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Vol. 1, 885-892, September 2002     Molecular Cancer Therapeutics
© 2002 American Association for Cancer Research

The Antisignaling Agent SC-{alpha}{alpha}{delta}9, 4-(Benzyl-(2-[(2,5-diphenyloxazole- 4-carbonyl)amino]ethyl)carbamoyl)- 2-decanoylaminobutyric Acid, Is a Structurally Unique Phospholipid Analogue with Phospholipase C Inhibitory Activity 1

Andreas Vogt2, Katharine E. Pestell, Billy W. Day, John S. Lazo2 and Peter Wipf

Departments of Pharmacology [A. V., K. E. P., J. S. L.], Pharmaceutical Sciences [B. W. D], and Chemistry [B. W. D., P. W.], University of Pittsburgh, Pittsburgh, Pennsylvania 15261


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Phospholipids and lipid second messengers mediate mitogenic signal transduction and oncogenesis, but there have been few successful examples of small molecules that affect biologically important phospholipid metabolism. Here we investigated the actions of a previously described antitumor agent, 4-(benzyl-(2-[(2,5-diphenyloxazole-4-carbonyl)amino]ethyl)carbamoyl)-2-decanoylaminobutyric acid (SC-{alpha}{alpha}{delta}9), which has antisignaling properties, on phospholipases. Although SC-{alpha}{alpha}{delta}9 had been shown to be a potent and selective inhibitor of the Cdc25 family of dual-specificity phosphatases, many of its cellular effects are not readily reconciled with phosphatase inhibition. Molecular modeling studies suggested that SC-{alpha}{alpha}{delta}9 shared several structural features with membrane phospholipids. Enzyme inhibition studies in vitro revealed that SC-{alpha}{alpha}{delta}9 was a potent inhibitor of phospholipase C (PLC; IC50 = 25 µM) but did not inhibit phospholipase D activity at concentrations up to 100 µM. In H-ras (Q61L)-transformed Rat-1 fibroblasts with constitutively elevated levels of phosphorylated extracellular signal-regulated kinase (Erk), SC-{alpha}{alpha}{delta}9 inhibited both proliferation and oncogenic Erk activation at concentrations that inhibited PLC in vitro. A SC-{alpha}{alpha}{delta}9 congener that lacked antiproliferative activity also did not inhibit PLC in vitro. In the PLC-dependent scratch wound healing model, SC-{alpha}{alpha}{delta}9 was 10-fold more potent than the phosphatidylcholine-specific PLC inhibitor D-609. We propose that the structural resemblance of SC-{alpha}{alpha}{delta}9 to phospholipids allows it to inhibit cellular PLC, thereby providing a possible molecular mechanism for SC-{alpha}{alpha}{delta}9’s effects on oncogenic Erk activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SC-{alpha}{alpha}{delta}93 is a dual-specificity phosphatase inhibitor that has in vivo antitumor activity (1, 2). In the synchronized tsFT210 mouse mammary carcinoma cell model, SC-{alpha}{alpha}{delta}9 caused dual G1 and G2 cell cycle block that correlated with increased phosphorylation of the cyclin-dependent kinases 1, 2, and 4 (3). Although perturbation of cell cycle progression through inhibition of Cdc25 phosphatases may contribute to SC-{alpha}{alpha}{delta}9’s antiproliferative activity, many of its effects in a variety of asynchronously growing cells are not readily reconciled with phosphatase inhibition. For example, treatment of MEFs transformed with the SV40 with SC-{alpha}{alpha}{delta}9 caused a decrease rather than an increase in global tyrosine phosphorylation levels (4). SC-{alpha}{alpha}{delta}9 inhibited oncogenic Erk activation in SV40 MEF (4) and prevented downstream consequences of IGF-1 receptor activation such as Cdc2 (cyclin-dependent kinase 1) expression, without appearing to directly affect receptor autophosphorylation (2). In MDA-MB-231 mammary carcinoma cells, which have constitutively activated Erk because of expression of a mutated Ki-ras oncogene (5), SC-{alpha}{alpha}{delta}9 also reduced Cdc2 expression and Erk activation (unpublished results).

We hypothesized that the molecular mechanism by which SC-{alpha}{alpha}{delta}9 affects oncogenic signaling through the Erk cascade might involve molecular targets that do not primarily regulate protein phosphorylation. Candidate targets for such an alternative mechanism of action are the phospholipid-metabolizing enzymes PLD and PLC or their downstream effectors PA and diacylglycerol. Both PLD and PLC appear to be transforming oncogenes whose actions have been placed downstream of Ras but upstream of the Ras effectors Raf-1 and Erk. Ras activation causes increased PC hydrolysis and levels of diacylglycerol, the primary reaction product of PLC-mediated PC or PI hydrolysis (6). Ectopic expression of PC-specific phospholipase C from Bacillus cereus or of PI-specific phospholipase C {gamma} transforms NIH3T3 cells and Rat 3Y1 fibroblasts, respectively (7, 8). NIH3T3 cells transformed with PC-PLC from B. cereus have constitutively elevated nuclear Erk activity, which can be inhibited by a dominant negative form of Raf-1 (7). Ectopic expression of PC-PLC bypasses the block to proliferation caused by a dominant negative mutant form of Ras but not Raf-1 (9). Furthermore, PLC {gamma} is overexpressed in breast (10, 11) and colorectal cancers (12). Elevated levels of PLD activity have been reported in renal (13) and gastric cancers (14). PLD activity is also high in ras-transformed NIH3T3 cells (15). Overexpression of PLD is usually toxic to cells but is tolerated in fibroblasts that overexpress epidermal growth factor receptor, where it renders cells transformed in the absence of epidermal growth factor (16). Recently, Min et al. (17) have successfully generated mouse fibroblast cells stably expressing PLD 1 or PLD 2. These cells grow in soft agar and form tumors in nude mice. Pharmacological PLC inhibitors have been described previously (18–20), some of which possess antitumor activity, but specific inhibitors of PLD are lacking.

Here we investigated a potential involvement of PLD and PLC in the antimitogenic and antiproliferative effects of SC-{alpha}{alpha}{delta}9 on Rat-1 fibroblasts transformed with a Ha-ras mutant oncogene (Q61L). SC-{alpha}{alpha}{delta}9 inhibited Ras-mediated oncogenic Erk activation and cell growth. Molecular modeling studies suggested significant structural similarity between SC-{alpha}{alpha}{delta}9 and phospholipids, and in vitro enzyme inhibition studies demonstrated that SC-{alpha}{alpha}{delta}9 selectively inhibited PLC but not PLD at concentrations consistent with the growth inhibitory activity. Finally, SC-{alpha}{alpha}{delta}9 prevented cell migration in the PLC-dependent scratch wound assay. We conclude that SC-{alpha}{alpha}{delta}9 is a novel PLC inhibitor that is structurally unrelated to other known PLC inhibitory agents and speculate that its antisignaling activities may be caused by cellular PLC inhibition.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents.
The synthesis and characterization of SC-{alpha}{alpha}{delta}9 and SC-{alpha}{alpha}{delta}4II have been described previously (1, 4, 21). The PLC inhibitor D-609 was from Biomol Research Laboratories (Plymouth Meeting, PA). The MEK1 inhibitor PD-98059 was from New England Biolabs (Beverly, MA). All other chemicals were from Sigma (St. Louis, MO). Antibodies used were: Erk1 K-23 (Santa Cruz Biotechnology, Santa Cruz, CA); phospho-Erk clone E10 (Cell Signaling Technologies, Beverly, MA); and pan-Ras AB-3 (Oncogene Research Products, Boston, MA).

Molecular Modeling.
Models were built with the Cerius2 version 4.5 (Accelrys, Inc., San Diego, CA) suite of software on a Silicon Graphics, Inc. O2 workstation. Atomic coordinates for PA were obtained from the X-ray crystallographic structure of USP (22). The structure of SC-{alpha}{alpha}{delta}9 was minimized with the MMFF94 force field using the ABNR approach. The models were then superimposed by iterative hand matching and rigid body superimpositions until 11 potentially superimposable atoms in each model were identified. This was followed by a flexible fit routine where torsion angles in SC{alpha}{alpha}{delta}9 were changed to maximize superimposition. The resulting structures were then reminimized with MMFF94/ABNR.

Cell Viability Assay.
Rat-1 fibroblasts transformed with an oncogenic mutant ras (Q61L; Ref. 23) were maintained in Dulbecco’s minimum essential medium containing 10% fetal bovine serum (HyClone, Logan, UT) and 1% penicillin-streptomycin (Life Technologies, Inc.) in a humidified atmosphere of 5% CO2 at 37°C. Cells (500–2,000/well) were plated in 96-darkwell microtiter plates (Packard ViewPlate) and treated the next day with vehicle SC-{alpha}{alpha}{delta}9 or SC-{alpha}{alpha}{delta}4II for 48 or 96 h, respectively. Cells were fixed with formaldehyde and stained with Hoechst 33342 to visualize nuclei. In some experiments, live cells were first incubated with a solution containing propidium iodide and CMFDA. Under these conditions, dead cells stain positive for propidium iodide because of loss of membrane integrity. Viable cells exclude propidium iodide but metabolize CMFDA to a fluorescent carboxylate that is trapped in the cytosol. Cells were then permeabilized and stained with Hoechst 33342. Images were acquired with a charge-coupled device camera and analyzed by multiparametric analysis on an ArrayScan II (Cellomics, Inc., Pittsburgh, PA) using excitation/emission wavelengths of 535/617 nm (propidium iodide), 492/516 nm (CMFDA), and 350/461 nm (Hoechst), respectively. A nuclear mask was generated from Hoechst 33342-stained nuclei, and cells were counted as positive for propidium iodide (dead) or CMFDA (live) if the intensity of the stain in an area defined by the nucleus in the appropriate channel exceeded a user-defined threshold.

Western Blots.
Rat-1 fibroblasts transformed with an oncogenic mutant ras (Q61L; Ref. 23) were plated in 100-mm cell culture dishes and treated for 24 h with vehicle or compounds. Cells were lysed, and lysates immunoblotted with anti-Erk1 (Santa Cruz) or antiphospho-Erk (E10; New England Biolabs) antibodies essentially as described previously (4). Positive antibody reactions were visualized using peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA) and an enhanced chemiluminescence detection system (Renaissance; NEN, Boston, MA) according to manufacturer’s instructions. For quantitation of protein expression levels, X-ray films were scanned on a Molecular Dynamics personal SI densitometer and analyzed using the ImageQuant software package (version 4.1; Molecular Dynamics, Sunnyvale, CA).

In Vitro PLD and PLC Assays.
PLD and PLC activities were determined in vitro using commercially available assay kits (Amplex Red; Molecular Probes, Inc., Eugene, OR), according to manufacturer’s instructions. The final reaction mixture for the PLD assay contained 0.25 units/ml-1 PLD from Streptomyces chromofuscus, 1 unit/ml-1 horseradish peroxidase, 0.1 units/ml-1 choline oxidase, 10 µM phosphatidyl choline (lecithin), and 50 µM Amplex Red (10-acetyl-3,7-dihydrophenoxazine) in 200 µl of assay buffer [5 mM CaCl2 and 50 mM Tris-HCl (pH 8.0)]. PLC assays contained 0.25 units/ml-1 PLC from B. cereus, 1 unit/ml-1 horseradish peroxidase, 4 units/ml-1 alkaline phosphatase, 0.1 units/ml-1 choline oxidase, 435 µM phosphatidyl choline (lecithin), 200 µM Amplex Red, 2 mM CaCl2, 0.14 M NaCl, 10 mM dimethylglutarate, and 50 mM Tris-HCl (pH 7.4). Inhibitors were resuspended in DMSO, and all reactions were performed in the presence of 1% DMSO. Reactions were initiated by addition of horseradish peroxidase, choline oxidase, phosphatidyl choline, and Amplex Red reagent.

Plates were incubated at 37°C in the dark, and fluorescence measured at 15-min intervals using a multiwell plate reader (excitation filter 530 ± 25 nm; emission filter 590 ± 35 nm; Cytofluor II, PerSeptive Biosystems, Framingham, MA). For both enzymes, increases in fluorescence were linear over time up to 75 min. Before inhibition studies, Kms for lecithin were determined from substrate saturation curves at time points where the reactions were proceeding at maximum velocity (15–45 min). The Kms for lecithin were calculated to be 11.9 ± 7.0 µM (mean ± SD, n = 4) for PLD and 431.5 ± 65.8 µM (mean ± SD, n = 3) for PLC, respectively, using nonlinear regression and fitting to Michaelis-Menten kinetics (GraphPad Software Inc., San Diego, CA). For all inhibition studies, reactions were allowed to proceed for 45 min in the presence of 10 M (PLD) or 435 µM (PLC) lecithin.

Alkaline Phosphatase Assay.
For control purposes, inhibition of alkaline phosphatase activity by SC-{alpha}{alpha}{delta}9 was determined using some components of the Amplex Red Phospholipase C Assay Kit. The final assay mixture contained 4 units/ml-1 alkaline phosphatase, 1 unit/ml-1 horseradish peroxidase, 0.1 units/ml-1 choline oxidase, 15 µM phosphocholine chloride (approximate Km) or 50 µM phosphocholine chloride (a concentration giving similar fluorescence readings to those seen when assaying PLC activity), 200 µM Amplex Red, 2 mM CaCl2, 0.14 M NaCl, 10 mM dimethylglutarate, and 50 mM Tris-HCl (pH 7.4). Reactions were allowed to proceed in the presence or absence of 100 µM SC-{alpha}{alpha}{delta}9 under the identical conditions as described for the PLC assay.

Migration Assay.
Cell migration was determined in a scratch wound assay similar to that described for breast carcinoma cells (24). Rat-1 Ras cells (1.5 x 106) were plated in the wells of a 6-well multiwell plate and allowed to form a confluent monolayer. Growth medium was replaced with serum-free medium for an additional 24 h, and a 1-mm wide wound was inflicted with a sterile 200-µl pipette tip. Cells were washed twice with sterile PBS and photographed at low magnification (10x objective) for initial gap width measurements. Cells were additionally incubated for up to 30 h in serum-free medium containing vehicle (DMSO) or inhibitors and washed twice with PBS. At the end of treatment, cells were photographed again and final wound size determined. Gap width was defined as the cell-free area between wound edges and was measured in Adobe PhotoShop at three arbitrarily chosen image sections along the wound edge. For morphology experiments, cells were incubated for 2 h after wounding in the presence or absence of inhibitors and stained with a solution of crystal violet as described previously (4). Morphology photomicrographs were taken using a 40x objective on an Olympus IX-70 inverted microscope attached to a Spot II charge-coupled device camera.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
SC-{alpha}{alpha}{delta}9 Is Structurally Similar to Phospholipids.
Fig. 1 shows the chemical structures of SC-{alpha}{alpha}{delta}9 and various phospholipids. We noticed that SC-{alpha}{alpha}{delta}9 appeared to share a number of structural elements with these phospholipids. Common features are a negatively charged head group, an aliphatic backbone assuming a turn conformation, and two hydrophobic side chains lending an overall lipophilic character to both molecules. Crystallographic data suggest that phospholipids occupy the binding pockets of proteins as monomeric entities (25). We therefore performed molecular modeling studies to investigate the extent of structural overlap between SC-{alpha}{alpha}{delta}9 and the model phospholipid metabolite PA. Fig. 2 shows the results from the molecular mechanics-based conformational analysis studies. For these studies, we used a previously described conformation of PA that was identified in the X-ray crystallographic structure of USP, the Drosophila homologue of the retinoid X receptor (22). A low, local minimum energy conformer of SC-{alpha}{alpha}{delta}9 with spatial and hydrophobic/hydrophilic characteristics similar to that of PA was identified by molecular mechanics. Both SC-{alpha}{alpha}{delta}9 and PA can adopt a U-turn conformation via hydrophobic collapse in local minimum energy structures. The negatively charged head-groups are exposed, and two lipophilic chains extend away from the glycerol and glutamate backbone, respectively. Fig. 2 shows the results of superimposition after matching respective hydrophobic and hydrophilic atoms in the two structures. The peripheries of both agents were, thus, very similar in terms of physicochemical properties, with the exception that PA possesses a longer, more extended lipophilic region. The structure of SC-{alpha}{alpha}{delta}9 showed an additional attribute in that one of the phenyl rings of its 2,5-diphenyloxazole substructure was tucked into the hydrophobic core of the flanking lipophilic systems. These in silico studies suggested that SC-{alpha}{alpha}{delta}9 could mimic the shape and hydrophobic/hydrophilic characteristics of PA in a biological environment but with attributes that might alter biochemical functions.



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  		Fig. 1. SC-{alpha}{alpha}{delta}9 resembles membrane phospholipids. Chemical structures of SC-{alpha}{alpha}{delta}9 and common phospholipids are shown.

 


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  		Fig. 2. SC-{alpha}{alpha}{delta}9 is structurally similar to PA. Models were built with the Cerius2 version 4.5 (Accelrys, Inc., San Diego, CA) suite of software on a Silicon Graphics, Inc. O2 workstation. Atomic coordinates for PA were obtained from the X-ray crystallographic structure of USP (22). The structure of SC-{alpha}{alpha}{delta}9 was minimized with the MMFF94 force field using the ABNR approach. The models were then superimposed by iterative hand matching and rigid body superimpositions until 11 potentially superimposable atoms in each model were identified. This was followed by a flexible fit routine where torsion angles in SC-{alpha}{alpha}{delta}9 were changed to maximize superimposition. The resulting structures were then reminimized with MMFF94/ABNR. PA is shown in atom-specific colors. SC{alpha}{alpha}{delta}9 is shown in yellow. The head group-protonated forms of both molecules were used in the molecular modeling.

 
SC-{alpha}{alpha}{delta}9 Inhibits ras-transformed Cell Growth.
SC-{alpha}{alpha}{delta}9 selectively inhibits the growth of SV40-transformed MEF in culture, and this growth inhibition is accompanied by inhibition of components of the Ras-Raf-Erk cascade (4). These cells owe their transformed phenotype at least, in part, to autocrine stimulation by IGF-1, but we were unable to document a direct inhibition of IGF-1 receptor activation by SC-{alpha}{alpha}{delta}9. Therefore, we chose cells transformed with a constitutively activated ras oncogene as a model for growth factor-independent oncogenic transformation. Rat-1 fibroblasts transformed with a GTPase-deficient mutant ras (Q61L; Ref. 23) were plated in a 96-well plate and treated with vehicle or SC-{alpha}{alpha}{delta}9 for 96 h. Cells were fixed, permeabilized, and stained with Hoechst 33342 fluorescent dye to visualize nuclei. Cell numbers were quantitated with the cell viability algorithm on the ArrayScan II as described in the "Materials and Methods" section. Fig. 3 shows that a 96-h continuous exposure to SC-{alpha}{alpha}{delta}9 caused a concentration-dependent decrease in cell numbers. The concentration of SC-{alpha}{alpha}{delta}9 required to inhibit cell growth by 50% (GI50) was 30 µM, which is similar to what we had previously observed in SV40-transformed cells (4). An analogue of SC-{alpha}{alpha}{delta}9, termed SC-{alpha}{alpha}{delta}4II, which differs from the parent compound only by a more hydrophilic side chain (4), was approximately three times less effective than SC-{alpha}{alpha}{delta}9 in this assay. In a separate experiment, live cells were incubated first with a solution of CMFDA and propidium iodide, followed by fixation and Hoechst 33342 staining. Microscopic evaluation of cells treated with 60 µM SC-{alpha}{alpha}{delta}9 after 48 h revealed that all cells metabolized the CMFDA live cell dye, whereas none stained positive for propidium iodide (data not shown), indicating that all cells that remained attached were viable.



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  		Fig. 3. SC-{alpha}{alpha}{delta}9 inhibits the growth of Ras-transformed cells. Rat-1 fibroblasts transformed with an oncogenic mutant Ras (Q61L) were plated in 96-darkwell microtiter plates and treated the next day with SC-{alpha}{alpha}{delta}9 ({blacksquare}) or SC-{alpha}{alpha}{delta}4II ({square}) for 96 h. Cells were fixed, permeabilized, and stained with Hoechst 33342. Fixed and stained cells were imaged on the ArrayScan II solid-phase cytometer (Cellomics, Inc.), and the number of nuclei/field were calculated using the cell viability algorithm as described in the "Materials and Methods" section

 
SC-{alpha}{alpha}{delta}9 Inhibits Oncogenic Erk Activation in Ras-transformed Rat-1 Fibroblasts.
We next asked whether SC-{alpha}{alpha}{delta}9 prevented Erk activation by oncogenic Ha-Ras. Compared with the parental cell line, Ras transformants had elevated levels of Ras and phospho-Erk, whereas overall Erk expression was unaltered (data not shown). Rat-1 Ras cells were treated with vehicle or inhibitors for 24 h, lysed, and Erk phosphorylation was analyzed by immunoblot analysis. Fig. 4 shows that SC-{alpha}{alpha}{delta}9 at a concentration that caused 50% inhibition of cell growth (30 µM) reduced Erk phosphorylation by 50% (Fig. 4, Lane 8). At higher concentrations (60 µM), complete Erk inactivation was observed (Fig. 4, Lane 7). A number of known nonspecific PLD inhibitors, namely ethanol, 1-butanol, and diphosphoglycerate (26), also caused significant inhibition of Erk activation by oncogenic H-Ras.



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  		Fig. 4. PLD inhibitors and SC-{alpha}{alpha}{delta}9 reduce Erk activation by oncogenic Ras. Rat-1 Ras cells were treated for 24 h with the indicated concentrations of known or suspected PLD inhibitors. Cells were lysed, lysates separated by SDS-PAGE, and immunoblotted with (top panel) anti-Erk1 or (bottom panel) antiphospho-Erk (E10) antibodies. A, immunoblot analysis. Lane 1, vehicle control; Lanes 2 and 3, 1 and 0.3% ethanol; Lanes 4 and 5, 0.3 and 0.1% 1-butanol; Lane 6, 50 µM PD-98059; Lanes 7 and 8, 60 and 30 µM SC-{alpha}{alpha}{delta}9; and Lane 9, 10 mM DPG. B, X-ray films were scanned and phospho-Erk band intensities quantified by densitometry. Data are from a single experiment that has been repeated twice with similar results. The MEK1 inhibitor PD-98059 was included as a positive control.

 
SC-{alpha}{alpha}{delta}9 Is an Inhibitor of PC-PLC but not of PC-PLD.
The molecular modeling studies described above suggested that SC-{alpha}{alpha}{delta}9 was structurally similar to phospholipids in general and that it most closely resembled PA, the reaction product of PLD. Because SC-{alpha}{alpha}{delta}9 and PLD inhibitors shared the ability to reduce Erk activation, we next asked whether SC-{alpha}{alpha}{delta}9 had in vitro PLD inhibitory activity and whether it had any selectivity for PLD over PLC. The only two PLC enzymes that were reported to be Ras effectors are PC-PLC from B. cereus (9) and mammalian PI-PLC-{epsilon} (27) and only the former has been shown to be transforming (7). Mammalian cells contain a PC-PLC activity that is cross-immunogenic and cross-reactive with the bacterial enzyme (28), but a mammalian homologue has not been cloned to date. We therefore performed our in vitro inhibition studies with SC-{alpha}{alpha}{delta}9 and its less toxic congener SC-{alpha}{alpha}{delta}4II on PC-PLC and PC-PLD from bacterial sources. Fig. 5A shows that at a concentration of SC-{alpha}{alpha}{delta}9 (100 µM) that was three times its GI50 in Rat-1 Ras cells, no inhibition of PLD was observed. The same concentration of SC-{alpha}{alpha}{delta}9, however, caused >70% inhibition of PLC, suggesting that SC-{alpha}{alpha}{delta}9 selectively inhibited PLC-catalyzed PC hydrolysis over PLD-catalyzed PC hydrolysis. The SC-{alpha}{alpha}{delta}9 congener SC-{alpha}{alpha}{delta}4II did not inhibit PC-PLC at concentrations as high as 300 µM, suggesting that a modification in one of the alkyl chains was sufficient to alter PLC inhibitory activity. Fig. 5B shows that inhibition of PLC by SC-{alpha}{alpha}{delta}9 was concentration dependent, with an IC50 of 25 µM. The maximum extent of PLC inhibition was <100% because at concentrations >= 100 µM, SC-{alpha}{alpha}{delta}9 exceeded its solubility limit and precipitated from the assay buffer.



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  		Fig. 5. SC-{alpha}{alpha}{delta}9 is an inhibitor of PLC but not PLD. In vitro phospholipase assays were performed with commercially available assay kits using purified PLC and PLD from bacterial sources and a fluorescent substrate (Amplex Red; Molecular Probes, Inc.). A, a single concentration of SC-{alpha}{alpha}{delta}9 (100 µM) inhibited PLC but did not affect PLD. The inactive analogue, SC-{alpha}{alpha}{delta}4II, lacked PLC inhibitory activity. B, concentration-dependent PLC inhibition by SC-{alpha}{alpha}{delta}9. Data are the averages ± SE of 3–8 (left panel) or 4 (right panel) data points.

 
Both the PLD and PLC assays are based on the quantification of free choline after enzymatic oxidation. The primary reaction product of PLC-mediated PC cleavage phosphocholine, however, is a poor substrate for choline oxidase and necessitates an additional alkaline phosphatase hydrolysis step in the PLC assay. Because SC-{alpha}{alpha}{delta}9 was originally designed based on serine/threonine phosphatase inhibitors, we determined whether SC-{alpha}{alpha}{delta}9 inhibited alkaline phosphatase by performing the PLC assay in the presence of all of the components of the PLC assay except enzyme but including the substrate for alkaline phosphatase, phosphocholine. Reactions were carried out exactly as described in the "Materials and Methods" section for the PLC assay. Under these conditions, we found no inhibition of alkaline phosphatase by SC-{alpha}{alpha}{delta}9 at a concentration that inhibited PLC by >70% (100 µM; data not shown).

SC-{alpha}{alpha}{delta}9 Inhibits Migration of Ras-transformed Cells.
Finally, we investigated whether SC-{alpha}{alpha}{delta}9 functionally affected PLC-mediated cellular events. PLC inhibitors have been shown to inhibit the invasive potential of HT-1080 cells (19), motility of T-cells (29), and adhesion of H-ras-transformed Baf3 cells (30). We therefore investigated the effects of SC-{alpha}{alpha}{delta}9 on cell migration by performing a scratch wound assay in confluent Rat-1 Ras cells. Cells were plated at high density in 100-mm dishes, allowed to grow to confluence overnight, then serum-starved for up to 3 days. A 1-mm wide wound was inflicted with a sterile pipette tip, and changes in gap width were measured over time. Cell migration was then measured in the presence or absence of SC-{alpha}{alpha}{delta}9 or the PC-PLC inhibitor, D-609. Two h after wounding, vehicle-treated cells at the wound edges had assumed a polarized morphology, formed broad-leading lamellae that pointed into the direction of migration, and began to move toward the center of the gap (Fig. 6A). Both D-609 and SC-{alpha}{alpha}{delta}9 prevented this change in morphology (Fig. 6, B and C). Twenty h after wounding, control cells had almost completely closed the wound (Fig. 7A), whereas D-609 and SC-{alpha}{alpha}{delta}9 significantly inhibited cell motility without causing appreciable cell detachment (Fig. 7, B and C). Higher concentrations of SC-{alpha}{alpha}{delta}9 (30 µM) also caused a significant loss of cell adhesion (data not shown). Quantitative measurements of the cell-free area between the wound edges using image analysis software revealed that SC-{alpha}{alpha}{delta}9 was 10-fold more potent than the known PC-PLC inhibitor, D-609 (Fig. 7E). To ensure that the wound closure was the result of cell migration rather than proliferation, we measured the proliferation rate of Rat-1 Ras cells in the absence and presence of serum. Under the conditions used in the migration assay, cells showed a dramatically reduced growth rate (doubling time ~4 days; data not shown). In contrast, wound closure commenced within a few hours of wounding and was essentially complete after 24 h. Furthermore, wound closure was linear over time, with a migration velocity of 40 µm/h (Fig. 7D), indicating that the observed effects were because of cell migration rather than cell division.



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  		Fig. 6. Effect of SC-{alpha}{alpha}{delta}9 on Ras-transformed cell morphology in a scratch wound assay. Rat-1 Ras cells were plated at confluence (1.5 x 106/100-mm dish), allowed to attach overnight, and growth arrested in serum-free medium for 24 h. A sterile pipette tip was used to inflict a 1-mm wide wound. Cells were washed twice with PBS and treated with vehicle or the indicated concentrations of PLC inhibitors in serum-free medium. Cells were stained with crystal violet 2 h after wounding/treatment and photographed using a 40x objective. A, vehicle-treated cells at the leading edge quickly assumed a polarized morphology and formed broad lamellae pointing into the direction of the gap (arrows). B and C, D-609 and SC-{alpha}{alpha}{delta}9 prevented cell polarization and lamellae formation after cell wounding.

 


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  		Fig. 7. SC-{alpha}{alpha}{delta}9 inhibits migration of Rat-1 Ras cells. Rat-1 Ras cells were plated, wounded, and treated with the indicated concentrations of D-609 or SC-{alpha}{alpha}{delta}9 as described in the legend of Fig. 6. Twenty h after wounding and treatment, photomicrographs were taken. A–C, phase-contrast images of treated cells using a 10x objective. D, kinetics of wound closure. Cells were treated with vehicle and scratch wounded. At the times indicated, gap width at three arbitrarily chosen image sections along the wound edge was measured using image analysis software. E, quantification of cellular response to PLC inhibitors. ({blacksquare}), DMSO; (), 10 µM SC-{alpha}{alpha}{delta}9; and ({square}), 100 µM D-609. Gap width is defined as the cell-free area between the wound edges. Initial wound size variations between experiments were 0.87 ± 0.59 mm (average CV = 5.35%, n = 3).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Phospholipases and their reaction products play important roles in cell proliferation, signaling, and survival. These enzymes catalyze the cleavage of membrane phospholipids to generate lipid second messengers, which bind to various signal transduction enzymes and thus mediate mitogenesis and survival. Furthermore, both PLC and PLD have been implicated in oncogenesis. For example, overexpression of either PLC or PLD transforms immortalized fibroblasts (7, 8, 17), and elevated levels of PLC and PLD have been found in several human cancers (10–14).

Despite the evidence suggesting that phospholipases may represent rational anticancer targets, selective inhibitors of PLC and PLD are rare. Examples of known PLC inhibitors with antitumor activity are steroid analogs (19), naturally occurring peptides (20), or the xanthate derivative D-609 (18). PLD has thus far not been successfully targeted for therapeutics, possibly because its oncogenic potential was only recently discovered (17). Perhaps, as a result very few small molecule inhibitors of PLD have been described. Examples of known but nonspecific PLD inhibitors are short-chain alcohols and diphosphoglycerate (26). More recently, it was discovered that calphostin-c, a perylenequinone natural product and highly potent protein kinase C inhibitor (31), also has PLD inhibitory activity (32).

We had previously shown that the structurally unique, synthetic antiphosphatase agent, SC-{alpha}{alpha}{delta}9, affects oncogenic signaling in MEFs transformed by SV40 (4). In these cells, which have constitutively activated Erk because of continuous autocrine stimulation of the IGF-1 receptor by IGF-1, SC-{alpha}{alpha}{delta}9 inhibits activation of Erk and lowers levels of Cdc2, suggesting that it interferes with IGF-1 receptor function. Interestingly, SC-{alpha}{alpha}{delta}9 does not appear to directly target the IGF-1 receptor, nor sequester growth factors (2). Because the above-mentioned cellular activities are not readily reconciled with phosphatase inhibition, we proposed that SC-{alpha}{alpha}{delta}9 might affect an intracellular target that was downstream of growth factor receptors, upstream of Erk, and did not primarily involve protein phosphorylation.

Inspired by our molecular modeling studies suggesting SC-{alpha}{alpha}{delta}9 was structurally similar to phospholipids, we hypothesized that SC-{alpha}{alpha}{delta}9, by virtue of its structural similarity to phospholipids, might affect phospholipase activity. We chose H-ras Q61L-transformed Rat-1 fibroblasts as a growth factor-independent but phospholipase-dependent model for oncogenic transformation and Erk activation. In this model, SC-{alpha}{alpha}{delta}9 and PLD inhibitors prevented Erk activation by oncogenic H-ras, consistent with an involvement of PLD in Ras signaling. In vitro enzyme inhibition studies, however, showed that SC-{alpha}{alpha}{delta}9 selectively inhibited PC-PLC but not PLD at concentrations that were growth inhibitory. This was a surprising result because SC-{alpha}{alpha}{delta}9 most closely resembled the PLD reaction product, PA, and known, albeit nonspecific, PLD inhibitors such as 1-butanol or ethanol also decreased Erk activation. On the basis of the observations of Bjorkoy et al. (33), who have shown that PC-PLC transformation leads to constitutive Erk activation, inhibition of PC-PLC alone would account for SC-{alpha}{alpha}{delta}9’s inhibition of oncogenic Erk activation. Despite the evidence suggesting that SC-{alpha}{alpha}{delta}9 may inhibit PC-PLC in the cell, however, the data do not rule out the possibility that SC-{alpha}{alpha}{delta}9 affects other targets involved in Erk activation. One such possibility would be that SC-{alpha}{alpha}{delta}9 might act as a PA analogue, thereby inhibiting Erk activation through a PA-dependent mechanism. Alternatively, SC-{alpha}{alpha}{delta}9 could affect PI-specific PLC-{epsilon}, which was recently shown to be a Ras effector (27). It seems unlikely that the effects we are observing on PLC and Erk activation with SC-{alpha}{alpha}{delta}9 in the Rat-1 cells are due to phosphatase inhibition because they occurred at concentrations that are less than those required for Cdc25 inhibition (3) and well below those needed to inhibit other phosphatases (4). Furthermore, recent work in our laboratory has suggested that inhibition of Cdc25 may enhance rather than decrease Erk activation (34).

PLCs have been implicated as important mediators of cell adhesion, migration, and tumor invasiveness. For example, overexpression of a dominant-negative fragment of PI-specific PLC {gamma} abrogates the invasiveness of DU-145 prostate cancer cells (35). H-Ras-mediated T-cell adhesion appears to be PI-PLC dependent (30). It has also been suggested that cell adhesion and migration may be differentially regulated by PI-specific and PC-specific PLC isoforms (29). Consistent with these findings, we found that the PC-PLC-specific inhibitor, D-609, exclusively inhibited cell migration but not adhesion of Rat-1 Ras cells. Similarly, low concentrations of SC-{alpha}{alpha}{delta}9 (10 µM) inhibited only cell migration, consistent with SC-{alpha}{alpha}{delta}9’s ability to inhibit PC-PLC in vitro. In contrast to D-609, however, higher concentrations of SC-{alpha}{alpha}{delta}9 (30 µM) also caused substantial cell detachment, suggesting that SC-{alpha}{alpha}{delta}9 may have PI-PLC inhibitory activity as well.

At the present time, our data are consistent with the hypothesis that the actions of SC-{alpha}{alpha}{delta}9 on oncogenic Erk activation are mediated by inhibition of PLCs. More studies are required to confirm the specificity of PLC inhibition by SC-{alpha}{alpha}{delta}9. Because a mammalian PC-PLC has not been cloned, cells lacking PC-PLC by targeted disruption are not available. Knockout mice have been generated for PLC-{gamma} 1 (36), PLC-{gamma} 2 (37), and PLC-ß 3 (38) and should be part of future investigations.

Despite these uncertainties, our data demonstrate that SC-{alpha}{alpha}{delta}9 represents a novel phospholipid analogue that is structurally unrelated to existing phospholipase inhibitors. It selectively inhibited PC-PLC but not PLD and affected PC-PLC-dependent cellular events at concentrations that inhibited PC-PLC in vitro. The data provide a novel molecular target for SC-{alpha}{alpha}{delta}9 and a molecular mechanism for its effects on oncogenic Erk signaling.


   Acknowledgments
 
We thank Dr. Guillermo Romero (University of Pittsburgh, Pittsburgh, PA) for Rat-1 fibroblasts transformed with H-Ras (Q61L) and critical review of this manuscript and Deepshikha Passey for excellent technical assistance.


   Footnotes
 
1 This work was supported by Institutional Research Grant 60-002-40-IRG from the American Cancer Society, The Fiske Drug Discovery Fund, and NIH Grants CA-78039 and 52995. Back

2 To whom requests for reprints should be addressed, at Department of Pharmacology, Biomedical Science Tower E-1017, University of Pittsburgh, Pittsburgh, PA 15261. Phone: (412) 383-9816; Fax: (412) 648-1945; E-mail: avogt{at}pitt.edu or at Department of Pharmacology, Biomedical Science Tower E-1340, University of Pittsburgh, Pittsburgh, PA 15261. Phone: (412) 648-9319; Fax: (412) 648-2229; E-mail: lazo{at}pitt.edu Back

3 The abbreviations used are: SC-{alpha}{alpha}{delta}9,4-(benzyl-(2-[(2,5-diphenyloxazole-4-carbonyl)amino]ethyl)carbamoyl)-2-decanoylaminobutyric acid; MEF, mouse embryonic fibroblast; SV40, simian virus 40 large T antigen; Erk, extracellular signal-regulated kinase; DPG, diphosphoglycerate; IGF-1, insulin-like growth factor 1; PLD, phospholipase D; PLC, phospholipase C; PA, phosphatidic acid; PC, phosphatidylcholine; PI, phosphatidylinositol; USP, Ultraspiracle; ABNR, Adopted Basis Newton-Raphson; CMFDA, carboxymethyl fluorescein diacetate; D-609, Tricyclodecan-9-yl xanthogenate, potassium salt. Back

Received 1/21/02; revised 6/24/02; accepted 7/10/02.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Wipf, P., Cunningham, A., Rice, R. L., and Lazo, J. S. Combinatorial synthesis and biological evaluation of library of small-molecule Ser/Thr-protein phosphatase inhibitors.Bioorg. Med. Chem. , 5:165 –177,1997 .[CrossRef][Medline]
  2. Vogt, A., Wang, A. S., Johnson, C. S., Fabisiak, J. P., Wipf, P., and Lazo, J. S. In vivo antitumor activity and induction of insulin-like growth factor-1-resistant apoptosis by SC-{alpha}{alpha}{delta}9.J. Pharmacol. Exp. Ther. , 292:530 –537,2000 .[Abstract/Free Full Text]
  3. Tamura, K., Rice, R. L., Wipf, P., and Lazo, J. S. Dual G1 and G2-M phase inhibition by SC-{alpha}{alpha}{delta}9, a combinatorially derived Cdc25 phosphatase inhibitor.Oncogene , 18:6989 –6996,1999 .[CrossRef][Medline]
  4. Vogt, A., Rice, R. L., Settineri, C. E., Yokokawa, F., Yokokawa, S., Wipf, P., and Lazo, J. S. Disruption of insulin-like growth factor-1 signaling and down-regulation of cdc2 by SC-{alpha}{alpha}{delta}9, a novel small molecule antisignaling agent identified in a targeted array library.J. Pharmacol. Exp. Ther. , 287:806 –813,1998 .[Abstract/Free Full Text]
  5. Kozma, S. C., Bogaard, M. E., Buser, K., Saurer, S. M., Bos, J. L., Groner, B., and Hynes, N. E. The human c-Kirsten ras gene is activated by a novel mutation in codon 13 in the breast carcinoma cell line MDA-MB231.Nucleic Acids Res. , 15:5963 –5971,1987 .[Abstract/Free Full Text]
  6. Lacal, J. C., Moscat, J., and Aaronson, S. A. Novel source of 1,2-diacylglycerol elevated in cells transformed by Ha-ras oncogene.Nature , 330:269 –272,1987 .[CrossRef][Medline]
  7. Johansen, T., Bjorkoy, G., Overvatn, A., Diaz-Meco, M. T., Traavik, T., and Moscat, J. NIH 3T3 cells stably transfected with the gene encoding phosphatidylcholine-hydrolyzing phospholipase C from Bacillus cereus acquire a transformed phenotype.Mol. Cell. Biol. , 14:646 –654,1994 .[Abstract/Free Full Text]
  8. Chang, J. S., Noh, D. Y., Park, I. A., Kim, M. J., Song, H., Ryu, S. H., and Suh, P. G. Overexpression of phospholipase C-{gamma}1 in rat 3Y1 fibroblast cells leads to malignant transformation.Cancer Res. , 57:5465 –5468,1997 .[Abstract/Free Full Text]
  9. Cai, H., Erhardt, P., Troppmair, J., Diaz-Meco, M. T., Sithanandam, G., Rapp, U. R., Moscat, J., and Cooper, G. M. Hydrolysis of phosphatidylcholine couples Ras to activation of Raf protein kinase during mitogenic signal transduction.Mol. Cell. Biol. , 13:7645 –7651,1993 .[Abstract/Free Full Text]
  10. Arteaga, C. L., Johnson, M. D., Todderud, G., Coffey, R. J., Carpenter, G., and Page, D. L. Elevated content of the tyrosine kinase substrate phospholipase C-{gamma}1 in primary human breast carcinomas.Proc. Natl. Acad. Sci. USA , 88:10435 –10439,1991 .[Abstract/Free Full Text]
  11. Noh, D. Y., Kang, H. S., Kim, Y. C., Youn, Y. K., Oh, S. K., Choe, K. J., Park, I. A., Ryu, S. H., and Suh, P. G. Expression of phospholipase C-{gamma}1 and its transcriptional regulators in breast cancer tissues.Anticancer Res , 18:2643 –2648,1998 .[Medline]
  12. Noh, D. Y., Lee, Y. H., Kim, S. S., Kim, Y. I., Ryu, S. H., Suh, P. G., and Park, J. G. Elevated content of phospholipase C-{gamma}1 in colorectal cancer tissues.Cancer , 73:36 –41,1994 .[CrossRef][Medline]
  13. Zhao, Y., Ehara, H., Akao, Y., Shamoto, M., Nakagawa, Y., Banno, Y., Deguchi, T., Ohishi, N., Yagi, K., and Nozawa, Y. Increased activity and intranuclear expression of phospholipase D2 in human renal cancer.Biochem. Biophys. Res. Commun. , 278:140 –143,2000 .[CrossRef][Medline]
  14. Uchida, N., Okamura, S., and Kuwano, H. Phospholipase D activity in human gastric carcinoma.Anticancer Res. , 19:671 –675,1999 .[Medline]
  15. Carnero, A., Cuadrado, A., del Peso, L., and Lacal, J. C. Activation of type D phospholipase by serum stimulation and ras-induced transformation in NIH3T3 cells.Oncogene , 9:1387 –1395,1994 .[Medline]
  16. Lu, Z., Hornia, A., Joseph, T., Sukezane, T., Frankel, P., Zhong, M., Bychenok, S., Xu, L., Feig, L. A., and Foster, D. A. Phospholipase D and RalA cooperate with the epidermal growth factor receptor to transform 3Y1 rat fibroblasts.Mol. Cell. Biol. , 20:462 –467,2000 .[Abstract/Free Full Text]
  17. Min, D. S., Kwon, T. K., Park, W. S., Chang, J. S., Park, S. K., Ahn, B. H., Ryoo, Z. Y., Lee, Y. H., Lee, Y. S., Rhie, D. J., Yoon, S. H., Hahn, S. J., Kim, M. S., and Jo, Y. H. Neoplastic transformation and tumorigenesis associated with overexpression of phospholipase D isozymes in cultured murine fibroblasts.Carcinogenesis , 22:1641 –1647,2001 .[Abstract/Free Full Text]
  18. Schutze, S., Potthoff, K., Machleidt, T., Berkovic, D., Wiegmann, K., and Kronke, M. TNF activates NF-{kappa}B by phosphatidylcholine-specific phospholipase C-induced "acidic" sphingomyelin breakdown.Cell , 71:765 –776,1992 .[CrossRef][Medline]
  19. Xie, W., Peng, H., Zalkow, L. H., Li, Y. H., Zhu, C., Powis, G., and Kunkel, M. 3[ß]-Hydroxy-6-aza-cholestane and related analogues as phosphatidylinositol specific phospholipase C (PI-PLC) inhibitors with antitumor activity.Bioorg. Med. Chem. , 8:699 –706,2000 .[CrossRef][Medline]
  20. Hill, S. R., Bonjouklian, R., Powis, G., Abraham, R. T., Ashendel, C. L., and Zalkow, L. H. A multisample assay for inhibitors of phosphatidylinositol phospholipase C: identification of naturally occurring peptide inhibitors with antiproliferative activity.Anticancer Drug Des. , 9:353 –361,1994 .[Medline]
  21. Rice, R. L., Rusnak, J. M., Yokokawa, F., Yokokawa, S., Messner, D. J., Boynton, A. L., Wipf, P., and Lazo, J. S. A targeted library of small-molecule, tyrosine, and dual-specificity phosphatase inhibitors derived from a rational core design and random side chain variation.Biochemistry , 36:15965 –15974,1997 .[CrossRef][Medline]
  22. Clayton, G. M., Peak-Chew, S. Y., Evans, R. M., and Schwabe, J. W. The structure of the ultraspiracle ligand-binding domain reveals a nuclear receptor locked in an inactive conformation.Proc. Natl. Acad. Sci. USA , 98:1549 –1554,2001 .[Abstract/Free Full Text]
  23. Der, C. J., Finkel, T., and Cooper, G. M. Biological and biochemical properties of human rasH genes mutated at codon 61.Cell , 44:167 –176,1986 .[CrossRef][Medline]
  24. Garrigues, J., Anderson, J., Hellstrom, K. E., and Hellstrom, I. Antitumor antibody BR96 blocks cell migration and binds to a lysosomal membrane glycoprotein on cell surface microspikes and ruffled membranes.J. Cell Biol. , 125:129 –142,1994 .[Abstract/Free Full Text]
  25. Fyfe, P. K., McAuley, K. E., Roszak, A. W., Isaacs, N. W., Cogdell, R. J., and Jones, M. R. Probing the interface between membrane proteins and membrane lipids by X-ray crystallography.Trends Biochem. Sci. , 26:106 –112,2001 .[CrossRef][Medline]
  26. Milella, M., Gismondi, A., Roncaioli, P., Palmieri, G., Morrone, S., Piccoli, M., Frati, L., Cifone, M. G., and Santoni, A. ß1 integrin cross-linking inhibits CD16-induced phospholipase D and secretory phospholipase A2 activity and granule exocytosis in human NK cells: role of phospholipase D in CD16-triggered degranulation.J. Immunol. , 162:2064 –2072,1999 .[Abstract/Free Full Text]
  27. Kelley, G. G., Reks, S. E., Ondrako, J. M., and Smrcka, A. V. Phospholipase C({epsilon}): a novel Ras effector.EMBO J. , 20:743 –754,2001 .[CrossRef][Medline]
  28. Clark, M. A., Shorr, R. G., and Bomalaski, J. S. Antibodies prepared to Bacillus cereus phospholipase C crossreact with a phosphatidylcholine preferring phospholipase C in mammalian cells.Biochem. Biophys. Res. Commun. , 140:114 –119,1986 .[CrossRef][Medline]
  29. Wooten, D. K., Teague, T. K., and McIntyre, B. W. Separation of integrin-dependent adhesion from morphological changes based on differential PLC specificities.J. Leukoc. Biol. , 65:127 –136,1999 .[Abstract]
  30. Shibayama, H., Anzai, N., Braun, S. E., Fukuda, S., Mantel, C., and Broxmeyer, H. E. H-Ras is involved in the inside-out signaling pathway of interleukin-3-induced integrin activation.Blood , 93:1540 –1548,1999 .[Abstract/Free Full Text]
  31. Kobayashi, E., Nakano, H., Morimoto, M., and Tamaoki, T. Calphostin C (UCN-1028C), a novel microbial compound, is a highly potent and specific inhibitor of protein kinase C.Biochem. Biophys. Res. Commun. , 159:548 –553,1989 .[CrossRef][Medline]
  32. Sciorra, V. A., Hammond, S. M., and Morris, A. J. Potent direct inhibition of mammalian phospholipase d isoenzymes by calphostin-c.Biochemistry , 40:2640 –2646,2001 .[CrossRef][Medline]
  33. Bjorkoy, G., Perander, M., Overvatn, A., and Johansen, T. Reversion of ras- and phosphatidylcholine-hydrolyzing phospholipase C-mediated transformation of NIH 3T3 cells by a dominant interfering mutant of protein kinase C {lambda} is accompanied by the loss of constitutive nuclear mitogen-activated protein kinase/extracellular signal-regulated kinase activity.J. Biol. Chem. , 272:11557 –11565,1997 .[Abstract/Free Full Text]
  34. Vogt, A., Takahito, A., Ducruet, A. P., Chesebrough, J., Nemoto, K., Carr, B. I., and Lazo, J. S. Spatial analysis of key signaling proteins by high-content solid-phase cytometry in Hep3B cells treated with an inhibitor of Cdc25 dual-specificity phosphatases.J. Biol. Chem. , 276:20544 –20550,2001 .[Abstract/Free Full Text]
  35. Turner, T., Epps-Fung, M. V., Kassis, J., and Wells, A. Molecular inhibition of phospholipase c{gamma} signaling abrogates DU-145 prostate tumor cell invasion.Clin. Cancer Res. , 3:2275 –2282,1997 .[Abstract/Free Full Text]
  36. Ji, Q. S., Winnier, G. E., Niswender, K. D., Horstman, D., Wisdom, R., Magnuson, M. A., and Carpenter, G. Essential role of the tyrosine kinase substrate phospholipase C-{gamma}1 in mammalian growth and development.Proc. Natl. Acad. Sci. USA , 94:2999 –3003,1997 .[Abstract/Free Full Text]
  37. Takata, M., Homma, Y., and Kurosaki, T. Requirement of phospholipase C-{gamma} 2 activation in surface immunoglobulin M-induced B cell apoptosis.J. Exp. Med. , 182:907 –914,1995 .[Abstract/Free Full Text]
  38. Wang, S., Gebre-Medhin, S., Betsholtz, C., Stalberg, P., Zhou, Y., Larsson, C., Weber, G., Feinstein, R., Oberg, K., Gobl, A., and Skogseid, B. Targeted disruption of the mouse phospholipase C ß3 gene results in early embryonic lethality.FEBS Lett. , 441:261 –265,1998 .[CrossRef][Medline]



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