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Experimental Therapeutics, Preclinical Pharmacology |
Department of Molecular Oncology, Division of Medicine, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 [A. M. E., T. S., M. M., G. B. M.], and Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan [J. A.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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26,800 women will be newly
diagnosed, and 14,500 will die from ovarian cancer (1)
.
The majority of patients are diagnosed with advanced epithelial ovarian
cancer with widespread metastatic disease. The dismal outcome for
ovarian cancer results from an inability to detect the tumor at an
early curable stage. As 90% of stage IA and 70% of stage II tumors
can be cured by current management, ovarian cancer diagnosed at an
early stage has a prognosis similar to breast cancer. The most likely
way to identify ovarian cancer at an early, curable stage and to
develop new, effective therapies for advanced ovarian cancers is to
improve our understanding of the processes leading to the initiation
and progression of this disease. Ascitic fluid from ovarian cancer patients, but not from patients with other cancers or with benign diseases such as hepatic disease, contains elevated levels of the phospholipid LPA4 (2, 3, 4, 5) . LPA levels are also significantly elevated in plasma from >90% of patients with ovarian cancer regardless of stage (6) . In contrast, LPA levels are not elevated in plasma of patients with breast cancer or leukemia or in healthy controls (6) . LPA levels are also increased in patients with endometrial cancer and cervix cancer (6) , multiple myeloma (7) , and renal dialysis (8) , all of which can be clinically distinguished from ovarian cancer. This suggests that LPA in plasma might provide a marker for diagnosis of ovarian cancer, establishing prognosis, or monitoring response to therapy. Because LPA levels are also elevated in the early stages of the disease (6) , the plasma LPA assay offers the possibility of earlier diagnosis of ovarian cancer, resulting in improved prognosis.
LPA displays a broad spectrum of biological activities (9, 10, 11, 12) . Its principle effects are growth related, such as induction of cellular proliferation and suppression of apoptosis, or involve the cytoskeleton or adhesive proteins contributing to aggregation, adhesion, contraction, secretion, and chemotaxis. LPA stimulates the growth (4 , 13) , prevents apoptosis (14) and anoikis (not presented), decreases sensitivity to chemotherapeutic drugs (15) , and increases invasiveness of ovarian cancer cells (12) . These effects are associated with increased phosphorylation of focal adhesion kinase, increased tyrosine phosphorylation of cellular proteins, increased intracellular calcium concentration, and increased MAPK activity after treatment with LPA (13) . In contrast, normal ovarian epithelial cells are resistant to the effects of LPA (16) ,5 ,6 suggesting that acquisition of LPA responsiveness is associated with transformation. LPA acts on G protein-coupled receptors encoded by the endothelial differentiation gene (Edg) subfamily (17) . The LPA and sphingosine-1-phosphate receptor Edg1 (18, 19, 20) is expressed at high levels in normal and immortalized ovarian epithelial cells but at low levels in most ovarian cancer cell lines (12) . The LPA receptor Edg2 (21) is expressed by both normal ovarian epithelial cells and ovarian cancer cell lines at varying levels (12 , 22 , 23) . In contrast, the LPA receptors Edg4 (24) and Edg7 (25) are expressed at relatively high levels in ovarian cancer cell lines but only at very low levels in normal and immortalized ovarian epithelial cells (12 , 22) . Binding of LPA to its receptor(s) activates pertussis toxin-sensitive (Gi) and -insensitive (Gq and G12/13) pathways (10 , 11) , leading to the expression of growth factor-regulated genes that contain serum response elements.
LPA is a normal constituent of serum (present at concentrations ranging from 1 to 5 µM), where it is produced and released by activated platelets (26) . LPA is also produced by growth factor-stimulated fibroblasts (27) , cytokine-stimulated leukocytes (11) , PMA-activated ovarian cancer cells (28) , and possibly by other cell types. Little is known, however, about LPA production in vivo and why LPA levels are elevated in ovarian cancer patients.
LPA may be synthesized by cells either de novo from glucose
through pathways of lipid metabolism in the endoplasmic reticulum or
through liberation of precursor phospholipids and subsequent enzymatic
conversions in membrane microvesicles (11
, 29
, 30)
. The
latter pathway is considered the principal source of production of free
and secreted LPA. PLD first converts phosphatidylcholine to PA. Two
distinct isoforms of PLD have been identified (31
, 32)
.
PLD1, but not PLD2, is activated by GTP-binding proteins and protein
kinase C. Both isoforms use phosphatidylinositol 4,5-bisphosphate as
cofactor. During the subsequent step in LPA synthesis,
PLA2 (or potentially phospholipase
A1) hydrolyzes the sn-2
(sn-1) ester bond of PA to generate LPA. Various
PLA2 enzymes displaying an exclusive or relative
selectivity for PA have been characterized (30)
. The
relative contribution of each PLA2 to LPA
synthesis is not known. On the basis of nucleotide sequence
comparisons, PLA2s have been divided into 10
groups. On the basis of biological properties,
PLA2s have been divided into three subgroups:
sPLA2, cPLA2, and
iPLA2 (33, 34, 35, 36, 37)
.
sPLA2s are low molecular mass proteins (14
kDa) with five to seven disulfide bonds that confer structural
rigidity. sPLA2s require millimolar calcium
concentrations for catalytic activity. cPLA2s are
high molecular mass proteins (85 kDa) that contain calcium- and
lipid-binding and pleckstrin homology domains that might confer
regulation by phosphatidylinositol 4,5-bisphosphate.
cPLA2s do not require calcium for their catalytic
mechanism, but in response to elevated calcium levels found in
stimulated cells, they translocate to membranes or membrane vesicles
where they encounter their phospholipid substrates. In addition, they
are regulated by phosphorylation on serine residues by
p38MAPK family members.
iPLA2s are also found in the cytosol and use a
similar catalytic mechanism as cPLA2s, but in
contrast to cPLA2s, iPLA2s
are not regulated by calcium. iPLA2s contain
ankyrin repeats that are involved in protein-protein interaction.
Recently, a PLA2-independent pathway for LPA
synthesis has been described. In addition to generating PA, PLD
directly generates LPA by hydrolysis of preexisting
lysophosphatidylcholine (38)
.
LPA has been demonstrated to activate PLD in a number of systems (39, 40, 41) and is a potent activator of increases in cytosolic calcium and of MAPKs in ovarian cancer cells (2 , 13) . Both increases in cytosolic calcium and MAPK activity activate cPLA2 (42) . In view of the higher levels of LPA in the ascites and plasma of ovarian cancer patients and the ability of LPA to activate the pathways mediating LPA production, we assessed basal and LPA-induced LPA production by ovarian cancer cells. We found that ovarian cancer cells, in contrast to normal ovarian epithelial cells or breast cancer cells, produce LPA either constitutively or in response to LPA. Both constitutive and LPA-induced LPA production exhibited PLD-dependent and -independent components. Constitutive LPA production was primarily dependent on group IB (pancreatic) sPLA2 and on cPLA2 and/or iPLA2, whereas LPA-induced LPA production was dependent on both group IB (pancreatic) and group IIA (synovial) sPLA2, but not cPLA2 or iPLA2.
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MATERIALS AND METHODS |
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Cell Lines and Media.
Cells were propagated in RPMI 1640 (Central Core Media Facility,
University of Texas M. D. Anderson Cancer Center) supplemented with
10% heat-inactivated FCS (Sigma) and 1000 units/ml
penicillin/streptomycin (Life Technologies, Inc., Grand Island, NY).
The ovarian cancer cell lines OVCAR-3 and SK-OV-3 were obtained from
the American Type Culture Collection (Rockville, MD). The ovarian
cancer cell line HEY was kindly provided by Dr. Ron Buick (University
of Toronto, Toronto, Ontario, Canada). A2780.6.3 is a subclone
of the ovarian cancer cell line A2780 (kindly provided by Dr. Thomas
Hamilton, Fox Chase Cancer Center, Philadelphia, PA) stably expressing
Edg-2 (23)
. The breast cancer cell lines MCF7, MDA-MB-231,
and MDA-MB-468 were kindly provided by Dr. Janet Price (University of
Texas M. D. Anderson Cancer Center). Normal ovarian epithelial cells
(NOE35) were obtained in-house, and immortalized ovarian epithelial
cells (IOSE29, IOSE80) were kindly provided by Dr. Nellie Auersperg
(University of British Columbia, Vancouver, British Columbia,
Canada).
In Vivo Labeling and Stimulation of Cells.
Cells (0.2–0.8 x 106) were plated in 60-mm
dishes in complete medium. After 2 days, at 80% confluency, the cells
were starved by removal of complete medium and addition of serum-free
medium. Twenty-four h later, the cells were washed with phosphate-free
medium and incubated in phosphate-free medium for 1 h. The cells
were again washed with phosphate-free medium and incubated with 0.1 mCi
[32P]Pi/ml in
phosphate-free medium. After 1 h, the labeling medium was removed,
and the cells were washed with serum-free medium and either treated
with inhibitor for 20 min or immediately stimulated with 25
µM LPA for 2 h. Preliminary time course experiments
had shown that maximum LPA production and release occurred after 2 h of LPA stimulation; therefore, this time point was used throughout.
LPA was added to the cells in a solution of 1% fatty acid-free BSA in
PBS. We therefore routinely tested fatty acid-free BSA for the presence
of trace amounts of LPA.
Lipid Extraction and Analysis of Phospholipids by TLC.
In thrombin-activated platelets, 90% of newly generated LPA is
released into the medium (26)
. Furthermore, preliminary
experiments showed that LPA produced by ovarian cancer cells was not
retained within the cells but released into the extracellular space.
Therefore, cell supernatants were used as source for extraction of
phospholipids. After stimulation, the cell supernatant was removed and
cleared by centrifugation at 14,000 x g for 5 min.
Acetic acid was added to the samples to a final concentration of 20
mM. The samples were then extracted with
1-butanol and centrifuged. The 1-butanol phase was removed, and the
aqueous phase was again extracted. The 1-butanol phases were combined
and washed twice with 1-butanol-saturated water. The extracted lipids
contained in the 1-butanol phases were dried, dissolved in
chloroform:methanol (1:1), and loaded onto TLC plates (precoated silica
gel 60 plates; EM Separations Technology, Gibbstown, NJ). Phospholipids
were separated by two-dimensional TLC with the first buffer system
containing chloroform:methanol:ammonium hydroxide (13:7:1.1) and the
subsequent buffer system containing chloroform:methanol:88% formic
acid:water (11:5.6:1:0.2). Phospholipids were detected by
autoradiography and identified by comigration with nonradioactive
marker lipids. Quantitation of LPA-containing spots was performed by
PhosphorImager. PhosphorImager units were normalized with respect to
the total amount of 32P-labeled phospholipids,
which minimizes variability in cell numbers or in
32P labeling. Each experiment was performed at
least twice, and the repeat experiment(s) yielded similar results. In
lipid extracts from SK-OV-3 cells, we routinely saw a second minor spot
running slightly further than the major LPA spot in the second
dimension, which may represent alkenyl-LPA and was included in the LPA
analysis.
Total RNA Preparation and Northern Blot Analysis.
Total cellular RNA was isolated from normal and immortalized ovarian
epithelial cells and various ovarian cancer cell lines using a RNeasy
Mini kit (Qiagen, Valencia, CA) according to the manufacturer’s
instructions. Equal amounts of total RNA were separated by
electrophoresis on denaturing 1% agarose gels and transferred to
Hybond N+ membranes (Amersham, Arlington Heights,
IL). Edg-7 and 18S RNA probes were radiolabeled by random-prime
labeling using the Redi-Prime labeling kit (Amersham). Membranes were
incubated with radiolabeled probes in 50% formamide, 10x Denhardt’s
solution, 0.1% SDS, 4x SSC, 10 mM EDTA, and 100 µg/ml
salmon-sperm single-strand DNA (Sigma) at 42°C for 18 h. The
blots were washed at room temperature in 1x SSC, 0.1% SDS for 20 min
three times and then at 50°C in 0.1x SSC, 0.1x SDS for 20 min three
times prior to autoradiography at -80°C for 1–2 days or analysis
with PhosphorImager. Quality and comparable loading of RNA were
confirmed by rehybridization of the membranes with radiolabeled 18S
RNA.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The other three ovarian cancer cell
lines (SK-OV-3, HEY, and A2780.6.3) constitutively produced LPA in the
absence of any exogenous stimulus, and LPA treatment did not further
increase the amount of LPA produced by these cell lines (Table 1)
. One
cell line, SK-OV-3, constitutively produced particularly high levels of
LPA (Table 1
and Fig. 1A
). In
these cells, LPA was the predominant phospholipid released into the
medium. In contrast, the breast cancer cell lines MCF7, MDA-MB-231, and
MDA-MB-468 produced only low levels of LPA, and treatment with LPA did
not increase LPA formation (Table 1
and Fig. 1B
). Indeed,
the SK-OV-3, HEY, and A2780.6.3 cell lines produced 5.6 times more LPA
than the three breast cancer cell lines (4708 units ± 1590
versus 838 units ± 169). In contrast to the ovarian
cancer cell lines tested, normal ovarian epithelial cells and
immortalized ovarian epithelial cells produced very low amounts of LPA
and could not be induced by LPA to produce more LPA (data not shown).
The effects of LPA were specific, because lysophosphatidylcholine, EGF,
and PDGF did not increase LPA production (presented herein).
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. We conclude that there
are PLD-dependent and -independent components of newly synthesized LPA
release by SK-OV-3 cells. PLD-independent synthesis might involve the
sequential action of PLC and diacylglycerol kinase (11)
.
|
. In contrast, OOEPC, at
a concentration that blocks group IB sPLA2
(49)
, reduced the amount of LPA being produced and
released by SK-OV-3 cells by 80% (Fig. 2C)
. This result
indicates that group IB sPLA2(s) play a major
role in the constitutive formation of LPA by SK-OV-3 cells, whereas
group IIA sPLA2 activity is not required for LPA
formation in SKOV-3 cells. Both manoalide and OOEPC altered the
distribution of phospholipids in cell supernatants, demonstrating
efficacy of the inhibitors (not presented). Recently, iPLA2s have been identified that display either an absolute specificity or a high selectivity for PA, therefore possibly playing a role in LPA synthesis (30) . Moreover, most cell types contain cPLA2 that are specific for arachidonic acid at the sn-2 position and that potentially are also involved in LPA formation (36) . The arachidonic acid analogue AACOCF3 inhibits both cPLA2 (IC50, 50 µM; Ref. 50 ) and iPLA2 (IC50, 15 µM; Ref. 51 ) and thus can be used to explore the function of both types of cytosolic PLA2s. SK-OV-3 cells were treated with AACOCF3 before measuring the amount of newly synthesized LPA released into the medium. AACOCF3 at 100 µM, a concentration that completely blocks both cPLA2 and iPLA2 (52) , markedly decreased the level of LPA being produced and released into the medium (90% inhibition). We conclude that cytosolic, calcium-dependent, and/or -independent PLA2s play a critical role in the constitutive production of LPA by SK-OV-3 cells.
LPA-induced LPA Production.
LPA markedly up-regulated LPA production in one of four ovarian
cancer cell lines tested (OVCAR-3; see Table 1
and Fig. 3
). This response was dose and time
dependent. Treatment with 3 µM LPA resulted in the
formation and release of a small amount of LPA. Treatment with 10 and
30 µM LPA, concentrations present in ascites of ovarian
cancer patients, however, caused the production and release of
substantial amounts of LPA (Fig. 4)
.
LPA formation in response to LPA was rapid; high levels of LPA were
detected in the supernatant of OVCAR-3 cells within 30 min of
incubation with LPA. Very little labeled LPA could be detected after
24 h of treatment with LPA (data not shown). Ovarian cancer
patients are potentially exposed to many different growth factors in
ascitic fluid, among them LPC, EGF, and PDGF (12
, 53)
. In
contrast to LPA, EGF (Fig. 5)
, PDGF (Fig. 5)
, and LPC (not presented) did not increase LPA production in OVCAR-3
cells.
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60% (Fig. 6A)
. Because basal LPA
production was also sensitive to the presence of 1-butanol (44%
inhibition), PLD appears to be involved in both the induced and basal
LPA production in OVCAR-3 cells. However, in each case there was also a
PLD-independent component of LPA production.
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40% (Fig. 6, B and C)
. This suggests that both types of secretory
PLA2 play a role in LPA-induced LPA production.
However, there remains considerable
sPLA2-independent LPA-induced LPA production. We
therefore assessed the participation of cPLA2 and
iPLA2 in LPA-induced LPA formation by using the
cPLA2 and iPLA2 inhibitor
AACOCF3. In contrast to its effect on constitutive LPA production,
AACOCF3 did not alter LPA-induced LPA production by OVCAR-3 cells (Fig. 6D)
. Therefore, cytosolic PLA2s (both
calcium-dependent and -independent) do not seem to participate in
LPA-induced production of LPA.
LPA Receptors Implicated in LPA-induced LPA Production.
We have demonstrated previously, by Northern blot analysis, that normal
and immortalized ovarian epithelial cells express the LPA receptor
Edg1, whereas ovarian cancer cell lines express only very low levels of
Edg1 (12)
. mRNA levels for Edg2 markedly vary among normal
and immortalized ovarian epithelial cells as well as among ovarian
cancer cell lines (12
, 22
, 23)
. Edg4 mRNA is expressed in
normal ovarian epithelial cells, and its mRNA levels are elevated in
ovarian cancer cells (12
, 22)
. The OVCAR-3 cell line
expresses moderately increased levels of Edg4 (12
, 22)
.
Recently, a novel LPA receptor, Edg7, was identified and cloned
(25)
. We determined its expression levels in normal and
immortalized ovarian epithelial cells as well as in ovarian cancer cell
lines by Northern blot analysis. Normal and immortalized ovarian
epithelial cells express barely detectable levels of Edg7 mRNA, whereas
ovarian cancer cells express Edg7 at varying levels (Fig. 7)
. Intriguingly, the highest level of
Edg7 expression is found in the OVCAR-3 cell line, which constitutively
produces very low levels of LPA and which produces markedly increased
levels of LPA in response to LPA. In OVCAR-3 cells, the change in Edg7
expression as compared with normal and immortalized ovarian epithelial
cells is much greater than the change in Edg4 expression (Fig. 7
;
19
, 22
). This suggests that Edg7 and potentially Edg4 may
play a role in LPA-induced LPA production by OVCAR-3 cells.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The two main types of enzymes involved in LPA synthesis are PLD, which contains at least two isoforms, and PLA2, which contains at least 10 different isoforms. PLD is involved in the formation of the LPA precursor PA, and as shown herein, PLD indeed plays a role in the production of LPA in ovarian cancer cells. Little is known about the role that the various PLA2 enzymes play in LPA formation. It has been shown that sPLA2 is inactive on intact membrane bilayers but requires membrane rearrangement and subsequent loss of membrane asymmetry to mediate LPA production (35 , 54, 55, 56) . Such loss of membrane asymmetry occurs during apoptosis or malignant transformation (57) . We have shown that LPA production by ovarian cancer cells requires group IB (pancreatic) sPLA2 activity, whereas group IIA (synovial) sPLA2 does not seem to play a role in constitutive LPA production by ovarian cancer cells and seems to play only a minor role in the induction of LPA by LPA. There seems to be a differential requirement for cPLA2 and/or iPLA2 phospholipase A2 by cells that constitutively produce LPA versus cells that are induced by LPA to produce LPA. Constitutive LPA production has an absolute requirement for cPLA2s and/or iPLA2s, whereas they do not seem to play a role in LPA induction by LPA.
The requirement for both secretory and cytosolic (calcium-dependent and/or -independent) PLA2 activity for constitutive LPA production might reflect a previously described cross-talk between sPLA2s and cPLA2. Functionally active cPLA2 may be required to activate sPLA2 and to mediate LPA production as cPLA2 activation precedes that of sPLA2 (58) . Furthermore, blocking cPLA2 with specific inhibitors leads to a pronounced reduction of arachidonic release from P388D1 macrophages, which is greater than the expected change, considering that sPLA2 is responsible for the majority of arachidonic acid released (58) . It has been postulated that an increase of free arachidonic acid brought about by cPLA2 catalysis activates sPLA2 (59) , possibly by resulting in the membrane rearrangement that appears to be required for sPLA2 activity (35 , 54, 55, 56) . In addition, AACOCF3, a cPLA2/iPLA2-specific inhibitor, markedly reduced interleukin 1/tumor necrosis factor-induced group IIA sPLA2 expression at the mRNA and protein level (59) . This suggests that arachidonic acid released by cPLA2 at the early stage of cytokine stimulation is required for the subsequent induction of group IIA sPLA2 expression. The addition of exogenous arachidonic acid only partially reversed the (indirect) inhibition of group IIA sPLA2 by AACOCF3, which might reflect the requirement of additional cPLA2 or iPLA2 metabolites for group IIA sPLA2 induction (59) . Interestingly, there seems to be a mutual interdependency between sPLA2 and cPLA2, because group IIA sPLA2 also increases the expression of cPLA2 (60) . Recently, it was reported that sPLA2 may indirectly regulate cPLA2 by activating p38MAPK (61) , which in turn phosphorylates cPLA2, contributing to its activation (42 , 62) . Taken together, these findings point to a complex interplay between sPLA2 and cPLA2, possibly also iPLA2. Specific PLA2 isoform inhibitors should allow further elucidation of the effects that the various PLA2 enzymes display on each other and on specific functions, such as LPA synthesis.
LPA has been previously demonstrated to activate PLD (39, 40, 41) . The mechanism(s) by which LPA regulates PLA2 have not, however, been explored. We have demonstrated previously that LPA induces rapid increases in cytosolic free calcium and activates MAPK in ovarian cancer cells (13) . It was thus somewhat surprising that cPLA2, which is activated by increases in cytosolic calcium (42) , does not seem to be involved in LPA-induced LPA production. We have shown recently that LPA activates p38MAPK in OVCAR-3 cells.5 This might be a mechanism by which constitutively produced LPA in ovarian cancer cells contributes to cPLA2 activation.
PLA1, which cleaves at the sn-1
position of a glycerophospholipid, may be involved in the production of
one particular species of LPA found in the ascites of ovarian cancer
patients. LPA found in ascites consists of a mixture of sn-1
and sn-2 species, with the sn-2 species
exhibiting greater bioactivity than the sn-1 counterpart
(4)
. PA, the precursor of LPA, is the preferred substrate
of both a membrane-bound (63)
and a cytosolic
PLA1 (64)
, thus further implicating
PLA1 in LPA synthesis. It will be interesting to
explore the contribution of PLA1 to LPA
production by ovarian cancer cells once inhibitors of
PLA1 become available. Interestingly, a recently
described isoform of cPLA2,
cPLA2-ß, prefers sn-1 cleavage to
sn-2 cleavage (65)
, whereas another isoform,
cPLA2-
, efficiently cleaves at both positions
(66)
. These cPLA2 enzymes could thus
also contribute to sn-2 LPA formation in the ascites of
ovarian cancer patients.
LPA levels in cell membranes are low (11) , reflecting rapid conversion or degradation of LPA. Reduced rates of conversion and/or degradation might contribute to the elevated levels of newly synthesized LPA in the supernatant of ovarian cancer cells as compared with breast cancer cells. This may also contribute to LPA-induced increases in LPA levels. LPA is converted back to PA by LPA acyltransferase, whereas PA phosphohydrolases and lysophospholipases rapidly degrade LPA (11) . Decreased expression or activity of these enzymes may contribute to the increased LPA levels in ovarian cancer patients.
In summary, we have shown that ovarian cancer cells, but not breast cancer cells or normal ovarian epithelial cells, release high levels of LPA into the extracellular medium. We have further shown that PLD plays a role in LPA synthesis by ovarian cancer cells, and that different PLA2 isoforms are required for constitutive and LPA-induced LPA production. These findings are clinically relevant because ascites and plasma of ovarian cancer patients, but not of patients with nongynecological tumors, contain elevated levels of LPA. LPA in ovarian cancer patients might be used as a marker for early diagnosis and as a molecular target for therapeutic intervention.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Grant PO1 CA64602 (to G. B. M.)
and by a sponsored research grant from Atairgin Technologies, Irvine,
California. 
2 Current address: Department of Nutritional
Science, Faculty of Health and Welfare Science, Okayama Prefectural
University, 111 Kuboki Soja, Okayama 719-1197, Japan.
3 To whom requests for reprints should be
addressed, at Department of Molecular Oncology, Division of Medicine,
Box 92, University of Texas M. D. Anderson Cancer Center, 1515
Holcombe Boulevard, Houston, TX 77030. Phone: (713) 792-7770; Fax:
(713) 794-1807; E-mail: gmills{at}notes.mdacc.tmc.edu
4 The abbreviations used are: LPA,
lysophosphatidic acid, 1-acyl-sn-glycerol-3-phosphate;
MAPK, mitogen-activated protein kinase; Edg, endothelial
differentiation gene; PMA, phorbol 12-myristate 13-acetate; PLD,
phospholipase D; PA, phosphatidic acid; PLA, phospholipase A;
sPLA2, secretory PLA2; cPLA2,
cytosolic PLA2; iPLA2, calcium-independent
PLA2; EGF, epidermal growth factor; PDGF, platelet-derived
growth factor; OOEPC, oleyloxyethylphosphocholine; AACOCF3,
arachidonyltrifluoromethyl ketone; LPC, lysophosphatidylcholine.
5 V. Estrella, T. Pustilnik, F. X. Claret, G. E.
Gallick, G. B. Mills, and J. R. Wiener. Lysophosphatidic acid
induction of urokinase plasminogen activator secretion requires
activation of the p38MAPK pathway, submitted for publication.
6 Y-L. Hu, E. Goetzl, G. B. Mills, N. Ferrara,
and R. B. Jaffe. Induction of vascular endothelial growth factor
expression by lysophosphatidic acid in normal and neoplastic ovarian
epithelial cells, submitted for publication.
Received 12/15/99; revised 2/22/00; accepted 2/23/00.
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, that is prenylated and contains homology to cPLA2. J. Biol. Chem., 273: 21926-19232, 1998.This article has been cited by other articles:
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 S. Yu, M. M. Murph, Y. Lu, S. Liu, H. S. Hall, J. Liu, C. Stephens, X. Fang, and G. B. Mills Lysophosphatidic Acid Receptors Determine Tumorigenicity and Aggressiveness of Ovarian Cancer Cells J Natl Cancer Inst, November 19, 2008; 100(22): 1630 - 1642. [Abstract] [Full Text] [PDF]
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T.-V. Do, J. C. Symowicz, D. M. Berman, L. A. Liotta, E. F. Petricoin III, M. S. Stack, and D. A. Fishman Lysophosphatidic Acid Down-Regulates Stress Fibers and Up-Regulates Pro-Matrix Metalloproteinase-2 Activation in Ovarian Cancer Cells Mol. Cancer Res., February 1, 2007; 5(2): 121 - 131. [Abstract] [Full Text] [PDF]
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F.-q. Wang, Y. Smicun, N. Calluzzo, and D. A. Fishman Inhibition of Matrilysin Expression by Antisense or RNA Interference Decreases Lysophosphatidic Acid-Induced Epithelial Ovarian Cancer Invasion Mol. Cancer Res., November 1, 2006; 4(11): 831 - 841. [Abstract] [Full Text] [PDF]
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 K.-S. Kim, S. Sengupta, M. Berk, Y.-G. Kwak, P. F. Escobar, J. Belinson, S. C. Mok, and Y. Xu Hypoxia Enhances Lysophosphatidic Acid Responsiveness in Ovarian Cancer Cells and Lysophosphatidic Acid Induces Ovarian Tumor Metastasis In vivo Cancer Res., August 15, 2006; 66(16): 7983 - 7990. [Abstract] [Full Text] [PDF]
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J. Ren, Y.-j. Xiao, L. S. Singh, X. Zhao, Z. Zhao, L. Feng, T. M. Rose, G. D. Prestwich, and Y. Xu Lysophosphatidic Acid is constitutively produced by human peritoneal mesothelial cells and enhances adhesion, migration, and invasion of ovarian cancer cells. Cancer Res., March 15, 2006; 66(6): 3006 - 3014. [Abstract] [Full Text] [PDF]
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 N. A. Neidlinger, S. K. Larkin, A. Bhagat, G. P. Victorino, and F. A. Kuypers Hydrolysis of Phosphatidylserine-exposing Red Blood Cells by Secretory Phospholipase A2 Generates Lysophosphatidic Acid and Results in Vascular Dysfunction J. Biol. Chem., January 13, 2006; 281(2): 775 - 781. [Abstract] [Full Text] [PDF]
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E. Iorio, D. Mezzanzanica, P. Alberti, F. Spadaro, C. Ramoni, S. D'Ascenzo, D. Millimaggi, A. Pavan, V. Dolo, S. Canevari, et al. Alterations of Choline Phospholipid Metabolism in Ovarian Tumor Progression Cancer Res., October 15, 2005; 65(20): 9369 - 9376. [Abstract] [Full Text] [PDF]
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H. Li, X. Ye, C. Mahanivong, D. Bian, J. Chun, and S. Huang Signaling Mechanisms Responsible for Lysophosphatidic Acid-induced Urokinase Plasminogen Activator Expression in Ovarian Cancer Cells J. Biol. Chem., March 18, 2005; 280(11): 10564 - 10571. [Abstract] [Full Text] [PDF]
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S. Miyamoto, M. Hirata, A. Yamazaki, T. Kageyama, H. Hasuwa, H. Mizushima, Y. Tanaka, H. Yagi, K. Sonoda, M. Kai, et al. Heparin-Binding EGF-Like Growth Factor Is a Promising Target for Ovarian Cancer Therapy Cancer Res., August 15, 2004; 64(16): 5720 - 5727. [Abstract] [Full Text] [PDF]
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D. Bian, S. Su, C. Mahanivong, R. K. Cheng, Q. Han, Z. K. Pan, P. Sun, and S. Huang Lysophosphatidic Acid Stimulates Ovarian Cancer Cell Migration via a Ras-MEK Kinase 1 Pathway Cancer Res., June 15, 2004; 64(12): 4209 - 4217. [Abstract] [Full Text] [PDF]
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T. Hiramatsu, H. Sonoda, Y. Takanezawa, R. Morikawa, M. Ishida, K. Kasahara, Y. Sanai, R. Taguchi, J. Aoki, and H. Arai Biochemical and Molecular Characterization of Two Phosphatidic Acid-selective Phospholipase A1s, mPA-PLA1{alpha} and mPA-PLA1{beta} J. Biol. Chem., December 5, 2003; 278(49): 49438 - 49447. [Abstract] [Full Text] [PDF]
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 C. Luquain, A. Singh, L. Wang, V. Natarajan, and A. J. Morris Role of phospholipase D in agonist-stimulated lysophosphatidic acid synthesis by ovarian cancer cells J. Lipid Res., October 1, 2003; 44(10): 1963 - 1975. [Abstract] [Full Text] [PDF]
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 J. L. Tanyi, Y. Hasegawa, R. Lapushin, A. J. Morris, J. K. Wolf, A. Berchuck, K. Lu, D. I. Smith, K. Kalli, L. C. Hartmann, et al. Role of Decreased Levels of Lipid Phosphate Phosphatase-1 in Accumulation of Lysophosphatidic Acid in Ovarian Cancer Clin. Cancer Res., September 1, 2003; 9(10): 3534 - 3545. [Abstract] [Full Text] [PDF]
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J. L. Tanyi, A. J. Morris, J. K. Wolf, X. Fang, Y. Hasegawa, R. Lapushin, N. Auersperg, Y. J. Sigal, R. A. Newman, E. A. Felix, et al. The Human Lipid Phosphate Phosphatase-3 Decreases the Growth, Survival, and Tumorigenesis of Ovarian Cancer Cells: Validation of the Lysophosphatidic Acid Signaling Cascade as a Target for Therapy in Ovarian Cancer Cancer Res., March 1, 2003; 63(5): 1073 - 1082. [Abstract] [Full Text] [PDF]
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J. Aoki, A. Taira, Y. Takanezawa, Y. Kishi, K. Hama, T. Kishimoto, K. Mizuno, K. Saku, R. Taguchi, and H. Arai Serum Lysophosphatidic Acid Is Produced through Diverse Phospholipase Pathways J. Biol. Chem., December 6, 2002; 277(50): 48737 - 48744. [Abstract] [Full Text] [PDF]
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H. Sonoda, J. Aoki, T. Hiramatsu, M. Ishida, K. Bandoh, Y. Nagai, R. Taguchi, K. Inoue, and H. Arai A Novel Phosphatidic Acid-selective Phospholipase A1 That Produces Lysophosphatidic Acid J. Biol. Chem., September 6, 2002; 277(37): 34254 - 34263. [Abstract] [Full Text] [PDF]
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 L. M. Baudhuin, K. L. Cristina, J. Lu, and Y. Xu Akt Activation Induced by Lysophosphatidic Acid and Sphingosine-1-phosphate Requires Both Mitogen-Activated Protein Kinase Kinase and p38 Mitogen-Activated Protein Kinase and Is Cell-Line Specific Mol. Pharmacol., September 1, 2002; 62(3): 660 - 671. [Abstract] [Full Text] [PDF]
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 L. T. Budnik and A. K. Mukhopadhyay Lysophosphatidic Acid and Its Role in Reproduction Biol Reprod, April 1, 2002; 66(4): 859 - 865. [Abstract] [Full Text] [PDF]
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G. Tigyi Selective Ligands for Lysophosphatidic Acid Receptor Subtypes: Gaining Control over the Endothelial Differentiation Gene Family Mol. Pharmacol., December 1, 2001; 60(6): 1161 - 1164. [Full Text] [PDF]
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