologies, Inc.-BRL) for 10 min. We used 100 µM tyrphostin 51 in our studies, which is in the same range as mentioned on the product data sheet of tyrphostin 51 (catalog no. T7665, Sigma), where 0–1 mM tyrphostin inhibited phosphorylation of rat hepatic leptin 1. In addition, the IC50 of tyrphostin 51 is 0.8 µM. We therefore decided to use a 100-µM concentration to inhibit phosphorylation of MAPK. The cells were lysed using the following buffer: 50 mM Tris-HC1 (pH 7.0), containing 1% Triton X-100, 1 mM EGTA, 1 mM EDTA, 50 mM NaF, 5 mM sodium pyrophosphate, 10 mM phenylmethylsulfonyl fluoride, 1 µg/ml each pepstatin, leupeptin, aprotinin, and trypsin inhibitor. The lysates were snap frozen in liquid nitrogen and stored frozen at –70 C until further use. Total protein was assayed using the bicinchoninic acid method (Pierce, Rockford, IL).

Radiochemical MAPK assay

MAPK activity in the luteinized granulosa cell lysates was determined by a radiochemical assay with myelin basic protein (MBP) as substrate using a MAPK assay kit (Upstate Biotechnology, Lake Placid, NY). Reaction mixtures (40 µl) containing 10 µl of total cell lysate, 500 mM ATP, 5 µCi {gamma}-32P-labeled ATP (NEN Life Science Products, Boston, MA), and 20 µg MBP were incubated at 30 C for 15 min. MAPK activity was determined by quantifying 32P incorporation into the MBP substrate using a scintillation counter (LS2800; Beckman, Irvine, CA).

Western blotting

Twenty-five micrograms of total protein were resolved through 12% SDS-PAGE (Bio-Rad, Hercules, CA) and transferred to nitrocellulose membranes. After blocking, the blots were probed for phosphorylated and total MAPK using polyclonal antibodies against phospho-p44/42 MAPK (New England Biolabs, Beverly, MA) or anti-ERK1/ERK2 MAPK (Oncogene, Cambridge, MA) respectively, at a 1:500 dilution. After washing with PBS containing 5% dry milk, the filters were incubated with the second antibody, alkaline phosphatase-conjugated antirabbit IgG (Sigma), at a 1:1000 dilution. MAPK was detected using the nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate method (Bio-Rad). For caspase-3 identification, the blots were incubated with anti-caspase-3 polyclonal antibody (1:1000; CPP32; PharMingen, San Diego, CA) and then with a horseradish peroxidase-conjugated second antibody (1:2000) and goat antirabbit IgG (Sigma). The blots were incubated with enhanced chemiluminescence detection solution, and caspase-3 was detected using the enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech, Piscataway, NJ). The immunoreactive bands were quantified by digital densitometric imaging (Gel Doc 1000 with model GS-700 Densitometer and Molecular Analyst Software; Bio-Rad).

Immunocytochemistry and confocal microscopy

Luteinized granulosa cells were treated with tyrphostin 51 and/or EGF as described above. The cells were fixed in methanol:acetone (1:1) and blocked with 5% BSA in Tris-HCl (pH 7.0). The cells were then incubated with anti-phosphorylated MAPK antibody (1:500 dilution). Subsequently, the cells were incubated for 30 min at room temperature with fluorescein isothiocyanate-conjugated antirabbit antibody (Sigma). The cells were washed and dehydrated in alcohol. Phosphorylated MAPK exhibiting green fluorescence was viewed using a Bio-Rad 1000 confocal microscope and a x60 lens.

Flow cytometry

Flow cytometry of isolated nuclei stained with propidium iodide was used to assess apoptotic fragmentation of DNA (20). Cultured cells were treated for 24 h as follows: 1) vehicle (1:1 dimethylsulfoxide:ethanol), 2) EGF, 3) tyrphostin 51, and 4) tyrphostin 51 plus EGF. After the treatments, cells were scraped from the plates; centrifuged at 200 x g for 5 min; and incubated overnight at 4 C in 500 µL of lysis buffer containing 50 µg/ml propidium iodide, 0.1% sodium citrate, and 0.1% Triton X-100. Flow cytometry was performed using a PerkinElmer FacsCalibur instrument (Norwalk, CT). Fluorescent events (1.0 x 104) were acquired at a low setting, and data were recorded and analyzed using the CellQuest software (PerkinElmer).

Statistical analysis

Data obtained were subjected to ANOVA using the CoStat package from CoHort Software (Berkley, CA). The Student-Neuman-Keuls test of mean separation was used for post-ANOVA analysis. The data are presented as mean ± SEM, and statistical significance was determined at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
EGF works through MAPK signaling pathway in luteinized granulosa cells

MAPK activity. MAPK activity in the lysates was assayed from the four treatment groups: 1) control, 2) EGF, 3) tyrphostin 51, and 4) tyrphostin 51 plus EGF. MAPK activity in EGF-treated cells was greater than in controls (Fig. 1Go). Treatment with tyrphostin 51 alone kept the MAPK activity to the level of control. Treatment with tyrphostin 51 in combination with EGF prevented the increase in MAPK activity above the baseline levels. The MAPK activities observed (mean ± SEM pmol/h 32P incorporation) for the different experimental groups were as follows: control, 100.68 ± 24.51; EGF, 208.70 ± 23.19; tyrphostin 51, 77.97 ± 15.97, and tyrphostin 51 + EGF = 111.15 ± 20.65 (n = 14 patients; P < 0.05).

Phosphorylation of ERK1/ERK2. Phosphorylated and total MAPK in the luteinized granulosa cells was determined by Western blotting. Treatment with EGF significantly increased the levels of phosphorylated MAPK. Tyrphostin 51 plus EGF treatment decreased the levels of phosphorylated MAPK, relative to EGF alone. Relative optical densities of the immunoreactive bands of the phosphorylated MAPK were as follows (Fig. 2AGo): control, 1.04 ± 0.34; EGF, 3.29 ± 0.47; tyrphostin 51, 1.04 ± 0.28; and tyrphostin 51 + EGF, 1.58 ± 0.45 (n = 5 patients; P < 0.05). As a control, we tested the levels of total MAPK and observed that they did not change with either EGF or tyrphostin 51 treatment (Fig. 2BGo).



View larger version (58K):
[in this window]
[in a new window]
  		FIG. 2. EGF-stimulated phosphorylation of MAPK. A, Representative Western blot of phosphorylated MAPK. Cell lysates from four treatment groups as described in Fig. 1Go were subjected to Western analysis using anti-phospho-p44/42 MAPK antibody. Immunoreactive bands for phosphorylated p44/42 MAPK are indicated. Densitometry of Western blots of phosphorylated p44/42 MAPK (n = 5 patients) demonstrated that EGF treatment increases phosphorylated MAPK compared with the other treatments (P < 0.05). The data presented are normalized. B, Representative Western blot of total MAPK. Shown is a Western blot analysis of cell lysates from four treatment groups using anti-total MAPK antibody. Overall, there was no difference in the levels of total MAPK in the different treatments.

 
Confocal microscopy and localization of MAPK Confocal microscopy of the immunostained luteinized granulosa cells was used to determine the phosphorylation and nuclear translocation of the MAPK (Fig. 3Go). We observed green fluorescence, indicating that phosphorylated MAPK was present in the cytoplasm and the nuclei of the control and EGF-treated cells (Fig. 3Go, A and B). Treatment with EGF enhanced the fluorescence staining at the nuclei of the luteinized cells (arrow, Fig. 3BGo). However, treatment with tyrphostin 51 alone or tyrphostin 51 in combination with EGF reduced the levels and nuclear translocation of phosphorylated MAPK (Fig. 3Go, C and D).



View larger version (71K):
[in this window]
[in a new window]
  		FIG. 3. EGF-stimulated nuclear translocation of activated MAPK; immunohistochemical confocal microscopy using anti-phospho-MAPK antibodies. A, Control cells treated with vehicle only. B, Cells treated with EGF. Arrow points to concentrated fluorescent staining surrounding the nucleus. C, Cells treated with tyrphostin 51, showing blockage of nuclear translocation of phosphorylated MAPK. D, Cells treated with tyrphostin 51 followed by EGF treatment.

 
Inhibition of EGF-R by tyrphostin 51 induces apoptosis in luteinized granulosa cells

Activation of caspase-3. Western analysis of cell lysates was performed to demonstrate the presence of activated caspase-3 in tyrphostin 51-treated luteinized granulosa cells (n = 3 patients). Distinct immunoreactive bands of pro- and active caspase-3 migrating at 32 and 17 kDa, respectively, were observed in tyrphostin-treated cells. However, only the pro- form of caspase-3 was seen in control cells (Fig. 4Go).



View larger version (69K):
[in this window]
[in a new window]
  		FIG. 4. Western analysis of activated caspase-3. Western analysis of lysates from control and tyrphostin 51-treated cells was performed using anti-caspase-3 antibodies (CPP32). A representative blot demonstrating the presence of a 32-kDa pro-form and 17-kDa active caspase-3 were observed in tyrphostin-treated luteinized granulosa cells. Only the pro- form was seen in control cells.

 
Apoptotic DNA fragmentation. Flow cytometry of isolated nuclei stained with propidium iodide was used to assess apoptotic fragmentation of DNA (Fig. 5Go). Tyrphostin 51 treatment resulted in a higher proportion of apoptotic nuclei. The percentages of apoptotic nuclei for different groups were as follows: control, 6.58 ± 1.36%; EGF, 7.26 ± 1.53%; tyrphostin 51, 11.63 ± 1.11%; and tyrphostin 51 plus EGF, 6.87 ± 0.79% (n = 5 patients; P < 0.05).



View larger version (16K):
[in this window]
[in a new window]
  		FIG. 5. Flow cytometry of tyrphostin 51-induced subdiploid apoptotic nuclei from the four treatment groups: control, EGF, tyrphostin 51, and tyrphostin 51 + EGF, conditions as detailed in Fig. 1Go legend. Granulosa cells from each patient (n = 5 patients) were isolated, aliquoted into wells, and treated with all four treatment conditions. The mean percentage of apoptotic nuclei for tyrphostin 51-treated cells is significantly greater than for other treatments (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Regulatory factors, such as EGF, that are present in the ovary have been reported to play diverse roles in the ovarian physiology. EGF-R autocrine pathways contribute to a number of processes important to cell proliferation and apoptosis (21). EGF-R tyrosine kinase activation is necessary for EGF-mediated cell proliferation. Blockage of receptor tyrosine kinase activation inhibits growth factor-mediated cell proliferation. EGF promotes proliferation and cell survival, and its actions on porcine granulosa cells have shown tyrosine phosphorylation of MAPK activity (22, 23). Reports indicated inhibition of MAPK activity during oxidative stress-induced apoptosis (24).

Thus, blockage of MAPK pathway has been shown in several instances to induce apoptosis. However, reports have been contradictory due to limitations of studies mainly of inhibition of cell growth in cell lines. Our present study involves inhibition of EGF-R activation by a specific tyrosine kinase inhibitor, tyrphostin 51, in human luteinized granulosa cells. Tyrphostin 51 is an EGF-R inhibitor with an IC50 of 0.8 mM. In our studies we blocked the EGF-R by incubating the cells for 1 h with 100 µM tyrphostin 51. We observed that treatment of luteinized granulosa cells with 100 µM tyrphostin 51 suppressed the MAPK activity and phosphorylation, as measured by radiochemical assay and Western blot analysis. Tyrphostin 51 also inhibited nuclear translocation of phosphorylated MAPK, suggesting that tyrphostin suppresses the MAPK in luteinized granulosa cells. Mitogenic stimuli phosphorylate the MAPK and shuttle these tyrosine kinases between the nucleus and cytoplasm, making the nucleus a critical site during signal transduction (25). Studies by other groups also demonstrated that subsequent to mitogenic stimulation by growth factors, MAPK translocated to the nucleus in fibroblast cells (26) and Chinese hamster ovary cells (27).

It has been reported that inhibition of EGF-R by its specific antibodies blocked activation of receptor tyrosine kinase, resulted in G1 cell cycle arrest, and induced apoptosis in human colorectal cell lines (28). In addition, studies have shown that inhibition of EGF-R by tyrphostin AG957 inhibited growth in leukemia cells (29) and tyrphostin B42 inhibited tyrosine phosphorylation in activated T cells (30). Tyrphostin AG1478 in combination with genistein, a general tyrosine kinase inhibitor, inhibited phosphorylation of EGF-R and induced apoptosis involving CPP32 (active caspase-3)-mediated poly(ADP-ribose) polymerase cleavage and DNA fragmentation in lung cancer cells (31). In our present study, we observed that treatment of luteinized granulosa cells with tyrphostin 51 resulted in activation of caspase-3, an executioner protease during apoptosis, and an increase in subdiploid apoptotic nuclei as observed by flow cytometry. Because treatment with tyrphostin 51 activated the caspase-3 and increased the subdiploid apoptotic DNA, we believe that activation of MAPK is required for survival of luteinized granulosa cells and its inhibition induces apoptosis. We demonstrated earlier that inhibition of the EGF-R signaling pathway by PD98059 (11), a MEK inhibitor, or with geldanamycin (12), a Raf-1 depletor, results in MAPK inhibition and induction of apoptosis in human luteinized granulosa cells. These data support the present findings of a direct correlation between inhibition of MAPK signaling and induction of apoptosis in luteinized granulosa cells. Our findings support our previous observations on granulosa cells obtained from in vitro fertilization patients, and we believe that it may be conceivable that the conclusions can also be applied to granulosa cells from a single follicle of an unstimulated cycle.

Our studies present for the first time a relationship between the EGF signaling pathway and apoptosis in luteinized granulosa cells. Our data support previous observations in cancer cells, that inhibition of EGF-R induces apoptosis, indicating that a similar mechanism may function in luteinized granulosa cells treated with tyrphostin 51. In addition, our findings support the hypothesis that EGF works through the MAPK pathway and its inhibition may induce apoptosis in luteinized granulosa cells. Thus, the EGF-R/MAPK signaling pathway may become an excellent potential target for understanding the molecular mechanisms of follicular atresia and corpus luteum regression. Further studies are required to define the role of the MAPK pathway in regulating ovarian physiology. The detailed mechanisms of ovarian cell apoptosis are not clearly understood. A better understanding of the relationship between MAPK signaling and apoptosis in luteinized granulosa cells could enable us to understand the process of apoptosis in ovarian physiology such as corpus luteum regression.


   Footnotes
 
This work was supported in part by NIH Grant HD 01016 (to J.Y.).

Results from this work were presented in part at the 46th Annual Meeting of the Society of Gynecological Investigation, Atlanta, Georgia, March 10–13, 1999, and at the 81st Annual Meeting of the Endocrine Society, San Diego, California, June 12–16, 1999.

First Published Online October 19, 2004

Abbreviations: EGF, Epidermal growth factor; EGF-R, EGF receptor; MBP, myelin basic protein; MEK, MAPK kinase.

Received March 5, 2004.

Accepted October 11, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Yeh J, Adashi E 1999 The ovarian life cycle. In: Yeh S, Jaffe R, Barbieri R, eds. Reproductive endocrinology. Philadelphia: WB Saunders; 153–190
  2. Khan-Dawood FS, Ayyagari RR, Dawood MY 1995 Epidermal growth factor receptors in human corpora lutea during the menstrual cycle and pregnancy. Hum Reprod 10:193–198[Abstract/Free Full Text]
  3. Huang JC, Khan-Dawood FS, Dawood MY, Yeh J 1995 Baboon corpus luteum: epidermal growth factor receptor messenger ribonucleic acid expression during early, midluteal, and late luteal phases. Fertil Steril 63:1318–1321[Medline]
  4. Kennedy TG, Brown KD, Vaughan TJ 1993 Expression of the genes for the epidermal growth factor receptor and its ligands in porcine corpora lutea. Endocrinology 132:1857–1859[Abstract]
  5. Gospodarowicz D, Ill CR, Birdwell CR 1977 Effects of fibroblast and epidermal growth factors on ovarian cell proliferation in vitro. II. Proliferative response of luteal cells to FGF but not EGF. Endocrinology 100:1121–1128[Abstract]
  6. Yarden Y, Sliwkowski MX 2001 Untangling the ErbB signalling network. Nat Rev Mol Cell Biol 2:127–137[CrossRef][Medline]
  7. Crews CM, Alessandrini A, Erikson RL 1992 The primary structure of MEK, a protein kinase that phosphorylates the ERK gene product. Science 258:478–480[Abstract/Free Full Text]
  8. Moyer JD, Barbacci EG, Iwata KK, Arnold L, Boman B, Cunningham A, DiOrio C, Doty J, Morin MJ, Moyer MP, Neveu M, Pollack VA, Pustilnik LR, Reynolds MM, Sloan D, Theleman A, Miller P 1997 Induction of apoptosis and cell cycle arrest by CP-358,774, an inhibitor of epidermal growth factor receptor tyrosine kinase. Cancer Res 57:4838–4848[Abstract/Free Full Text]
  9. Lei W, Mayotte JE, Levitt ML 1998 EGF-dependent and independent programmed cell death pathways in NCI-H596 nonsmall cell lung cancer cells. Biochem Biophys Res Commun 245:939–945[CrossRef][Medline]
  10. Gilmore AP, Valentijn AJ, Wang P, Ranger AM, Bundred N, O’Hare MJ, Wakeling A, Korsmeyer SJ, Streuli CH 2002 Activation of BAD by therapeutic inhibition of epidermal growth factor receptor and transactivation by insulin-like growth factor receptor. J Biol Chem 277:27643–27650[Abstract/Free Full Text]
  11. Oliver RH, Khan SM, Leung BS, Yeh J 1999 Induction of apoptosis in luteinized granulosa cells by the MAP kinase kinase (MEK) inhibitor PD98059. Biochem Biophys Res Commun 263:143–148[CrossRef][Medline]
  12. Khan SM, Oliver RH, Dauffenbach LM, Yeh J 2000 Depletion of Raf-1 protooncogene by geldanamycin causes apoptosis in human luteinized granulosa cells. Fertil Steril 74:359–365[CrossRef][Medline]
  13. Izawa M, Nguyen PH, Kim HH, Yeh J 1998 Expression of the apoptosis-related genes, caspase-1, caspase-3, DNA fragmentation factor, and apoptotic protease activating factor-1, in human granulosa cells. Fertil Steril 70:549–552[CrossRef][Medline]
  14. Robles R, Tao XJ, Trbovich AM, Maravel DV, Nahum R, Perez GI, Tilly KI, Tilly JL 1999 Localization, regulation and possible consequences of apoptotic protease-activating factor-1 (Apaf-1) expression in granulosa cells of the mouse ovary. Endocrinology 140:2641–2644[Abstract/Free Full Text]
  15. Boone DL, Tsang BK 1998 Caspase-3 in the rat ovary: localization and possible role in follicular atresia and luteal regression. Biol Reprod 58:1533–1539[Abstract/Free Full Text]
  16. Matsui T, Manabe N, Goto Y, Inoue N, Nishihara S, Miyamoto H 2003 Expression and activity of Apaf1 and caspase-9 in granulosa cells during follicular atresia in pig ovaries. Reproduction 126:113–120[Abstract]
  17. Cole JA 1999 Parathyroid hormone activates mitogen-activated protein kinase in opossum kidney cells. Endocrinology 140:5771–5779[Abstract/Free Full Text]
  18. Ben-Bassat H, Rosenbaum-Mitrani S, Hartzstark Z, Levitzki R, Chaouat M, Shlomai Z, Klein BY, Kleinberger-Doron N, Gazit A, Tsvieli R, Levitzki A 1999 Tyrphostins that suppress the growth of human papilloma virus 16-immortalized human keratinocytes. J Pharmacol Exp Ther 290:1442–1457[Abstract/Free Full Text]
  19. Twaddle GM, Turbov J, Liu N, Murthy S 1999 Tyrosine kinase inhibitors as antiproliferative agents against an estrogen-dependent breast cancer cell line in vitro. J Surg Oncol 70:83–90[CrossRef][Medline]
  20. Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C 1991 A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 139:271–279[CrossRef][Medline]
  21. Ciardiello F, Tortora G 2001 A novel approach in the treatment of cancer: targeting the epidermal growth factor receptor. Clin Cancer Res 7:2958–2970[Abstract/Free Full Text]
  22. Keel BA, Hildebrandt JM, May JV, Davis JS 1995 Effects of epidermal growth factor on the tyrosine phosphorylation of mitogen-activated protein kinases in monolayer cultures of porcine granulosa cells. Endocrinology 136:1197–1204[Abstract]
  23. Keel BA, Davis JS 1999 Epidermal growth factor activates extracellular signal-regulated protein kinases (ERK) in freshly isolated porcine granulosa cells. Steroids 64:654–658[CrossRef][Medline]
  24. Schaeffer C, Vandroux D, Thomassin L, Athias P, Rochette L, Connat JL 2003 Calcitonin gene-related peptide partly protects cultured smooth muscle cells from apoptosis induced by an oxidative stress via activation of ERK1/2 MAPK. Biochim Biophys Acta 1643:65–73[Medline]
  25. Volmat V, Camps M, Arkinstall S, Pouyssegur J, Lenormand P 2001 The nucleus, a site for signal termination by sequestration and inactivation of p42/p44 MAP kinases. J Cell Sci 114:3433–3443[Abstract/Free Full Text]
  26. Lenormand P, Sardet C, Pages G, L’Allemain G, Brunet A, Pouyssegur J 1993 Growth factors induce nuclear translocation of MAP kinases (p42mapk and p44mapk) but not of their activator MAP kinase kinase (p45mapkk) in fibroblasts. J Cell Biol 122:1079–1088[Abstract/Free Full Text]
  27. Hulleman E, Bijvelt JJ, Verkleij AJ, Verrips CT, Boonstra J 1999 Nuclear translocation of mitogen-activated protein kinase p42MAPK during the ongoing cell cycle. J Cell Physiol 180:325–333[CrossRef][Medline]
  28. Wu X, Fan Z, Masui H, Rosen N, Mendelsohn J 1995 Apoptosis induced by an anti-epidermal growth factor receptor monoclonal antibody in a human colorectal carcinoma cell line and its delay by insulin. J Clin Invest 95:1897–1905
  29. Carlo-Stella C, Regazzi E, Sammarelli G, Colla S, Garau D, Gazit A, Savoldo B, Cilloni D, Tabilio A, Levitzki A, Rizzoli V 1999 Effects of the tyrosine kinase inhibitor AG957 and an anti-Fas receptor antibody on CD34+ chronic myelogenous leukemia progenitor cells. Blood 93:3973–3982[Abstract/Free Full Text]
  30. Bright JJ, Du C, Sriram S 1999 Tyrphostin B42 inhibits IL-12-induced tyrosine phosphorylation and activation of Janus kinase-2 and prevents experimental allergic encephalomyelitis. J Immunol 162:6255–6262[Abstract/Free Full Text]
  31. Lei W, Mayotte JE, Levitt ML 1999 Enhancement of chemosensitivity and programmed cell death by tyrosine kinase inhibitors correlates with EGFR expression in non-small cell lung cancer cells. Anticancer Res 19:221–228[Medline]



This article has been cited by other articles:


Home page
 Hum ReprodHome page
I. Ben-Ami, L. Armon, S. Freimann, D. Strassburger, R. Ron-El, and A. Amsterdam
EGF-like growth factors as LH mediators in the human corpus luteum
Hum. Reprod., October 3, 2008; (2008) den359v1.
[Abstract] [Full Text] [PDF]

Home page
 Hum ReprodHome page
K.N. Fru, M. Cherian-Shaw, M. Puttabyatappa, C.A. VandeVoort, and C.L. Chaffin
Regulation of granulosa cell proliferation and EGF-like ligands during the periovulatory interval in monkeys
Hum. Reprod., May 1, 2007; 22(5): 1247 - 1252.
[Abstract] [Full Text] [PDF]

Home page
 Reproductive SciencesHome page
J. Yeh and B. Kim
Increasing Blunting of Inhibin Responses to Dynamic Ovarian Challenge Is Associated With Reproductive Aging in the Rat
Reproductive Sciences, January 1, 2007; 14(1): 10 - 19.
[Abstract] [PDF]

Home page
 Mol Hum ReprodHome page
R. Haimov-Kochman, A. Ravhon, D. Prus, C. Greenfield, Z. Finci-Yeheskel, D. S.Goldman-Wohl, S. Natanson-Yaron, R. Reich, S. Yagel, and A. Hurwitz
Expression and regulation of Sprouty-2 in the granulosa-lutein cells of the corpus luteum
Mol. Hum. Reprod., August 1, 2005; 11(8): 537 - 542.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
90/1/469    most recent
Author Manuscript (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Khan, S. M.
Right arrow Articles by Yeh, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Khan, S. M.
Right arrow Articles by Yeh, J.
Related Collections
Right arrow Female Endocrinology

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals