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in MCF-10A Human Breast Cells Engenders Dramatic Alterations in Morphology, Proliferation, and Motility1
Department of Chemistry and Biochemistry, Queens College, City University of New York, Flushing, New York 11367
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Abstract |
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 TOP Abstract IntroductionResultsDiscussionMaterials and MethodsReferences |
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(PKC) so as to investigate a role for this isoform in the metastatic phenotype. PKC transfectants (clone 26) expressed an 8-fold higher level of PKC protein without compensatory alterations in other isoforms. Clone 26 proliferated slowly (accumulating in G1 of the cell cycle) but exhibited pronounced increases in motility and adhesion. Elevated expression of cell cycle inhibitor p27 and focal adhesion proteins was observed, whereas E-cadherin expression decreased to undetectable levels. These observations were consistent with the morphology of PKC transfectants (large, disaggregated, and flat, with lamellipodia and extensive actin fibers) and control cells (small, aggregated, and refractile). Treatment with PKC inhibitors or transfection of a dominant negative (dn) mutant of Rac1, but neither dn RhoA nor dn cdc42, reduced the motility of clone 26, implicating PKC catalytic activity and endogenous Rac1, respectively, in the PKC-induced phenotype. Overall, PKC overexpression suppresses proliferation while endowing MCF-10A cells with properties consistent with the metastatic phenotype. |
Introduction |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
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Investigation of the relationship between PKC and breast cancer is prompted by observations that identified elevated PKC activity in human breast tumors (3
, 4)
. Using the overexpression approach with MCF-7 cells, a human breast cell model, transfection of PKC led to enhanced proliferation, anchorage independence, loss of epithelioid appearance, and increased tumorigenicity in nude mice (5)
. By contrast, a similar study found that overexpression of PKC in MCF-7 cells induced a less aggressive phenotype marked by suppressed proliferation and diminished tumor formation (6)
. In both studies, PKC overexpression was observed to cause up-regulation of PKCß1, suggesting that the expression levels of the two PKC isoforms are mutually regulating in these cells. Furthermore, both studies found that activation of PKC by TPA, a phorbol ester tumor promoter, was required to observe the PKC-induced phenotype. The apparently conflicting role of PKC in the two studies could reflect the significant biological variation previously found to exist among MCF-7 cell lines used by different laboratories (7)
.
To clarify the role of PKC in breast cell biology and to extend previous studies with this isoform, we used MCF-10A cells, which are an immortalized, nontransformed, nontumorigenic human breast epithelial cell line introduced in 1990 (8
, 9)
. MCF-10A cells are regarded as an excellent model system for analyzing the effect on healthy cell behavior by genetically engineering the overexpression of a protein of interest. Previous work with MCF-10A cells focused on derivatives overexpressing H-ras, revealing the significance of H-ras and other GTP-binding proteins to cell adhesion, contractility, and tyrosine protein phosphorylation (10
, 11)
.
In light of observations that correlate PKC activity and expression levels with the metastatic phenotype of breast cells (3
, 4)
, the present study focuses on the integrated activities of cell adhesion and motility (12)
. Although the precise mechanistic role(s) of PKC in these phenomena remains undefined, other avenues of research have identified key PKC substrates in the actin cytoskeleton, such as MARCKS (13
, 14)
and fascin (15)
, whose phosphorylation by PKC alters their ability to bind actin. In particular, MARCKS phosphorylation by specific PKC isoforms (, 
, and 
; Ref. 16
) leads to reversible binding to actin (17)
and, consequently, to the reorganization of actin structure, membrane ruffling, and adhesion, all of which are coordinated to produce cell movement (18)
. In the past several years, the small GTP-binding proteins of the Rho family (Rho, Rac1, and cdc42) have been assigned pivotal roles in actin stress fiber formation (19
, 20)
, membrane ruffling (21)
, and cell movement (19
, 22)
. The precise sequence of events by which these proteins are regulated by accessory-binding proteins and by specific signaling pathways is currently under active study (23)
. There is some recent evidence that kinases functioning downstream of PKC, namely tyrosine protein kinases (24)
, phosphatidylinositol 3'-kinase (25)
, and MAP kinase (26)
, participate in the regulation of the Rho family, although a direct role of PKC has never been addressed.
In the present work, we examine the effect of PKC overexpression on the proliferation, adhesion, and motility of human breast MCF-10A cells. Our findings reveal a profound influence of PKC on these phenomena, especially with regard to cell proliferation, actin structure, and the steady-state levels of specific proteins involved in adhesion and cell-cell contacts. The far-reaching pleiotropic effects governed by PKC overexpression in these cells underscore the importance of this enzyme as a therapeutic target in human breast cells.
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Results |
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Transfectants of MCF-10A Cells., MCF-10A cells were transfected with a plasmid encoding wild-type PKC or a control plasmid. Clones expressing high levels of PKC were identified by Western blot analysis. Relative to parental cells or cells transfected with the control plasmid 8C, transfectants were identified that expressed either an intermediate level of PKC expression (clone 17) or higher expression levels of PKC (clones 11 and 26), as shown in Fig. 1A
. Quantitative measurements using two-dimensional scanning densitometry recorded PKC expression levels for clones 11 and 26 as 8- and 9-fold higher, respectively, than that of the control clone 8C. Subcellular fractionation of clone 26 showed that, in contrast to control cells, distribution of PKC was equivalent between the soluble and particulate fractions and represented an enrichment of PKC in both cellular compartments. Although PKC expression was doubled in the soluble fraction of clone 26, the particulate fraction contained a 5-fold elevation of PKC protein relative to the particulate fractions of control cells (either 8C or parental cells; Fig 1B
). An assay of PKC activity was carried out in vitro with samples that had been partially purified by DEAE-Sephacel chromatography (Fig. 1C)
. The 5-fold higher in vitro PKC catalytic activity measured for clones 11 and 26 as well as the intermediate level of activity measured for 17 (Fig. 1C)
correlated well with their relative abundance (Fig. 1A)
.
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in clones 26 and 8C and parental MCF-10A cells (ß1, 
, , 
, 
, and 
) revealed no significant compensatory changes in the expression levels of these isoforms. PKCß1 levels were only slightly detectable in 26, 8C, and parental cells, a finding that contrasts with the increased levels observed for PKCß1 as a consequence of PKC overexpression in MCF-7 cells (5
, 6)
.
Rates of proliferation of PKC transfectants were compared with those of the parental MCF-10A cells and transfection control 8C cells (Fig. 2A
; Table 1
). An analysis revealed that high-level expression of PKC (clone 26) lengthened the doubling time by almost 3-fold, whereas an intermediate level of expression (clone 17) produced an intermediate effect on doubling time. It was noted that a slightly lower proliferative rate and saturation density were observed for 8C cells relative to parental cells. Control experiments indicated that this effect was likely due to the antibiotic G418 (125 µg/ml) present in the growth medium of transfectant cells.
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cells. An examination of Table 1
and Fig. 2B
indicates that the increased doubling time of 26 cells corresponds to an accumulation of cells in G1 and can be attributed to an increase in the time required for the G1 transition. Inhibition of cell cycle progression through G1 by a specific PKC isoform has been demonstrated previously only with ectopically expressed PKC- (27)
. Expression levels of p21 and p27, two cell cycle-related proteins that inhibit passage through G1 (28
, 29)
, were analyzed by Western blot. Densitometry of the resulting signals demonstrated that p27 expression was increased by 33-fold in cells expressing very high levels of PKC (clones 11 and 26), as shown in Fig. 2
C. In contrast, p21 was present in very low but equivalent abundance in control, 17, 11, and 26 cells (data not shown).
Microscopic examination of PKC transfectants revealed radical alterations in cellular morphology. In contrast to the parental and control transfectants, both of which consisted of small, rounded cells that were tightly aggregated, PKC transfectant cells (clones 11 or 26) were large, nonaggregated, irregularly shaped cells with prominent lamellipodia and filopodia (Fig. 3)
. Immunostaining of 26 cells with an anti-PKC antibody (Fig. 4A)
highlighted the lamellipodia and membrane ruffling of these cells and revealed that PKC was located in the perinuclear region of the cytoplasm and the plasma membrane. However, parental (10A) and vector control cells (8C) were similar in that the PKC signal was perinuclear but was more diffusely distributed throughout the cell. Staining clone 26 with rhodamine-phalloidin revealed a profoundly different actin structure in the PKC transfectants (Fig. 4B)
, which consisted of an extensive and well-defined organization of actin stress fibers, in contrast with the diffuse staining pattern observed in parental cells (10A) and control transfectant cells (clone 8C).
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Transfectants. expression in transfectants of MCF-10A cells (Fig. 1A)
was proportionate with the extent of motile behavior. As shown in Fig. 5
, both 11 and 26 cells displayed a >40-fold higher motility than control transfectants, whereas clone 17 was intermediate in this activity (Fig. 5A)
. Adhesion was elevated only in cells expressing the highest levels of PKC (clones 11 and 26; Fig. 5B
).
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catalytic activity in motility and adhesion was tested with tools that are known to modulate PKC catalytic activity. When tested for 1 h with 1 µM TPA, a potent PKC activator, migration of 26 cells was not further stimulated, whereas the motility of 8C cells was stimulated by 5-fold (Fig. 6A)
, suggesting that in 26 cells, PKC was being activated endogenously. Evidence that this was the case was also given by the antagonistic effects on migration and adhesion by two different PKC inhibitors, calphostin C or Go6976, each of which recognizes a different functional domain of PKC. Calphostin C, a fluorescent light-activated inhibitor of PKC with nanomolar specificity (IC50 = 75 nM in vitro), is targeted to the phorbol ester binding site of the PKC regulatory domain (30)
. Because inhibition by calphostin C was previously shown to require the presence of phosphatidylserine/Ca2+, it may inhibit only PKC that is localized specifically in the membrane compartment (30)
. In contrast, the indolocarbazole Go6976 recognizes a target site in the PKC catalytic domain and represents the most potent inhibitor available for inhibition of PKC activity (IC50 = 2.3 nM in vitro; Ref. 31
). Treatment of 26 cells with either inhibitor at 1 µM and a subsequent assay of migration (Fig. 6B)
and adhesion (Fig. 6C)
showed that each inhibitor, especially calphostin C, significantly diminished these activities. When activated by fluorescent light, 1 µM calphostin C reduced the motility of 26 cells by >90% while decreasing the adhesion to collagen IV by 60%. Although less effective, 1 µM Go6976 nonetheless produced reductions of motility and adhesion by 40% and 30%, respectively.
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overexpression causes this functional phenotype could be related both to its catalytic activity and to an ability to up-regulate proteins associated with cell movement. In this regard, Western blot analysis of cell lysates obtained from parental cells and clones 8C and 26 revealed altered expression levels of specific adhesion proteins, as shown in Fig. 7
. In particular, adhesion proteins that are associated with focal adhesion plaques (paxillin, integrin ß1, FAK) were significantly elevated in clone 26 relative to control cells. Adhesion proteins for which no changes in expression were observed in clone 26- included -, ß-, and 
-catenin and dynamin (data not shown). A striking characteristic of clone 26 was the absence of detectable E-cadherin, a protein known to participate in cell-cell contacts and to suppress cell motility (32)
. Down regulation of E-cadherin in 26 cells was verified by both Western blot (Fig. 7)
and confocal microscopy (Fig. 4C)
; in contrast, both parental (10A) and control transfectants (8C) expressed abundant levels of E-cadherin. The absence of E-cadherin in PKC-overproducing cells is consistent with the loss of cell aggregation (Fig. 3)
and the dramatic enhancement of cell motility (Fig. 5A
; Ref. 32
). A similar effect by overexpression of a constitutively active form of PKC was recently found to occur in intestinal cells (33)
.
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-generated Phenotype. on cell motility was addressed. Because PKC overexpression induced the formation of lamellipodia and membrane ruffling (Figs. 3
and 4A)
, the participation of specific GTP-binding proteins (G proteins) in the PKC-generated phenotype was considered. Rac1, RhoA, and cdc42 were of interest due to their known involvement in cell movement (21
, 22)
. Western blot analysis of 10A, 8C, 17, 11, and 26 cells established that PKC overexpression did not significantly alter the expression levels of Rac1, RhoA, or cdc42 (data not shown).
To test whether the small G proteins play a functional role in the PKC-induced phenotype, a dn mutant of each G protein (or a control plasmid) was transfected individually into 26 cells. The mutant constructs used for these studies were used previously with E1A-ras cotransformed primary epithelial cells (34)
. After transient transfection with the plasmids and a 48-h incubation period, the motility of 26 cells was tested. It was observed that dn Rac1 consistently impaired the motility of 26 cells by 40%, but neither dn cdc42 nor dn RhoA had any effect (Fig. 8A)
. Compared with the plasmid control neo, transient transfection of each of the three dn mutant G proteins suppressed the formation of stress fibers induced by PKC overexpression (Fig. 8B)
. These findings specifically implicate endogenous Rac1 as a participant in the PKC-induced motility of 26 cells, whereas all three G proteins contribute to stress fiber formation in these cells.
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Discussion |
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overexpression in MCF-10A cells on growth properties and activities ascribed to the metastatic phenotype. The demonstration that a diminished proliferative rate occurs in parallel with dramatically increased motility underscores the critical role of PKC in redirecting cellular resources to drive metastatic behavior. Our findings with clonal populations that express low, intermediate, or high levels of PKC indicate that several aspects of the phenotype, namely proliferative rate, motility, and morphology, all correlate with the level of PKC expression. Recent observations by others (33)
revealed that overexpression of a constitutively active mutant of PKC in intestinal cells also caused reduced proliferation coincident with increased cell invasion. Our findings with human breast cells suggest certain possible mechanisms by which PKC orchestrates these pronounced pleiotropic effects: (a) modulation of the cell cycle, as evidenced by the appearance of larger-sized cells, the accumulation of cells in G1, and the up-regulation of p27 protein; (b) alteration of steady-state levels of focal adhesion proteins (FAK, paxillin, ß1 integrin) and E-cadherin; (c) engagement of the small G protein Rac1; and (d) the immediate action communicated by PKC catalytic activity, as demonstrated by the decrease in motility by PKC inhibitors (calphostin C or Go6976).
Limited information has previously linked PKC with the regulation of the cell cycle. A recent study demonstrated a role for ectopically expressed PKC- in NIH3T3 cells that consequently accumulated in the G1 phase of the cell cycle. The mechanism was proposed to involve elevated expression of cyclin E and cyclin-dependent kinase (inhibitors p21Waf1 and p27Kip1 (27)
. Accordingly, PKC-- induced elevation of these cyclin-dependent kinase inhibitors could suppress cyclin E-associated protein kinase activity, thereby stalling the cell cycle in G1 (28)
. In this regard, a recent study reported that p27Kip1 overexpression in human epithelial breast cells (MCF-10F) correlated with a longer G1 phase and an increased doubling time (35)
, which is consistent with our observations of elevated p27 levels in PKC-overproducing MCF-10A cells. The present study, however, is the first evidence that links PKC specifically with elevated expression of p27 and cell cycle progression.
These studies also provide the first indication that a small G protein participates in PKC-directed signaling. In testing the motility of PKC-overproducing cells transfected with dn forms of Rac1, RhoA, or cdc42, we observed that only dn Rac1 suppressed motility (by 40%), thereby implicating a contribution by wild-type Rac1 in the PKC-generated phenotype. The observation that all three dn mutants of the G proteins eliminated stress formation (Fig. 8B)
suggests that the three wild-type proteins have a common role in promoting stress fiber formation in 26 cells. Taken together, only Rac1 activity in PKC-expressing cells correlates with both enhanced stress fiber formation and motility in these cells.
PKC is a known component of growth factor-activated pathways, which include the MAP kinase cascade and pathways involving Ras p21 GTP-binding proteins. Previous studies with Rat1 fibroblasts demonstrated that transformation by oncogenic Ras absolutely requires the participation of Rac and that this discrete pathway acts in parallel with MAP kinase pathways to produce the oncogenic phenotype (36)
. Similarly, in MCF-10A cells transformed by overexpression of the oncogenic Ha-v-ras-encoded protein (Ha-Ras), others have observed a decrease in cell motility after cotransfection of dn analogues of Rac1 and RhoA (11
, 37)
, implying that Ha-Ras activates these small G proteins during transformation. In our studies with MCF-10A cells (which were not transformed with Ras), the fact that overexpression of PKC also caused increased motility that could be inhibited somewhat by a dn form of Rac1 implied that PKC also activates endogenous Rac1. Because other studies have shown that Ras p21 activates PKC (38
, 39)
, it is likely that PKC functions downstream of Ras p21 in MCF-10A cells. Overall, our findings support the idea that overproduction of PKC in MCF-10A cells disposes it to behave as an activated component of the Ras signaling pathway, leading to effects on Rac1 activity, cell motility, and a more highly organized actin structure. The endogenous activation of PKC in 26 cells is discussed below.
An important question prompted by our results with this engineered MCF-10A cell system is whether the overproduced PKC acts as an activated protein kinase or as a binding protein present at a high dosage. Previous studies of cellular PKC used TPA, a potent tumor promoter and specific PKC activator, to achieve increased adhesion and motility. For example, with cultured metastatic human breast cells (MDA-MB-435), activation of PKC by short-term TPA treatment was required for increased adhesion to type IV collagen, an event that was blocked by calphostin C (40)
. In other studies, short-term TPA treatment of MCF-7 cells (41)
or transfection of a constitutively active PKC mutant into intestinal HT-29 cells (33)
was necessary to observe a dramatic increase in invasiveness and motility. However, our studies with PKC transfectants of MCF-10A cells did not require TPA to observe these phenotypic effects, suggesting that PKC is being activated endogenously by some unknown mechanism. The reason for TPA independence does not lie with the PKC protein itself because another study using the same wild-type PKC-encoding plasmid for transfection of NIH3T3 fibroblasts showed that TPA was also required to observe the phenotypic effects (2)
. Observations that support the idea of endogenous activation of PKC in 26 cells are as follows: (a) addition of TPA to 26 cells was not necessary to achieve the dramatic 40-fold increase in motility (Fig. 5)
. When 26 cells were treated with TPA, no further effect on cell motility occurred, whereas treatment of 8C cells with TPA stimulated cell motility 5-fold (Fig. 6A)
; (b) in the absence of TPA, we observed a 5-fold elevation of PKC in the particulate fraction of 26 cells (Fig. 1B)
, which is a characteristic of activated PKC; (c) when 26 cells were treated with the PKC inhibitors Go6976 or calphostin C, significant decreases in motility and adhesion were observed (Fig. 6, B and C)
; and (d) inhibition of proliferation (produced in the absence of TPA) by PKC overexpression in MCF-10A cells (Fig. 2)
contrasts with the requirement for short-term TPA treatment to achieve a similar effect in MCF-7 cells (6
, 42)
.
Whereas endogenously activated PKC may account for the observed phenotypic effects, it is possible that the expression level of PKC protein in 26 cells is sufficiently high to titrate an unknown endogenous PKC inhibitory factor or to saturate the binding sites of PKC-binding proteins (e.g., cytoskeletal scaffolding proteins and PKC substrate proteins) that are known to mediate changes in molecular architecture, adhesion, and motility (1
, 43) . Binding interactions of PKC with several such proteins are known to involve the PKC regulatory domain (44)
and/or catalytic domain (45)
. Therefore, it is plausible that the 8-fold higher level of PKC expression in 26 cells, irrespective of its catalytic activity, may be a critical determinant of the observed phenomena.
Overexpression of PKC and its phenotypic impact on human breast cells take on clinical significance in view of reports that malignant human breast tumors can express higher levels of PKC activity (3
, 4)
. In the present work, we demonstrated that heightened PKC expression correlates with drastically elevated cell motility, an aspect of the metastatic phenotype (46)
. A potential therapeutic application of a PKC-overproducing MCF-10A cell line resides in its use as a model for testing PKC-targeted modalities, thereby improving upon the limited predictive value of an in vitro assay. The foregoing experiments showed that Go6976, a potent and specific PKC inhibitor in vitro that is targeted to the ATP-binding site of Ca2+-dependent PKC isoforms (31)
, only partially decreased the functional phenotype of 26 cells, whereas calphostin C, whose action may be specifically restricted to the membrane compartment where it interacts with the PKC regulatory domain (30)
, was far more effective in reducing cell motility. In this regard, others have shown that calphostin C also proved effective in inhibiting PKC-regulated localization of cytoskeletal proteins and phosphorylation of FAK (47)
. A unique advantage of 26 cells is that they provide an appropriate model system for evaluating the ability of a drug to inhibit PKC in its native cellular compartment and to suppress the larger constellation of measurable events engendered by this isoform.
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Materials and Methods |
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, p21, and p27 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-RhoA and anti-cdc42 were purchased from Cytoskeleton, Inc. (Denver, CO), and all other antisera were from Transduction Laboratories (Lexington, KY). Rhodamine-phalloidin was obtained from Molecular Probes, Inc. (Eugene, OR). Go6976 and calphostin C were purchased from Calbiochem Novabiochem Corp. (San Diego, CA). Matrigel and collagen IV were purchased from Becton Dickinson (Bedford, MA) and Sigma (St. Louis, MO), respectively.
Cell Culture.
Mid-passage MCF-10A cells, human breast epithelial cells obtained from the Barbara Ann Karmanos Cancer Institute (passage 120), were cultured in DMEM:F12 media (1:1) supplemented with 15% equine serum, insulin (10 µg/ml), epidermal growth factor (20 ng/ml), cholera toxin (100 ng/ml), and hydrocortisone (0.5 µg/ml). Parental cells were maintained with penicillin (100 units/ ml), streptomycin (100 µg/ml), and fungizone (0.5 µg/ml). Cells were passaged at 1:3 to 1:6 every 3–4 days.
Cell Transfection.
The PKC- expression plasmid (pMTH-) and control plasmid (pMTH) were kindly provided by Dr. F. Mushinski (NIH, Bethesda, MD). The plasmid contains the mouse metallothionein promoter and neomycin resistance gene and has been used by others for stable transfection of PKC cDNA (2
, 48)
. MCF-10A cells were transfected with either pMTH or pMTH- using LipofectAMINE Plus (Life Technologies, Inc.). Forty-eight h after transfection was initiated, cells were replated in complete medium at a 1:5 ratio. Selection for neomycin resistance was started the following day by the addition of the antibiotic G418 (Sigma) to 400 µg/ml. After 10–14 days, G418-resistant clones were isolated and subsequently maintained in complete growth medium containing 125 µg/ml G418. Stable transfectants were cultured for up to 20 passages.
High expression mammalian plasmids conferring neomycin resistance and bearing dn mutant cDNA of Rac1, cdc42, or RhoA whose expression is driven by a long terminal repeat (LTR) promoter (34)
were transiently transfected into PKC-overproducing cells (clone 26). Twenty-four h after cells were seeded into T75 flasks (60–70% confluence), transfection was carried out in serum-free medium in the absence of antibiotics. Plasmid cDNA plus Lipofectamine reagent (5 µg) was added to each flask with gentle mixing, and the flasks were incubated at 37°C in a 5% CO2 atmosphere for 3 h. Serum was added to a final concentration of 15%, and the cells were incubated for an additional 8 h, followed by replacement of the transfection medium with fresh, complete medium containing G418 (125 µg/ml). Cells were incubated in complete medium for 48 h, after which an analysis of cell migration in vitro was carried out (see below).
Analysis of PKC Expression by Western Blot.
For analysis of PKC expression cells were harvested and lysed in lysis buffer [50 mM Tris (pH 7.4), 5 mM EDTA, 5 mM EGTA, 5 mM 2-mercaptoethanol, and 0.1% Triton X-100] containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor, 10 µg/ml calpain inhibitor II, and 10 µl/ml aprotinin), and the protein content was determined (Bio-Rad protein reagent) using BSA as a standard. Fractionation of cell lysates was conducted using a method described previously (49)
. Samples that had been normalized for protein content were applied to the gel (5 µg/lane) and subjected to electrophoresis in 9% SDS-PAGE using a Bio-Rad mini-gel apparatus. After electrophoresis, transfer to a nitrocellulose membrane (Pharmacia) was carried out by an established method (50)
. Immunochemical analysis for PKC was conducted with a rabbit anti-PKC and horseradish peroxidase-conjugated secondary antibody. PKC was detected by chemiluminescence (Amersham), and signals were quantified by two-dimensional scanning densitometry (Molecular Dynamics).
Assay of PKC Catalytic Activity.
Lysates of each clone were partially purified by column chromatography on DEAE-Sephacel using an established procedure (51)
. Ca2+/phospholipid/diacylglycerol-dependent protein kinase activity was measured in column eluates by the transfer of 32P from [-32P]ATP to a peptide substrate (RFARKGSLRQKNV) modeled on the pseudosubstrate sequence of PKC, as described previously (49)
.
Adhesion Assay.
Twenty-four-well tissue culture plates were coated with 200 µl of either collagen IV (2 µg/cm2) or Matrigel (43.5 µg/cm2). Plates were incubated at 37°C in 5% CO2 for 1 h, followed by washing with PBS. Immediately before use, the coated wells were overlaid with 1% BSA for 30 min, washed five times with PBS, and dried for 30 min at room temperature in the tissue culture hood. Cells were applied to individual wells at 1.5 x 105 cells/well and incubated for 40 min at 37°C in 5% CO2. Nonadherent cells were removed by aspiration and three additional washes with PBS. Adherent cells were counted visually using a Nikon Diaphot-TMD inverted microscope at a magnification of x400. In each well, cells were counted in eight randomly chosen fields and numerically averaged. Each experimental group was assayed in a minimum of three wells. Statistical significance was determined using Student’s paired t test.
Motility Assay.
A 12-mm Costar transwell with a 12-µm pore size (Fisher Scientific) was used to measure cell movement across a porous polycarbonate membrane. The bottom surface was coated with 35 µg of Matrigel (Becton Dickinson Labware) for 1 h at 37°C in 5% CO2. Cells were then seeded into the upper chamber of the transwell at 1.5 x 105 cells/well and incubated at 37°C in 5% CO2. After a 15-h incubation, the upper chamber was carefully wiped with a cotton swab to remove cells that remained on the upper membrane surface. Those cells that had migrated to the lower membrane surface were fixed and stained by the H&E method (Fisher Diagnostics Leukostat). Eight fields of adherent cells visualized with a Nikon Diaphot-TMD inverted microscope at x400 magnification, were randomly counted in each well and numerically averaged. Each condition was carried out in triplicate transwells. Statistical significance was determined using Student’s paired t test.
Cell Treatment with TPA or PKC Inhibitors.
Twenty-four h before treatment, cells were seeded into 10-cm dishes. For each experiment, cells were washed twice with PBS and incubated in serum-free medium containing the indicated PKC inhibitor under the following conditions. Treatment with either 1 µM TPA or 1 µM Go6976 was carried out for 1 h at 37°C in 5% CO2. Cells treated with 1 µM calphostin C were immediately exposed to fluorescent light in the tissue culture hood for 15 min, followed by incubation in the dark at 37°C in 5% CO2 for 30 min. After each treatment, cells were washed twice with PBS and resuspended to 3 x 105 cells in serum-free medium for the subsequent assay of adhesion or migration.
Fluorescence Microscopy.
Parental and transfectant cells were seeded onto coverslips 24 h before the addition of the fluorophore. On the day of the experiment, cells were washed three times with PBS and treated for 10 min with freshly prepared fixative [4% formaldehyde, 0.2% Triton X-100, 60 mM PIPES, 25 mM HEPES, 10 mM EGTA, and 3 mM MgCl2 (pH 6.1)]. The coverslips were washed with PBS and dried at room temperature. Coverslips were incubated with rhodamine-phalloidin (1.4 units/ml PBS) for 30 min at room temperature, followed by three washes with PBS. Gel mounting medium (Biomeda Corp., Foster City, CA) was applied to the coverslips and allowed to dry overnight, and cells were examined for fluorescence. For detection of either PKC or E-cadherin, coverslips bearing the fixed cells were blocked with 1% BSA for 30 min and treated with anti-E-cadherin (6 µg/ml) for 45 min or anti-PKC (3 µg/ml) for 4 h at room temperature, followed by washing with PBS, a 30-min treatment with the appropriate FITC-conjugated secondary antibody (20 µg/ml), and the application of mounting solution. Images were visualized using a Meridian Ultima confocal microscope equipped with an argon ion laser and 530/30 and 580/30 band pass filters.
Flow Cytometric Analysis.
Ethidium bromide-stained nuclei were isolated from exponentially growing parental and transfectant cells as described previously (52)
. Each nuclei preparation was filtered through nylon mesh (40–80-µm pore size) and analyzed for DNA content on a FACScan flow cytometer (Becton Dickinson). The percentage of cells in different phases of the cell cycle was determined using Multicycle 2.5 software (Phoenix Flow Systems).
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Acknowledgments |
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-encoding plasmid. We also thank Dr. Crislyn D’Souza-Schorey (University of Notre Dame, Notre Dame, IN) and Dr. Harish Radhakrishna (NIH) for suggesting the involvement of Rac1 in the phenotype of 26 cells. Fluorescence-activated cell-sorting analysis was carried out by Thomas Delohery (Flow Cytometry Core Facility, Memorial Sloan-Kettering Cancer Center). Jeannette M. Schaefer, of the Cellular Imaging Analysis Core of Queens College, provided expert technical assistance in operating the confocal microscope. S. A. R. thanks Dr. Corinne Michels for stimulating discussions. |
Footnotes |
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1 Supported by grants (to S. A. R.) from the Gustavus and Louise Pfeiffer Research Foundation and NIH Grant CA 60618. 
2 To whom requests for reprints should be addressed, at Department of Chemistry and Biochemistry, Queens College, City University of New York, 65-30 Kissena Boulevard, Flushing, NY 11367. Phone: (718) 997-4133; Fax: (718) 997-5531; E-mail: susan_rotenberg{at}qc.edu
3 The abbreviations used are: PKC, protein kinase C; TPA, 12-O-tetradecanoylphorbol-13-acetate; dn, dominant negative; MAP, mitogen-activated kinase; FAK, focal adhesion kinase; MARCKS, myristoylated alanine-rich C-kinase substrate.
Received for publication 10/20/98. Revision received 2/ 5/99. Accepted for publication 3/15/99.
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References |
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  T. P. Abeyweera, X. Chen, and S. A. Rotenberg Phosphorylation of {alpha}6-Tubulin by Protein Kinase C{alpha} Activates Motility of Human Breast Cells J. Biol. Chem., June 26, 2009; 284(26): 17648 - 17656. [Abstract] [Full Text] [PDF]
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 J. M. Atienza, N. Yu, X. Wang, X. Xu, and Y. Abassi Label-Free and Real-Time Cell-Based Kinase Assay for Screening Selective and Potent Receptor Tyrosine Kinase Inhibitors Using Microelectronic Sensor Array J Biomol Screen, September 1, 2006; 11(6): 634 - 643. [Abstract] [PDF]
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 W. G. Tharp, R. Yadav, D. Irimia, A. Upadhyaya, A. Samadani, O. Hurtado, S-Y. Liu, S. Munisamy, D. M. Brainard, M. J. Mahon, et al. Neutrophil chemorepulsion in defined interleukin-8 gradients in vitro and in vivo J. Leukoc. Biol., March 1, 2006; 79(3): 539 - 554. [Abstract] [Full Text] [PDF]
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 P. Chaudhuri, S. M. Colles, P. L. Fox, and L. M. Graham Protein Kinase C{delta}-Dependent Phosphorylation of Syndecan-4 Regulates Cell Migration Circ. Res., September 30, 2005; 97(7): 674 - 681. [Abstract] [Full Text] [PDF]
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 T. Hara, Y. Saito, T. Hirai, K. Nakamura, K. Nakao, M. Katsuki, and K. Chida Deficiency of Protein Kinase C{alpha} in Mice Results in Impairment of Epidermal Hyperplasia and Enhancement of Tumor Formation in Two-Stage Skin Carcinogenesis Cancer Res., August 15, 2005; 65(16): 7356 - 7362. [Abstract] [Full Text] [PDF]
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M. Zhan, D. Yu, J. Liu, J. Hannay, and R. E. Pollock Transcriptional Repression of Protein Kinase C{alpha} via Sp1 by Wild Type p53 Is Involved in Inhibition of Multidrug Resistance 1 P-Glycoprotein Phosphorylation J. Biol. Chem., February 11, 2005; 280(6): 4825 - 4833. [Abstract] [Full Text] [PDF]
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 J. Martinez, O. Vogler, J. Casas, F. Barcelo, R. Alemany, J. Prades, T. Nagy, C. Baamonde, P. G. Kasprzyk, S. Teres, et al. Membrane Structure Modulation, Protein Kinase C{alpha} Activation, and Anticancer Activity of Minerval Mol. Pharmacol., February 1, 2005; 67(2): 531 - 540. [Abstract] [Full Text] [PDF]
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I. T. Makagiansar, S. Williams, K. Dahlin-Huppe, J.-i. Fukushi, T. Mustelin, and W. B. Stallcup Phosphorylation of NG2 Proteoglycan by Protein Kinase C-{alpha} Regulates Polarized Membrane Distribution and Cell Motility J. Biol. Chem., December 31, 2004; 279(53): 55262 - 55270. [Abstract] [Full Text] [PDF]
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S. Tsushima, M. Kai, K. Yamada, S.-i. Imai, K. Houkin, H. Kanoh, and F. Sakane Diacylglycerol Kinase {gamma} Serves as an Upstream Suppressor of Rac1 and Lamellipodium Formation J. Biol. Chem., July 2, 2004; 279(27): 28603 - 28613. [Abstract] [Full Text] [PDF]
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 C. Cataisson, E. Joseloff, R. Murillas, A. Wang, C. Atwell, S. Torgerson, M. Gerdes, J. Subleski, J.-L. Gao, P. M. Murphy, et al. Activation of Cutaneous Protein Kinase C{alpha} Induces Keratinocyte Apoptosis and Intraepidermal Inflammation by Independent Signaling Pathways J. Immunol., September 1, 2003; 171(5): 2703 - 2713. [Abstract] [Full Text] [PDF]
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 T. Yu, I. Morita, K. Shimokado, T. Iwai, and M. Yoshida Amlodipine Modulates THP-1 Cell Adhesion to Vascular Endothelium via Inhibition of Protein Kinase C Signal Transduction Hypertension, September 1, 2003; 42(3): 329 - 334. [Abstract] [Full Text] [PDF]
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K. D. Kikawa, D. R. Vidale, R. L. Van Etten, and M. S. Kinch Regulation of the EphA2 Kinase by the Low Molecular Weight Tyrosine Phosphatase Induces Transformation J. Biol. Chem., October 11, 2002; 277(42): 39274 - 39279. [Abstract] [Full Text] [PDF]
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X. Zeng, H. Xu, and R. I. Glazer Transformation of Mammary Epithelial Cells by 3-Phosphoinositide-dependent Protein Kinase-1 (PDK1) Is Associated with the Induction of Protein Kinase C{alpha} Cancer Res., June 1, 2002; 62(12): 3538 - 3543. [Abstract] [Full Text] [PDF]
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 A. B. da Rocha, D.R.A. Mans, A. Regner, and G. Schwartsmann Targeting Protein Kinase C: New Therapeutic Opportunities Against High-Grade Malignant Gliomas? Oncologist, February 1, 2002; 7(1): 17 - 33. [Abstract] [Full Text] [PDF]
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 G. Stapleton, A. Malliri, and B. W. Ozanne Downregulated AP-1 activity is associated with inhibition of Protein-Kinase-C-dependent CD44 and ezrin localisation and upregulation of PKC theta in A431 cells J. Cell Sci., January 7, 2002; 115(13): 2713 - 2724. [Abstract] [Full Text] [PDF]
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 I. Eude, E. Dallot, F. Ferre, and M. Breuiller-Fouche Protein Kinase C{alpha} Is Required for Endothelin-1-Induced Proliferation of Human Myometrial Cells Biol Reprod, January 1, 2002; 66(1): 44 - 49. [Abstract] [Full Text]
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 N. D. Zantek, J. Walker-Daniels, J. Stewart, R. K. Hansen, D. Robinson, H. Miao, B. Wang, H.-J. Kung, M. J. Bissell, and M. S. Kinch MCF-10A-NeoST: A New Cell System for Studying Cell-ECM and Cell-Cell Interactions in Breast Cancer Clin. Cancer Res., November 1, 2001; 7(11): 3640 - 3648. [Abstract] [Full Text] [PDF]
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R. M. Sullivan, M. Stone, J. F. Marshall, F. Uberall, and S. A. Rotenberg Photo-Induced Inactivation of Protein Kinase Calpha by Dequalinium Inhibits Motility of Murine Melanoma Cells Mol. Pharmacol., October 1, 2000; 58(4): 729 - 737. [Abstract] [Full Text]
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 B. Liu, S. A. Rotenberg, and M. V. Mirkin Scanning electrochemical microscopy of living cells: Different redox activities of nonmetastatic and metastatic human breast cells PNAS, August 29, 2000; 97(18): 9855 - 9860. [Abstract] [Full Text] [PDF]
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 J. C. Adams, J. D. Clelland, G. D.M. Collett, F. Matsumura, S. Yamashiro, and L. Zhang Cell-Matrix Adhesions Differentially Regulate Fascin Phosphorylation Mol. Biol. Cell, December 1, 1999; 10(12): 4177 - 4190. [Abstract] [Full Text]
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), transfectant control cells (8C; 
), and PKC
) and 26
), as described in "Materials and Methods." Each data point is the average of triplicate measurements that were within 10% error. The results are representative of three independent experiments. B, fluorescence-activated cell-sorting analysis of ethidium bromide-stained nuclei isolated from 10A, 8C, and 26
