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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 9 3370-3375
Copyright © 2000 by The Endocrine Society

Original Studies

A Gonadotropin-Releasing Hormone-Responsive Phosphatase Hydrolyses Lysophosphatidic Acid within the Plasma Membrane of Ovarian Cancer Cells

Atsushi Imai, Tatsuro Furui, Teruhiko Tamaya and Gordon B. Mills

Department of Obstetrics and Gynecology, Gifu University School of Medicine (A.I., T.F., T.T.), Gifu 500-8705, Japan; and Department of Molecular Therapeutics, University of Texas, M. D. Anderson Cancer Center (G.B.M.), Houston, Texas 77030

Address all correspondence and requests for reprints to: Dr. Atsushi Imai, Department of Obstetrics and Gynecology, Gifu University School of Medicine, Tsukasamachi, Gifu 500-8705, Japan. E-mail: atsushi{at}cc.gifu-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Lysophosphatidic acid (LPA) mediates pleomorphic effects on multiple cell lineages, including an increased proliferative response of ovarian cancer cells both in vitro and in vivo, at least in part through the novel expression of LPA receptors. Thus, LPA hydrolysis is necessary to limit the duration of LPA’s action on multiple cell types, including ovarian cancer cells. We determined the principal mechanism of LPA hydrolysis by ovarian cancer cells and its regulation by GnRH, which is known to have antiproliferative actions on ovarian carcinomas. LPA-hydrolyzing activity in cell membranes of ovarian cancer specimens was assessed by measuring the conversion of exogenous [3H-oleoyl]LPA to [3H]oleic acid or mono[3H-oleoyl]glycerol. Approximately 98% of LPA hydrolysis could be accounted for by the dephosphorylation of LPA to yield monoglyceride, with the deacylation reaction accounting for less than 1% of LPA hydrolysis. The phosphatase activity in the plasma membrane ovarian cancer cells was approximately 2.5- and 8-fold higher than those in microsome and homogenate fractions, respectively. The membrane phosphatase was Mg2+ independent and insensitive to inhibition by N-ethylmaleimide, characteristics suggestive of phosphatidic acid phosphatase activity. Incubation of membranes from GnRH receptor-positive ovarian cancer specimens with the GnRH agonist, buserelin, induced a dose-dependent increase in LPA phosphatase activity, with a half-maximal effect occurring with 30 nmol/L buserelin. The stimulatory action of buserelin could be neutralized by displacement of GnRH from its receptor by the GnRH antagonist, antide. The plasma membranes from GnRH receptor-negative ovarian cancer specimens did not respond to GnRH stimulation. LPA phosphatase activity was also increased when the ovarian cancer cell line Caov-3 was exposed to GnRH agonist in intact cells before assay of cell membranes. These data demonstrate that LPA is hydrolyzed in the plasma membrane of ovarian cancer cells by the action of LPA phosphatase and provide initial evidence for functional coupling of LPA phosphatase to GnRH receptor occupancy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LYSOPHOSPHATIDIC acid (LPA), the simplest phospholipid, mediates a wide range of functions, including enhanced cell growth, decreased cell death, increased cancer cell invasion, and improved wound healing and regeneration (1, 2, 3). Previous reports have shown that ascetic fluid and plasma from patients with ovarian cancer contain high levels of LPA (4, 5, 6). LPA stimulates the proliferation of ovarian cancer cells; increases intracellular calcium release and tyrosine phosphorylation; activates multiple kinases, including mitogen-activated protein kinase and focal adhesion kinase; and increases the production of proteases involved in invasion and increases production of LPA itself (7, 8). LPA also decreases chemotherapy-induced ovarian cancer cell death (9). The effects of LPA on ovarian cancer cells may be mediated in part by the novel expression of LPA receptors by ovarian cancer cells (10). Thus, LPA contributes to the pathophysiology and likely outcome of ovarian cancer. The contribution of LPA to multiple functions in ovarian cancer cells suggests that LPA levels may be tightly regulated in vivo. There are two possible LPA-hydrolyzing pathways, one via deacylation by (lyso)phospholipase and the other via dephosphorylation by (lyso)phosphatase (11). Alternatively, LPA can also be converted to PA, which is also bioactive, through the action of endopilin (12). It is, however, unclear which pathways contribute to LPA metabolism in vivo.

GnRH agonists inhibit the proliferation of ovarian cancer cells both in vivo and in vitro (13, 14). GnRH-responsive ovarian carcinomas express functional GnRH receptors; however, the molecular mechanisms linking GnRH receptor occupancy to the growth-inhibiting activity remain unclear (15, 16). We have shown that GnRH agonists decrease the phosphorylation of a membrane protein substrate through activation of a phosphoprotein phosphatase that is known to counteract protein phosphorylation and to promote cell growth (17, 18). The objectives of this investigation were to define the pathway(s) mediating LPA degradation and determine whether the growth inhibitory activity of GnRH agonists on ovarian cancer cells was associated with alterations in LPA metabolism in surgically removed ovarian carcinomas and cloned ovarian cancer cell lines.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

The GnRH agonists, buserelin and leuprolide, were gifts from Hoechst Japan (Tokyo, Japan) and Takeda Pharmaceutical (Osaka, Japan), respectively. The GnRH antagonist, antide, TRH, oleolyl LPA, N-ethylmaleimide (NEM), orthovanadate (Na3VO4), sphingosine 1-phosphate, ceramide 1-phosphate, guanosine-5'-O-(2-thiodiphosphate) (GDP-ß-S), and guanosine-5'-O-(3-thiotriphosphate) (GTP-{gamma}-S) were purchased from Sigma (St. Louis, MO). Mono-oleoyl-[9,10-3H]LPA (1.9 TBq/mmol) was obtained from NEN Life Science Products (Boston, MA). Silica gel 60 plates were products of Merck & Co., Inc. (Darmstadt, Germany). All other chemicals were of reagent grade.

Tissue collection and cell cultures

Ovarian carcinoma specimens were placed in ice-cold phosphate-buffered saline immediately after surgical removal, and representative portions were excised for histological frozen sections. These tissue samples were washed and immediately used or stored in liquid nitrogen. Only tissues obtained at initial surgery were used for analyses. We screened the surgical samples for the presence of GnRH-binding sites and GnRH receptor messenger ribonucleic acid as described previously (16). Of a total of 12 samples (5 mucinous cystadenocarcinomas, 5 serous cystadenocarcinomas, and 2 clear cell carcinomas), 10 (5 mucinous cystadenocarcinomas, 4 serous cystadenocarcinomas, and 1 clear cell carcinoma) had obvious GnRH receptor expression. Clinical data and the results of GnRH receptor analyses are summarized in Table 1Go. The investigation had the approval of the Gifu University research ethics committee, and all patients gave informed consent to the disposition of their surgically removed tissues. Aside from the diagnosis of ovarian carcinoma, these patients were free of endocrine or systemic diseases.


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  		Table 1. Clinical data and LPA phosphatase activities of the surgically removed ovarian carcinomas

 
The human ovarian carcinoma cell line, Caov-3, was obtained from American Type Culture Collection (Manassas, VA); this cell line is GnRH receptor positive (16, 19). The cells were grown at 37 C in an appropriate medium containing FBS in a humidified atmosphere of 5% CO2-95% air. To examine hormonal effects, the cells were grown to confluence in multiwell plates and treated with the indicated concentrations of test agents for the desired time intervals.

Plasma membrane isolation

The specimens (tumors surgically removed and cell pellet) were homogenized in 10 vol ice-cold lysis buffer (0.5 mmol/L dithiothreitol, 1 mmol/L ethyleneglycol tetraacetic acid, 1 mmol/L NaHCO3, and 10 mmol/L HEPES, pH 7.9) by brief sonication. Highly purified plasma membrane fractions from the homogenates were obtained as described previously (17, 18). The isolated plasma membranes finally were resuspended in the lysis buffer and immediately submitted to the following experiments. Marker enzyme analyses were performed using 5'-nucleotidase as a marker for the plasma membrane and succinate dehydrogenase for mitochondria (20, 21). Protein was determined according to Lowry’s method (22), using BSA as a standard. The specific activity of 5'-nucleotidase in the plasma membrane fraction was increased approximately 6- to 9-fold, compared to that of the homogenate (1–3 µmol/mg protein·h); the specific activity of succinate dehydrogenase was decreased to approximately 1/10th that of the homogenate (650 pmol/mg protein·min).

Where indicated, the plasma membranes isolated from different ovarian carcinomas were pooled; pool 1 contained samples from cases 1–3, pool 2 from cases 4–6, and pool 3 from cases 7–9 and 10. Pool 4 contained plasma membranes of tumors from cases 10 and 12 for GnRH receptor-negative specimens.

LPA hydrolysis assay

LPA metabolism was assessed using exogenous [3H-oleoyl]LPA by measuring [3H]oleic acid and mono-[3H-oleoyl]glycerol production. The reaction mixture consisted of 50 mmol/L HEPES (pH 7.5), 1 mmol/L ethyleneglycol tetraacetic acid, 50 µmol/L [3H]LPA (2 x 106 dpm), and the indicated amount of enzyme (10–100 µg protein) as a final concentration (23). In some experiments, the concentration of LPA was altered by the addition of nonradiolabeled oleoyl LPA. LPA was added after dispersal by brief sonication. The incubation was started by adding enzyme protein and was incubated at 37 C for the desired time intervals in the presence of the ligands to be tested. For the experiments with sphingosine 1-phosphate or ceremide 1-phosphate, these lipids were added after dispersal by brief sonication. The reaction was terminated by adding 4 vol chloroform-methanol (1:2, vol/vol), followed by 1 vol chloroform and 1 vol water. After the two phases were separated, the lower phases were dried under N2 and applied to silica gel 60 plates. Plates were developed in chloroform-acetone (96:4, vol/vol) (20, 21). Dried plates were exposed to iodine vapor to localize free fatty acid and monoglyceride fractions by comparison with authentic standards. The areas corresponding to individual lipids were scraped, and the radioactivity was determined. Under these conditions, less than 2% of the added substrate LPA was consumed. All experiments were performed in triplicate determinations.

Statistics

Statistical analysis was performed using one-way ANOVA in combination with Scheffe’s test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LPA hydrolysis by phosphatase activity within plasma membrane

When the plasma membrane fraction isolated from ovarian carcinoma surgical specimens was incubated with exogenous [3H-oleoyl]LPA, there was a time-dependent increase in mono-[3H-oleoyl]glycerol (Fig. 1Go, upper panel). Monoglyceride production persisted for at least 60 min. The apparent activity per mg protein in the plasma membrane was approximately 2.5- and 8-fold higher than those found in the microsome (crude membrane) and homogenate fractions, respectively. Table 1Go summarizes the clinical data and LPA phosphatase activity of the ovarian carcinomas tested. All surgically removed specimens had significant LPA phosphatase activity.



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  		Figure 1. Time courses of [3H-oleoyl]LPA hydrolysis to mono-[3H-oleoyl]glycerol and [3H]oleic acid in plasma membranes, microsomes, and homogenates from ovarian carcinomas. Plasma membrane, microsome, and homogenate fractions from surgically removed ovarian carcinomas were incubated with [3H-oleoyl]LPA (50 µmol/L) at 37 C for the indicated times. The reaction was terminated with chloroform-methanol. The radioactivity in lipids was determined by thin layer chromatography followed by scraping and scintillation counting. Each point represents the mean ± SD of four different membrane pools. *, P < 0.01 vs. plasma membrane fractions.

 
Exposure of exogenous oleoyl LPA to plasma membranes resulted in a time-dependent increase in oleic acid levels. However, phospholipase activity was approximately 100-fold less than LPA phosphatase activity (Fig. 1Go, lower panel). Hence, the predominant pathway of LPA metabolism by ovarian cancer cell membranes was dephosphorylation by LPA phosphates.

Kinetics and catalytic properties of LPA phosphatase

The membrane LPA phosphatase hydrolyzed [3H-oleoyl]- LPA to mono-[3H-oleoyl]glycerol with typical Michaelis-Menten kinetics (Fig. 2Go). Analysis of the results with a double reciprocal plot yielded a maximum velocity of 6.41 nmol/mg protein·h and a Km for LPA phosphatase of 43.7 µmol/L.



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  		Figure 2. Kinetics of LPA phosphatase activity in the plasma membrane of ovarian carcinomas. The ability of the plasma membranes to hydrolyze [3H-oleoyl]LPA to mono-[3H-oleoyl]glycerol was determined at the substrate concentrations indicated. Each point represents the mean ± SD of four different membrane pools. Inset, A double reciprocal plot of the data.

 
We then compared the sensitivity of the LPA phosphatase activity in ovarian cancer cell membranes to that reported for phosphatidic acid (PA) phosphohydrolase (PAP) (23) or lipid phosphatase activity in subcellular fractions (23, 24, 25). Ca2+ and Mn2+ inhibited LPA dephosphorylation; both exhibited a 90% inhibitory effect at 2 mmol/L. Mg2+ had no effect on enzyme activity up to 2 mmol/L, suggesting that the LPA phosphatase is Mg2+ independent (Fig. 3Go). Orthovanadate, a phosphatase transition state analog, inhibited LPA phosphatase activity by 90% when added at 10 mmol/L (Fig. 3Go). NEM, known to inhibit purified PAP1 (23, 24, 25), had no effect on phosphatase activity up to 5 mmol/L (Fig. 3Go).



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  		Figure 3. Effects of various cations, inhibitors, sphingosine 1-phosphate, and ceramide 1-phosphate on LPA phosphatase activity. The plasma membrane (50 µg protein) was incubated with [3H-oleoyl]LPA (50 µmol/L) for 60 min in the presence of the indicated concentrations of Mg2+, Mn2+, Ca2+, NEM, orthovanadate, sphingosine 1-phosphate, or ceramide 1-phosphate. Each point is expressed as a percentage of the control value and represents the mean ± SD of four different membrane pools. *, P < 0.01 vs. control.

 
To explore substrate specificity, the effects of other lipid substrates were examined (Fig. 3Go). Sphingosine 1-phosphate and ceramide 1-phosphate had no effect on LPA hydrolysis at a 4-fold higher concentration (200 µmol/L) of the LPA substrate.

GnRH agonist stimulation of LPA phosphatase

The GnRH agonist, buserelin, increased LPA phosphatase activity in plasma membranes of GnRH receptor-positive ovarian cells in the presence of the nonhydrolyzable GTP analog GTP-{gamma}-S (Fig. 4Go). The effect of buserelin on monoglyceride production was dose dependent, with a half- maximal effect occurring with 30 nmol/L buserelin. GnRH agonist in the absence of GTP-{gamma}-S had no effect on LPA phosphatase activity. GDP-ß-S completely reversed the stimulated phosphatase activity (Fig. 4Go). Similar results were obtained with another GnRH agonist, leuprolide (data not shown). Control peptide, TRH (1 µmol/L), had no effect in the presence of GTP-{gamma}-S (Fig. 4Go, upper panel). LPA phosphatase activity in plasma membranes of GnRH receptor- negative tumors (cases 10 and 12, Table 1Go) was not altered by GnRH agonist (Fig. 4Go, lower panel), providing further evidence of specificity.



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  		Figure 4. Effects of a GnRH agonist on LPA phosphatase activity in the plasma membrane of GnRH receptor-positive and -negative ovarian carcinomas. Upper panel, The plasma membrane (50 µg protein) was incubated with [3H-oleoyl]LPA (50 µmol/L) for 60 min in the presence of various concentrations of buserelin with or without 1 mmol/L GTP-{gamma}-S. {odot}, Buserelin (1 µmol/L), GTP-{gamma}-S (1 mmol/L), plus GDP-ß-S (200 µmol/L). {blacktriangleup}, TRH (1 µmol/L) plus GTP-{gamma}-S (1 mmol/L). Each point is expressed as a percentage of the control value and represents the mean ± SD of three different membrane pools from GnRH receptor-positive specimens (pools 1–3). *, P < 0.01 vs. control. Lower panel, The activity was determined in the plasma membrane from GnRH receptor-negative ovarian carcinomas (pool 4). Each point is expressed as a percentage of the control value and represents the mean ± SD of triplicate determinations.

 
Ovarian cancers contain heterogeneous cell types, including cancer cells, stromal cells, lymphocytes, endothelial cells, and platelets. To assess whether GnRH analogs increase LPA phosphatase activity in ovarian cancer cells and whether the effects of activation of the GnRH receptor observed in cell membranes might occur in intact cells, LPA phosphatase activity was examined in membranes from Caov-3 ovarian cancer cells pretreated with or without the GnRH agonist buserelin (Table 2Go). LPA phosphatase activity was approximately 2.1-fold higher in membranes isolated from Caov-3 cells incubated with buserelin before lysis than in membranes from control cells.


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  		Table 2. LPA phosphatase activities in plasma membranes isolated from Caov-3 cells exposed to a GnRH agonist

 
LPA phosphatase activation associated with GnRH receptor occupancy

To determine whether GnRH receptor occupancy was specifically linked to increased LPA hydrolysis, we used antide, a drug that displaces bound GnRH from its receptors and serves as a competitive antagonist of GnRH action. As shown in Fig. 5Go, after 30 min of incubation with buserelin, the addition of 10 µmol/L antide, a dose that displaces virtually all of the GnRH agonist under these conditions (26, 27), abolished buserelin-stimulated LPA dephosphorylation. These findings suggest that continued GnRH receptor occupancy was necessary for ongoing LPA hydrolysis.



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  		Figure 5. Effects of GnRH antagonist antide on buserelin-stimulated LPA phosphatase activity in plasma membrane from ovarian carcinomas. Plasma membranes were incubated with [3H]LPA in the presence (•) or absence ({circ}) of buserelin (1 µmol/L) plus GTP-{gamma}-S (1 mmol/L). At 30 min, antide (10 µmol/L) was added to portions of the membrane suspension previously exposed to buserelin ({odot}). Each point represents the mean ± SD of three different membrane pools (pools 1–3). *, P < 0.01 vs. GnRH-stimulated cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
LPA degradation may be crucial to the termination of its activity on both normal and malignant cells. LPA can be degraded by PAPs and by (lyso)phospholipases (11). In clinical specimens of surgically removed ovarian cancer, we first found that a LPA phosphatase in the plasma membrane was the predominant mediator of LPA degradation, and LPA lysophospholipase activity was very low. Further the GnRH receptor couples to the LPA phosphatase activity in membranes from GnRH receptor-positive cells.

LPA phosphatase activity in ovarian cell membrane most closely resembles PAP (23, 28). To date, two LPA/PA phosphatase families have been identified. PAP type 1 (PAP1) is predominantly present in cytosol and microsomes (29, 30), whereas PAP type 2 (PAP2) is localized in the plasma membrane (24, 31, 32). Three PAP2 isozymes have been cloned and demonstrated to hydrolyze LPA (32, 33). PAP1 requires Mg2+ and is completely inhibited by NEM (23, 24, 25). In contrast, PAP2 does not require Mg2+, and its activity is not inhibited by NEM (23, 24, 25). As ovarian cancer plasma membrane LPA phosphatase activity was Mg2+ independent and NEM insensitive, PAP2 is likely to be the predominant enzyme(s) responsible for LPA hydrolysis in ovarian cancer membrane. PAP2 is proposed to place the catalytic site in the extracellular space (34, 35). The likely transmembrane topology of the PAP2 may be consistent with its physiological role to attenuate cell proliferation through the conversion of exogenous LPA to its dephosphorylated counterpart.

The GnRH receptor is a member of the family of GTP-binding protein-coupled receptors that possess seven transmembrane domains (36). There are striking differences in the signal transduction pathways activated by GnRH receptors in ovarian cancer cells and anterior pituitary cells; the GnRH receptor in ovarian cancer couples to the Gi family (17, 26), whereas the GnRH receptor in pituitary gonadotrophs cells couples to the subfamily (37, 38) of G proteins. In the cell-free system, hormones that bind to a specific GTP-binding protein coupled receptor require the copresence of a nonhydrolyzable GTP analog to reveal their hormonal effects (39). In the plasma membrane of GnRH receptor-bearing ovarian cancer, we found that 1) GnRH agonists increase LPA phosphatase activity; 2) GTP-{gamma}-S was essential for the GnRH agonist to stimulate LPA phosphatase activity, and non- hydrolyzable GDP-ß-S reversed the GnRH action; and 3) membranes from GnRH receptor-negative specimens did not demonstrate increased LPA phosphatase activity in response to GnRH stimulation. GnRH-stimulated LPA dephosphorylation was neutralized by the competitive antagonist, antide. Detergents such as Triton X-100 (0.1%) caused severalfold increases in the basal activity of LPA dephosphorylation (data not shown), as previously described (23, 24, 25). However, GnRH had no activating effect in the presence of the detergent. This may be caused by nonspecific perturbation of the structure of the plasma membranes by detergents.

Ovarian cancers contain multiple cell types, including cancer cells, stromal cells, and blood vessels. Our specimens may also contain platelets, which can produce and release LPA (40). Ovarian cancer cells can produce LPA, at least in vitro (41). Further, the elevated LPA levels in ovarian cancer patient plasma suggest that ovarian cancer cells may contribute to LPA production in vivo. We demonstrated that treatment of intact Caov-3 cells with GnRH agonists increased LPA phosphatase activity in cell membranes. Thus, ovarian cancer cells might display GnRH-responsive LPA phosphatase activity in vivo. As GnRH agonists decrease the growth of GnRH receptor-positive ovarian cancer cells, and LPA can increase the growth of ovarian cancer cells, increased LPA hydrolysis may contribute to the effects of GnRH on ovarian cancer cells. These findings also allowed us to draw two important conclusions: 1) activation of the GnRH receptor can increase LPA phosphatase activity; and 2) continued receptor occupancy was necessary for ongoing LPA hydrolysis.

Lastly, we demonstrated that hormonal activation of phosphatase may contribute to the majority of LPA loss at the level of plasma membrane in GnRH receptor-expressing ovarian carcinoma. Membranes from ovarian cancers have low levels of LPA lysophospholipase activity; however, this was less than 1% of the LPA phosphatase activity. The LPA phosphatase activation linked to GnRH receptor occupancy may at least partially mediate the antiproliferative action of GnRH agonist by release of LPA stimulation to promote cell growth. Although our data were derived from experiments that used surgically removed ovarian cancers as well as an ovarian cancer cell line in vitro, GnRH-induced LPA hydrolysis may play a role in LPA homeostasis and effects of GnRH in vivo.

Received March 21, 2000.

Revised May 4, 2000.

Accepted May 22, 2000.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Moolenaar W. 1995 Lysophosphatidic acid; a multifunctional phospholipid messenger. J Biol Chem. 270:12949–12952.[Free Full Text]
  2. Moolenaar W, Kranenburg O, Postma F, Zondag G. 1997 Lysophosphatidic acid: G-protein signalling and cellular responses. Curr Opin Cell Biol. 9:168–173.[CrossRef][Medline]
  3. Westermann A, Beijnen J, Moolenaar W, Rodenhuis S. 1997 Growth factors in human ovarian cancer. Cancer Treat Rev. 23:113–131.[CrossRef][Medline]
  4. Xu Y, Shen Z, Wiper D, et al. 1998 Lysophosphatidic acid as a potential biomarker for ovarian and other gynecologic cancers. JAMA. 280:719–723.[Abstract/Free Full Text]
  5. Mills G, May C, McGill M, Roifman C, Mellors A. 1988 A putative new growth factor in ascitic fluid from ovarian cancer patients: identification, characterization, and mechanism of action. Cancer Res. 48:1066–1071.[Abstract/Free Full Text]
  6. Mills G, May C, Hill M, Campbell S, Shaw P, Marks A. 1990 Ascitic fluid from human ovarian cancer patients contains growth factors necessary for intraperitoneal growth of human ovarian adenocarcinoma cells. J Clin Invest. 86:851–855.
  7. Xu Y, Fang X, Casey G, Mills G. 1995 Lysophospholipids activate ovarian and breast cancer cells. Biochem J. 309:933–940.
  8. Xu Y, Gaudette D, Boynton J, et al. 1995 Characterization of an ovarian cancer activating factor in ascites from ovarian cancer patients. Clin Cancer Res. 1:1223–1232.[Abstract]
  9. Frankel A, Mills G. 1996 Peptide and lipid growth factors decrease cis-diamminedichloplatinum-induced cell death in human cancer cells. Clin Cancer Res. 2:1307–1313.[Abstract]
  10. Goetzl E, Dolezalova H, Kong Y, et al. 1999 Distinctive expression and functions of the type 4 endothelial differentiation gene-encoded G protein-coupled receptor for lysophosphatidic acid in ovarian cancer. Cancer Res. 59:5370–5375.[Abstract/Free Full Text]
  11. Brindley D, Waggoner D. 1998 Mammalian lipid phosphate phosphohydrolases. J Biol Chem. 273:24281–24284.[Free Full Text]
  12. Schmidt A, Wolde M, Thiele C, et al. 1999 Endophilin I mediates synaptic vesicle formation by transfer of arachidonate to lysophosphatidic acid. Nature. 401:133–141.[CrossRef][Medline]
  13. Emons G, Ortmann O, Schulz K. 1996 GnRH analogues in ovarian, breast and endometrial cancers. In: Lunenfeld B, Insler V, ed. GnRH analogues: the state of the art. New York: Parthenon; 95–120.
  14. Imai A, Horibe S, Takagi H, Fuseya T, Tamaya T. 1996 Signal transduction of GnRH receptor in the reproductive tract tumor. Endocr J. 43:249–260.[Medline]
  15. Imai A, Tamaya T. 2000 GnRH receptor and apoptotic signaling. Vitam Horm. 59:1–33.[Medline]
  16. Imai A, Ohno T, Iida K, Fuseya T, Furui T, Tamaya T. 1994 Gonadotropin-releasing hormone receptor in gynecologic tumors: frequent expression in adenocarcinoma histologic types. Cancer. 74:2555–2561.[CrossRef][Medline]
  17. Imai A, Horibe S, Takagi A, Tamaya T. 1997 Gi protein activation of gonadotropin-releasing hormone-mediated protein dephosphorylation in human endometrial carcinoma. Am J Obstet Gynecol. 176:371–376.[CrossRef][Medline]
  18. Imai A, Takagi H, Furui T, Horibe S, Fuseya T, Tamaya T. 1996 Evidence for coupling of phosphotyrosine phosphatase to gonadotropin-releasing hormone receptor in ovarian carcinoma membrane. Cancer. 77:132–137.[CrossRef][Medline]
  19. Imai A, Takagi A, Horibe S, Takagi H, Tamaya T. 1998 Evidence for tight coupling of gonadotropin-releasing hormone receptor to stimulated Fas ligand expression in reproductive tract tumors: possible mechanism for hormonal control of apoptotic cell death. J Clin Endocrinol Metab. 83:427–431.[Abstract/Free Full Text]
  20. Imai A, Gershengorn MC. 1987 Independent phosphatidylinositol synthesis in rat pituitary plasma membrane and endoplasmic reticulum. Nature. 325:725–728.
  21. Imai A, Gershengorn MC. 1987 Measurement of lipid turnover in response to TRH. Methods Enzymol. 141:100–111.[Medline]
  22. Lowry OH, Rosenbrough N, Farr AL, Randall RJ. 1951 Protein measurement with the Folin phenol reagent. J Biol Chem. 193:265–275.[Free Full Text]
  23. Hiroyama M, Takenawa T. 1998 Purification and characterization of a lysophosphatidic acid-specific phosphatase. Biochem J. 336:483–489.
  24. Dillon D, Chen X, Zeimetz G, et al. 1997 Mammalian Mg2+-independent phosphatidate phosphatase (PAP2) displays diacylglycerol pyrophosphate phosphatase activity. J Biol Chem. 272:10361–10366.[Abstract/Free Full Text]
  25. Kanoh H, Imai S, Yamada K, Sakane F. 1992 Purification and properties of phosphatidic acid phosphatase from porcine thymus membranes. J Biol Chem. 267:25309–25314.[Abstract/Free Full Text]
  26. Imai A, Takagi H, Horibe S, Fuseya T, Tamaya T. 1996 Coupling of gonadotropin-releasing hormone receptor to Gi protein in human reproductive tract tumors. J Clin Endocrinol Metab. 81:3249–3253.[Abstract]
  27. Imai A, Iida K, Tamaya T. 1992 Tight coupling of gonadotropin-releasing hormone receptor to stimulated phosphoinositide turnover and antigonadotropic action in granulosa cells. Gynecol Obstet Invest. 33:36–41.[CrossRef][Medline]
  28. Thompson F, Clark M. 1994 Purification of a lysophosphatidic acid-hydrolysing lysophospholipase from rat brain. Biochem J. 300:457–561.
  29. Martin A, Gomez-Munoz A, Jamal Z, Brindley D. 1991 Characterization and assay of phosphatidate phosphatase. Methods Enzymol. 197:553–563.[Medline]
  30. Gomez-Munoz A, Hatch G, Martin A, Jamal Z, Vance D, Brindley D. 1992 Effects of okadaic acid on the activities of two distinct phosphatidate phosphohydrolases in rat hepatocytes. FEBS Lett. 301:103–106.[CrossRef][Medline]
  31. Frank D, Waechter C. 1998 Purification and characterization of a polyisoprenyl phosphate phosphatase from pig brain; possible dual specificity. J Biol Chem. 273:11791–11798.[Abstract/Free Full Text]
  32. Sciorra V, Morris A. 1999 Sequential actions of phospholipase D and phosphatidic acid phosphorylase 2b generate diglyceride in mammalian cells. Mol Biol Cell. 10:3863–3876.[Abstract/Free Full Text]
  33. Roberts R, Sciorra V, Morris A. 1998 Human type 2 phosphatidic acid phosphohydrolases. Substrate specificity of the type 2a, 2b, and 2c enzymes and cell surface activity of the 2a isoform. J Biol Chem. 273:22059–22067.[Abstract/Free Full Text]
  34. Jasinska R, Zhang Q, Pilquil C, et al. 1999 Lipid phosphate phosphohydrolase-1 degrades exogenous glycerolipid and sphingolipid phosphate esters. Biochem J. 340:677–686.
  35. Waggoner D, Xu J, Singh I, Jasinska R, Zhang Q, Brindley D. 1999 Structural organization of mammalian lipid phosphate phosphatases: implications for signal transduction. Biochim Biophys Acta. 1439:299–316.[Medline]
  36. Stojilkovic SS, Reinhart J, Catt KJ. 1994 Gonadotropin-releasing hormone receptors: structure and signal transduction pathways. Endocr Rev. 15:462–499.[CrossRef][Medline]
  37. Kaiser U, Katzenellenbogen R, Conn P, Chin W. 1994 Evidence that signalling pathways by which thyrotropin-releasing hormone and gonadotropin-releasing hormone act are both common and distinct. Mol Endocrinol. 8:1038–1048.[Abstract]
  38. Hsieh K, Martin T. 1992 Thyrotropin-releasing hormone and gonadotropin-releasing hormone receptors activate phospholipase C by coupling to guanosine triphosphate-binding proteins Gq and G11. Mol Endocrinol. 6:1673–1681.[Abstract]
  39. Strader CD, Fong TM, Tota MR, Underwood D, Dixon RAF. 1994 Structure and function of G protein-coupled receptors. Annu Rev Biochem. 63:101–132.[CrossRef][Medline]
  40. Eichholitz T, Jalink K, Fahrenfort I, WH M. 1993 The bioactive phospholipid lysophosphatidic acid is released from activated platelets. Biochem J. 291:677–680.
  41. Shen Z, Belinson J, Morton R, Xu Y. 1998 Phorbol 12-myristate 13-acetate stimulates lysophosphatidic acid secretion from ovarian and cervical cancer cells but not from breast or leukemia cells. Gynecol Oncol. 71:364–368.[CrossRef][Medline]



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