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Biochemistry and Biophysics |
and -µ
Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina 27709
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and PKCµ, two of the novel PKC isozymes, from the cytosol to the membrane. In contrast, a conventional PKC isozyme, PKC
, as well as the atypical isozymes, PKC
and PKC
, did not translocate after cis-PUFA treatment. Function-blocking antibodies specific for 1, 2, and ß1 integrin subunits inhibited cell adhesion to collagen IV, whereas antibodies to 3 and 5 did not. No increase in the expression of these integrins on the cell surface was detected after the incubation of cells with cis-PUFAs, suggesting that there is an increase in the activity, but not in the amount, of these ß1 integrins. Altogether, these data suggest that cis-PUFAs enhance human breast cancer cell adhesion to collagen IV by selectively activating specific PKC isozymes, which leads to the activation of ß1 integrins. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Both n-3 and n-6 cis-PUFAs affect mammary tumorigenesis (21)
and metastasis (22, 23, 24)
in experimental animal models. At the cellular level, cis-PUFAs and their metabolites can alter the metastatic phenotype of various tumor cells in vitro and in vivo (24
, 25)
. Nevertheless, little is known about the molecular mechanisms involved in the effects of cis-PUFAs on metastasis, in particular, in mammary tumor cells. AA and its precursor, -LA, which belong to the n-6 family, are common dietary FAs that are available to cells in the extracellular environment or through release from membrane phospholipids by the action of phospholipases. They are potential substrates for cyclooxygenases, lipoxygenases, and cytochromes P-450 (26, 27, 28)
and are metabolized to a plethora of biologically active molecules, including the prostaglandins, leukotrienes, and thromboxanes.
There is substantial evidence that PKC activity is related to the metastatic potential of tumor cells in vivo (12 , 29, 30, 31, 32) . PKCs have been shown to be involved in the activation of integrins (33, 34, 35) and formation of focal contacts (36) and, consequently, in cell adhesion and cell spreading in vitro (11 , 12 , 37, 38, 39, 40, 41) . The PKCs comprise a family of at least 11 isozymes that have different properties; the role of these different isozymes in cell adhesion and metastasis is not yet known. Interestingly, cis-PUFAs have been shown to activate directly some PKC isozymes in vitro (42) ; other studies have reported that essential FAs alter the ability of carcinoma cells to attach to extracellular proteins (43, 44, 45) . These data stress the importance of clarifying the role of PKCs in cis-PUFA effects.
We have previously shown that lipoxygenase and PKC activities are involved in a rapid increase in the adhesion of a metastatic human breast cancer cell line, MDA-MB-435, to collagen IV after either intracellular calcium mobilization or the supply of exogenous AA (39) . Thus, FAs released from the cell membrane or present in the serum may affect the capacity of human mammary carcinoma cells to adhere to extracellular matrix proteins.
In this study, we report the effect of various dietary FAs in the modulation of metastatic human breast cancer cell adhesion to collagen IV. Furthermore, we describe the importance of PKC activity in this process and the involvement of specific PKC isozymes expressed in these cells. Finally, we describe the role and the expression of integrins involved in cis-PUFA-induced cell adhesion.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2 (clone P1E6), 3 (clone P1B5), and ß1 (clone P4C10) were obtained from Life Technologies, Inc. (Grand Island, NY). Mouse antihuman CD16 monoclonal Ab was from PharMingen (San Diego, CA). Ab to 1 integrin (clone FB12) was from Chemicon International, Inc. (Temecula, CA). Fluorescein-conjugated secondary Ab (goat Fab antimouse IgG) was from Calbiochem (La Jolla, CA). Mouse monoclonal Abs against PKC, 
, , , µ, and and the horseradish peroxidase-conjugated goat polyclonal antimouse IgG were from Transduction Laboratories (Lexington, KY). Rabbit polyclonal Abs against PKCß1, ß2, , 
, and were a generous gift from Dr. William C. Wetsel (Duke University, Durham, NC). The horseradish peroxidase-conjugated goat polyclonal antirabbit IgG was from Transduction Laboratories or Kirkegaard & Perry Laboratories, Inc. (Gaithersburg, MD).
Materials.
IBR, penicillin, streptomycin, glutamine, N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid], and PBS were from Life Technologies, Inc. FCS was from Hyclone (Logan, UT). Human collagen IV and poly-D-lysine were from Becton Dickinson (Bedford, MA). BSA (FA-free; <0.01%), crystal violet, TPA, and EDTA were from Sigma Chemical Co. (St. Louis, MO). Calphostin C was from Calbiochem. Oleic acid, LA, AA, and trans-LA were from Cayman Chemical Co. (Ann Arbor, MI). Methyl-AA was from Nu-Check-Prep, Inc. (Elysian, MN). The 96-well cell culture dishes were from Corning Inc. (Acton, MA). The protease inhibitors leupeptin and 4-(2-aminoethyl)benzenesulfonyl fluoride were from Boehringer Mannheim (Indianapolis, IN). 1,4-DTT was from ICN (Costa Mesa, CA). Protein molecular weight standards and 2x Tris-glycine/SDS sample buffer were from Novex (San Diego, CA). The modified Bradford protein assay kit and a premixed solution of acrylamide and bis-acrylamide were from Bio-Rad Laboratories (Hercules, CA). Immobilon-P transfer membranes were from Millipore (Bedford, MA). Ultra chemiluminescent substrate and Triton X-100 were from Pierce (Rockford, IL).
Cell Preparation.
The human mammary adenocarcinoma cell line MDA-MB-435 (46)
was the kind gift of Dr. Janet Price (University of Texas M. D. Anderson Comprehensive Cancer Center, Houston, TX) and was maintained in culture as described previously (39)
. Subconfluent cells were harvested by washing with calcium- and magnesium-free PBS and incubating the cells for 1–2 min with 1 ml of 2 mM EDTA per 175-cm2 flask at room temperature. The EDTA solution was neutralized by adding 25 ml of serum-free IBR medium containing 20 mM N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] at pH 7.4. The cells were washed once with IBR medium and resuspended at a final concentration of 8.5 x 105 cells/milliliter of medium.
Cell Adhesion Assay.
Tissue culture dishes (96-well plates) were coated with human collagen IV (50 µl at 3.2 µg/ml) as described previously (39)
. Nonspecific binding sites on both collagen IV-coated and uncoated wells were blocked by overlaying each well with 200 µl of a 1% solution of heat-denatured BSA. Potassium salts of the various FAs at different concentrations were freshly prepared in 10% ethanol before each experiment (except for MeAA, which was prepared in 95% ethanol). FA solutions or ethanol as a solvent control was added to cell suspensions in polypropylene tubes; the final ethanol concentration never exceeded 0.1%. The cells were mixed briefly at room temperature, and 100 µl of cell suspension were transferred into each protein-coated well. Incubations were carried out at 37°C in a humidified 5% CO2 atmosphere for 45 min, unless indicated otherwise. In some experiments, a 1:1000 dilution of function-blocking monoclonal Abs was added to cell suspensions before adding the FAs. When appropriate, cell suspensions were preincubated in the presence of calphostin C or its solvent, 0.033% DMSO, for 10 min at room temperature before adding the stimuli. The preincubation and incubation steps with calphostin C were carried out under fluorescent light to activate the inhibitor (47)
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Cell adhesion was quantified using a colorimetric adhesion assay as described previously (39) . Briefly, after incubation, nonadherent cells were removed by aspiration and two additional washes. Adherent cells were stained with 0.5% crystal violet in 20% methanol. Absorbance was determined at 595 nm after solubilizing the dye with 1% SDS. The results were standardized as a percentage of adhesion to polylysine.
Flow Cytometry Analysis.
The cells were prepared as described above but were resuspended at a final concentration of 1 x 106 cells/milliliter of IBR. Cell suspensions were incubated in polypropylene tubes in the presence of FAs or their solvent for 60 min at 37°C in a humidified 5% CO2 atmosphere. The cells were collected at 350 x g for 5 min at 4°C and washed twice with PBS containing 1% FCS and 0.1% sodium azide (PBS-FCS). Monoclonal Abs to 1, 2, 3, or ß1 integrins were added to cell suspensions (5 x 106 cells/ml) in PBS-FCS at a concentration of 10 µg/ml. As a negative control, cells were incubated with an isotype- and species-matched control mouse IgG. After a 30-min incubation on ice, the cells were washed two times with PBS-FCS and incubated with goat antimouse fluorescein-conjugated secondary Ab for 30 min on ice. The cells were then washed two times with PBS-FCS, fixed, and kept at 4°C until they were analyzed by flow cytometry (less than 24 h). The fluorescent signal of 10,000 cells was then quantified with a Becton Dickinson FACScan flow cytometer. These conditions allowed us to detect higher expression of integrins on other cell types (data not shown), demonstrating that we could have detected an increase in expression of integrins on the MDA-MB-435 cells if it had occurred.
Immunoblot Analysis of PKC Isozymes.
Cell suspensions (1 x 106 cells/milliliter of IBR) were incubated in polypropylene tubes in the presence of various FAs, TPA, or their solvent at 37°C in a humidified 5% CO2 atmosphere. The cells (20 x 106) were then centrifuged at 350 x g for 5 min at 4°C and washed once with ice-cold calcium- and magnesium-free PBS. The pellets were resuspended in 125 µl of homogenization buffer [20 mM Tris-HCl (pH 7.5), 5 mM DTT, 250 mM sucrose, 2 mM EDTA, 10 mM ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, and 10 µg/ml leupeptin]. Cell suspensions were sonicated and centrifuged at 105,000 x g for 45 min at 4°C. The supernatants were collected and used as cytosolic fractions. Proteins were extracted from the pellet fractions by sonication in 250 µl of homogenization buffer containing 1% Triton X-100. These samples were centrifuged, and the supernatants were collected and saved as the membrane fractions. The supernatants and resuspended pellets were mixed with 1 volume of 2x Tris-glycine SDS sample buffer. The protein concentration in each fraction was determined by a modified Bradford protein assay. The samples (250 µg of total protein) were resolved by electrophoresis with a 10% SDS-polyacrylamide gel and transferred to Immobilon-P transfer membranes. The membranes were probed with either monoclonal mouse Abs or rabbit polyclonal Abs following two different protocols. To probe with the monoclonal Abs, the membranes were incubated in blocking solution (5% dry milk in Tris-HCl containing 100 mM NaCl and 0.1% Tween 20) overnight at 4°C. The blots were then incubated with Abs for PKC, , , µ, , and for 1 h in Tris-HCl blocking solution. After washing membranes with Tris-HCl containing 0.1% Tween 20, the membranes were incubated for 1 h with a 1:5,000 dilution of a horseradish peroxidase-conjugated goat antimouse IgG. Finally, the membranes were washed again with Tris-HCl containing 0.1% Tween 20. To probe with the polyclonal Abs, the membranes were incubated in blocking solution (5% dry milk in PBS solutions containing 0.03% Tween 20) overnight at 4°C. The blots were then incubated with rabbit polyclonal Abs to PKCß1, ß2, , , and for 1 h in PBS blocking solution supplemented with 0.03% Tween 20. After washing membranes with PBS containing 0.03% Tween 20, membranes were incubated for 90 min with a 1:100,000 dilution of a horseradish peroxidase-conjugated goat antirabbit Ab. Finally, the membranes were washed with PBS containing 0.1% Tween 20. Immunoreactivity was detected using the Ultra chemiluminescent substrate.
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RESULTS |
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. A substantial and maximal increase in cell adhesion was observed at 
45 min of incubation in the presence of either LA or AA (data not shown). Although AA induced greater adhesion than LA, the overall dose responses of AA and LA were similar. In contrast, these cis-PUFAs did not induce significant effects on cell adhesion to wells coated with BSA alone (Fig. 1)
, which suggests that the FA modulation of cell adhesion to wells coated with collagen IV before blocking with BSA involves specific adhesion molecules.
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To test the specificity of the FA modulation of adhesion, cells were treated with OA, trans-LA, or MeAA. Neither trans-LA nor MeAA significantly affected MDA-MB-435 cell adhesion to collagen IV compared to the control (Fig. 2)
. Oleic acid induced only a slight increase in cell adhesion. Oleic acid, trans-LA, and MeAA did not affect nonspecific cell adhesion to polylysine at the concentrations used in these experiments. These data demonstrate that only cis-PUFAs substantially affect cell adhesion in these cells. The other types of FAs, which are known to alter membrane fluidity (48, 49, 50, 51)
but are not substrates for lipoxygenase and cyclooxygenase, were ineffective at modulating cell adhesion.
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. Calphostin C induced only a modest inhibition of the nonstimulated adhesion compared to the cis-PUFA-stimulated cell adhesion. Furthermore, calphostin C did not substantially alter cell adhesion to polylysine (data not shown) and did not affect cell viability.
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and PKCµ to the membrane fraction but did not induce translocation of other PKC isozymes found to be highly expressed in these cells (Fig. 4)
. Translocation of the cytosolic PKC and PKCµ to the membrane fraction occurred within 2 min of incubation in the presence of 30 µM AA. PKC was found in the membrane fraction after 10 and 60 min of incubation with AA whereas PKCµ disappeared from the membrane fraction after 2 min of treatment, reappearing in the cytosol fraction at 10 and 60 min after cis-PUFA treatment. In contrast, the distribution of PKC and PKC was not affected after 2, 10, or 60 min of incubation with AA. As reported in other cell types, 100 nM TPA stimulated the translocation of both PKC and PKC but did not induce the translocation of PKC and PKCµ after 30 min of incubation (Fig. 4)
. Furthermore, we found that LA also induced the translocation of cytosolic PKC to the membrane fraction (Fig. 5)
. Overall, these data demonstrate that cis-PUFA-induced cell adhesion is PKC dependent and that cis-PUFAs selectively stimulate the translocation of PKC and possibly PKCµ in these cells.
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2, 3, and ß1 integrins were easily detectable, whereas 1 appeared to be present at lower levels and perhaps only on a subset of cells (Fig. 6)
. Therefore, we assessed the role of the expressed integrin collagen receptors with function-blocking monoclonal Abs. A monoclonal Ab that recognizes the 1 subunit inhibited the basal adhesion to collagen IV but did not block AA-stimulated adhesion (Fig. 7)
. Blocking Ab specific to 2 did not significantly reduce the basal adhesion but inhibited the AA-stimulated binding. Monoclonal Ab to 3 (Fig. 7)
and to 5 (data not shown) did not significantly inhibit cell adhesion. Ab to ß1 integrins strongly inhibited all MDA-MB-435 cell adhesion to collagen IV in both the presence and absence of AA (Fig. 7)
. These data suggest that the 1ß1 and 2ß1 integrin complexes may play a crucial role in the attachment of these cells to collagen IV induced by cis-PUFAs and that 2ß1 is most likely involved in the adhesion stimulated by FAs.
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. MDA-MB-435 cells incubated with AA for 45 min showed no significant change in surface integrin expression when probed for 1, 2, 3, or ß1 (Fig. 8)
. Furthermore, neither LA nor eicosapentaenoic acid induced any increase in cell surface integrin expression (data not shown), indicating that the cis-PUFAs that induce adhesion appear not to function by altering integrin expression. These data suggest that activation of some step in adhesion rather than an increased expression of integrins on the cell surface is responsible for the enhancement of cell adhesion observed after exposure to cis-PUFAs.
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DISCUSSION |
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2ß1 integrin that is mediated by a selective effect of cis-PUFAs on PKC and PKCµ translocation. Dietary cis-PUFAs that are potential substrates for lipoxygenases and cyclooxygenases, but not other FAs, induced a rapid increase in cell adhesion to collagen IV, suggesting a mechanism by which dietary components may influence tumor cell behavior. Previous studies have suggested that the effects of cis-PUFAs on various cellular processes are a consequence of their membrane fluidity-enhancing effects (48, 49, 50, 51) . These conclusions were based on the correlation between biological and physical phenomena. Other unsaturated FAs, such as OA (48, 49, 50, 51) and the methyl ester of OA (50) , have been shown to induce membrane perturbations that lead to altered cellular behavior. It has also been shown that LA and OA change platelet membrane fluidity similarly, whereas trans-LA does not (48 , 49) . However, in our assays, OA failed to reproduce the effects of cis-PUFAs, and MeAA did not induce any significant increase in cell adhesion at concentrations up to 120 µM. Thus, our results suggest that although cis-PUFAs may alter membrane fluidity, this alteration is not sufficient to induce MDA-MB-435 cell adhesion to collagen IV.
Both AA and LA are potential substrates for both the cyclooxygenases and lipoxygenases (26 , 27) . Our data suggest that the activation of specific signal transduction pathways is dependent on the cis-polyunsaturation of FAs, perhaps because of the formation of enzymatically oxidized metabolites generated by lipoxygenases. Consistent with this hypothesis is the finding that AA-induced cell adhesion is significantly reduced by a lipoxygenase inhibitor (39) . In addition, Liu et al. (53) have shown that a lipoxygenase inhibitor also blocks the LA-stimulated invasive capacity of MDA-MB-435 cells through a reconstituted basement membrane. Altogether, these data suggest that cis-PUFAs may trigger specific intracellular events, perhaps through lipoxygenase metabolism, in metastatic human breast tumor cells that enhance their ability to adhere to collagen IV and further their ability to metastasize.
We assessed the role of PKC activity in the cis-PUFA-induced cell adhesion by treating the MDA-MB-435 cells with calphostin C, a selective PKC inhibitor (52)
. This inhibition was more dramatic in the presence of the cis-PUFAs than for nonstimulated cell adhesion. These data argue for the requirement of PKC activity in cis-PUFA-induced cell adhesion. Furthermore, our results suggest that cis-PUFAs induce cell adhesion through a diacylglycerol-dependent PKC activation. Indeed, it has previously been shown that calphostin C inhibits phorbol ester binding to the diacylglycerol/phorbol ester-binding region in the regulatory domain (52)
. Thus, calphostin C preferentially inhibits the diacylglycerol-dependent PKCs, such as PKC and PKC, over the diacylglycerol-independent PKCs, such as PKC (54)
. Moreover, cis-PUFAs do not interact with the diacylglycerol/phorbol ester-binding region (55)
, which means that calphostin C should not affect PKC-cis-PUFA interactions. Therefore, the finding that calphostin C inhibits adhesion induced by cis-PUFAs suggests an indirect activation of the PKC, possibly mediated by diacylglycerol. In support of this suggestion, some studies have described a synergistic action between diacylglycerol and unsaturated FAs for PKC activation (55
, 56)
, which might sustain the enzyme activated in the presence of low concentrations of diacylglycerol (57)
. Moreover, other studies have shown that unsaturated FAs stimulate phosphatidylinositol-specific phospholipase C
(58
, 59)
, which generates intracellular diacylglycerol. Furthermore, it has been shown that lipoxygenase products modulate murine melanoma cell adhesion by activating PKC through an increase in diacylglycerol (60)
.
It is now well established that PKC activation involves a redistribution of the enzymes, frequently through interactions with membrane receptor proteins (61
, 62)
. We found that the PKC, , µ, and isozymes were highly expressed in MDA-MB-435 cells. After a brief incubation of the cells with AA, only PKC and PKCµ were translocated from the cytosol to the particulate fraction. PKC remained in the particulate fraction for at least 60 min, whereas PKCµ quickly reappeared in the cytosol. The fact that cis-PUFAs induce a PKC-dependent cell adhesion to collagen IV, whereas PKC and PKCµ are the only PKC isozymes found to translocate, suggests that one or both of these isozymes are involved in the modulation of cell adhesion to collagen IV by AA and LA. These data are consistent with those from other reports. Chun et al. (40)
found that only PKC was translocated during HeLa cell adhesion to a gelatin substratum, suggesting that PKC is involved in carcinoma cell attachment to extracellular matrix proteins. The mechanism by which PKC induces this attachment is not yet known, although it is intriguing that at least two groups have shown that PKC can bind to F-actin in diverse model systems (63
, 64)
, consistent with early work that identified PKC activity attached to cytoskeletal components (65)
. Previous studies revealed changes in cytoskeletal organization after treatment with TPA, which generates AA (41)
, or treatment with lipoxygenase-derived metabolites of AA (66)
. Increased PKC activity generated through phorbol ester treatment (67, 68, 69)
or activation of muscarinic acetylcholine receptors (70)
has also been linked to various cell adhesion-related processes, including increased motility, migration, and protease expression.
The locations to which the PKCs translocate are probably critical to their ultimate function (71)
, but these sites may vary from cell to cell. Shirai et al. (72)
have used an overexpressed, green fluorescent protein-linked PKC construct to show that high concentrations of AA induced a translocation of PKC from the cytoplasm to the Golgi network in Chinese hamster ovary cells. Studies with cardiac myocytes have shown that AA stimulates a specific translocation of PKC from the cytoplasm to a filament/nuclear fraction, and confocal microscopy was used to show PKC localized near the Z-line where actin filaments are anchored and where transverse tubules are closely apposed to the myofilaments (73)
. We are currently investigating the precise location of PKC and µ in the MDA-MB-435 cells after stimulation with cis-PUFAs.
The relative sensitivity of different PKC isozymes to cis-PUFAs or their metabolites may explain the selective activity of AA or its metabolites on PKC as compared to PKC and PKC. Interestingly, PKC has been described as "greatly activated by AA" (74)
. Furthermore, the fact that novel PKC isoforms are activated by diacylglycerol, in contrast to diacylglycerol nonactivated PKC, but do not require calcium, in contrast to calcium-dependent conventional PKC, might be responsible for the difference in responses to cis-PUFAs.
To characterize the effects of cis-PUFAs on specific cell adhesion receptors, we evaluated the role of the relevant receptors that bind to collagen IV and are expressed on the MDA-MB-435 cell surface. We found that function-blocking Abs to ß1, 1, and 2 integrin subunits, but not Abs to 3, inhibited cell adhesion to collagen IV. These results suggest that specific ß1 integrin complexes mediate MDA-MB-435 cell-collagen IV interactions. Furthermore, we showed by flow cytometry that neither 1, 2, 3, nor ß1 expression on the cell surface increased after cis-PUFA treatment. These data suggest that the functional activity of the ß1 integrin complexes, rather than the total surface expression, is enhanced by exposure of the cells to FAs. Precedence for this hypothesis comes from reports that the functional up-regulation of 2ß1 integrins in human T cells and human melanoma cells after TPA treatment was not associated with changes in integrin expression (38
, 75) . There is also evidence that the 2 integrin cytoplasmic domain regulates the constitutive and TPA-stimulated adhesive properties of human tumor cell lines to collagen (76
, 77)
.
The requirement for PKC activity and apparent activation of ß1 integrins suggests that intracellular signals involving PKC activity play a central role in the functional up-regulation of 2ß1 collagen receptors. The precise mechanism by which a PKC enhances ß1 integrin function has not yet been described. However, phosphorylation of other integrins has been reported, including recent evidence that phosphorylation of a ß3 integrin threonine residue generates a docking site for proteins containing a Src homology 2 domain (78)
. Phosphorylation of the PKC substrate, myristoylated alanine-rich C-kinase substrate, has been implicated in the increased diffusion of ß2 integrin molecules, thus increasing the chance that these integrins will interact with their ligands (79)
. We are currently attempting to define the role of PKC translocation and activation in ß1 integrin-mediated adhesion to the extracellular matrix. It has been proposed that the ability to modulate integrin functions increases migration capability (80)
and contributes to a more metastatic phenotype (13)
. The biochemical mechanisms underlying this modulation are not yet delineated, although we have recently shown that the mitogen-activated protein kinase-activated protein kinase 2 is activated in AA-treated MDA-MB-435 cells (81)
. Further work will be required to characterize the pathways that lead from exposure to cis-PUFAs to increased cell adhesion in metastatic human tumor cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Present address: SmithKline Beecham Biologicals, Extramural R & D, Rixensart, Belgium. 
2 To whom requests for reprints should be addressed, at Mail Drop C2-14, Laboratory of Molecular Carcinogenesis, P. O. Box 12233, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709. Phone: (919) 541-5023; Fax: (919) 541-7784, E-mail: Roberts1{at}niehs.nih.gov
3 The abbreviations used are: Ab, antibody; AA, arachidonic acid; collagen IV, type IV collagen; FA, fatty acid; IBR, IBR modified Dulbecco’s Eagle medium; LA, linoleic acid; MeAA, arachidonic acid methyl ester; OA, oleic acid; PKC, protein kinase C; cis-PUFA, polyunsaturated FA; TPA, 12-tetradecanoyl phorbol 13-acetate; trans-LA, linoelaidic acid (trans isomer of linoleic acid).
Received 6/19/00. Accepted 1/17/01.
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  L. Guan, K. Song, M. A. Pysz, K. J. Curry, A. A. Hizli, D. Danielpour, A. R. Black, and J. D. Black Protein Kinase C-mediated Down-regulation of Cyclin D1 Involves Activation of the Translational Repressor 4E-BP1 via a Phosphoinositide 3-Kinase/Akt-independent, Protein Phosphatase 2A-dependent Mechanism in Intestinal Epithelial Cells J. Biol. Chem., May 11, 2007; 282(19): 14213 - 14225. [Abstract] [Full Text] [PDF]
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P. A. Nony, S. B. Kennett, W. C. Glasgow, K. Olden, and J. D. Roberts 15(S)-Lipoxygenase-2 Mediates Arachidonic Acid-stimulated Adhesion of Human Breast Carcinoma Cells through the Activation of TAK1, MKK6, and p38 MAPK J. Biol. Chem., September 9, 2005; 280(36): 31413 - 31419. [Abstract] [Full Text] [PDF]
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S. B. Kennett, J. D. Roberts, and K. Olden Requirement of Protein Kinase C{micro} Activation and Calpain-mediated Proteolysis for Arachidonic Acid-stimulated Adhesion of MDA-MB-435 Human Mammary Carcinoma Cells to Collagen Type IV J. Biol. Chem., January 30, 2004; 279(5): 3300 - 3307. [Abstract] [Full Text] [PDF]
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) or their solvent for 45 min at 37°C. *, P < 0.05 compared to the control (no FAs).
) for 45 min at 37°C. The results are expressed as the mean ± SE of three experiments. *, P < 0.05 as compared to the samples with no calphostin C added (defined as 100%).
). Cells were then assayed for adhesion to collagen IV as described in "Materials and Methods." Each condition was tested in triplicate, and the data shown are representative of two or three experiments.