Phospholipase C δ-4 overexpression upregulates ErbB1/2 expression, Erk signaling pathway, and proliferation in MCF-7 cells
© Leung et al; licensee BioMed Central Ltd. 2004
Received: 31 December 2003
Accepted: 13 May 2004
Published: 13 May 2004
The expression of the rodent phosphoinositide-specific phospholipase C δ-4 (PLCδ4) has been found to be elevated upon mitogenic stimulation and expression analysis have linked the upregulation of PLCδ4 expression with rapid proliferation in certain rat transformed cell lines. The human homologue of PLCδ4 has not been extensively characterized. Accordingly, we investigate the effects of overexpression of human PLCδ4 on cell signaling and proliferation in this study.
The cDNA for human PLCδ4 has been isolated and expressed ectopically in breast cancer MCF-7 cells. Overexpression of PLCδ4 selectively activates protein kinase C-φ and upregulates the expression of epidermal growth factor receptors EGFR/erbB1 and HER2/erbB2, leading to constitutive activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathway in MCF-7 cells. MCF-7 cells stably expressing PLCδ4 demonstrates several phenotypes of transformation, such as rapid proliferation in low serum, formation of colonies in soft agar, and capacity to form densely packed spheroids in low-attachment plates. The growth signaling responses induced by PLCδ4 are not reversible by siRNA.
Overexpression or dysregulated expression of PLCδ4 may initiate oncogenesis in certain tissues through upregulation of ErbB expression and activation of ERK pathway. Since the growth responses induced by PLCδ4 are not reversible, PLCδ4 itself is not a suitable drug target, but enzymes in pathways activated by PLCδ4 are potential therapeutic targets for oncogenic intervention.
Phosphoinositide-specific phospholipase C (PI-PLC) plays a role in the inositol phospholipid signaling by hydrolyzing phosphatidylinositol-4,5-bisphosphate (PIP2). This reaction produces two intracellular second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), which cause the increase of intracellular calcium concentration and the activation of protein kinase C (PKC), respectively [1–3]. In addition to hydrolyzing PIP2, PI-PLC can also utilize phosphatidylinositol (PI) or PI-4-phosphate as substrates.
The PLC family in murine or human species is comprised of 11 subtypes. On the basis of their structure, they have been divided into four classes, β (β1, 2, 3 and 4), γ (γ1 and 2), δ (δ1, 3 and 4), ε, and ζ types. Positive regulation mechanisms of PLC by association with membrane receptors are well characterized in β- and γ-type isozymes . β-type isozymes are activated by the Gα or Gβγ subunit released from heterotrimeric Gαβγ proteins after ligand stimulation. γ-type isozymes are activated by the phosphorylation of specific tyrosine residues through the activation of receptor or nonreceptor tyrosine kinases. ε-type enzymes possess both PLC and ras dependent guanine nucleotide exchange (RasGEF) activity. As such, PLCε may mediate the effects of G protein-coupled receptors through two divergent pathways involving PI hydrolysis as well as direct activation of the Ras/MAP kinase pathway [4–6]. PLCζ, a sperm protein that shows similarity to a truncated PLCδ with the pleckstrin homology (PH) domain deleted, is involved in the triggering of Ca++ oscillations in eggs ; however, it remains to be documented whether PLCζ does have PLC enzymatic activity.
The regulation of δ-type PLC activity is less understood; however, certain isoforms of δ-type PLC such as δ1 can be regulated through interaction with a dual function G-protein (Gh) that also has transglutaminase activity [8, 9]. Another δ-type PLC isozyme known as PLCδ4 has been implicated to have a key role in cell proliferation, as its mRNA is expressed in higher levels in regenerating liver than in normal resting liver and in tumor cells such as hepatoma and src-transformed cells than in non-transformed cells. Western blot analysis and immunohistochemical staining showed that the murine PLCδ4 is predominantly present in nuclei with its expression level markedly induced by serum in serum-starved murine cells, whereas the amounts of PLCβ1, PLCγ1, and PLCδ1 do not change significantly after serum stimulation . The rat PLCδ4 level has also been found to be markedly elevated in a fast proliferating hepatoma H3924A cell line comparing to a slow growing hepatoma H7795 line; however, immunohistochemical staining and western analysis of subcellular fractions show rat PLCδ4 is mainly expressed in the cytoplasmic fraction . These results suggest that PLCδ4 is expressed in response to mitogenic stimulation and plays important roles in cell growth and tumorigenesis. Splice variants of rat PLCδ4 with enzymatic PLC activities or with dominant-negative activity [12, 13], and the promoter region of murine PLCδ4  have also been described. Gene knockout by homologous recombination shows PLCδ4-/- sperms are not able to initiate the acrosome reaction required for egg fertilization .
Despite the extensive characterization of the murine or rat PLCδ4 enzyme, the effect of overexpression of the human form of PLCδ4 in cells has not been characterized. This paper reports the molecular cloning of human PLCδ4 and examines the signaling pathways that are affected by ectopic expression of PLCδ4 in human breast cancer MCF-7 cells.
Human PLCδ4 expression in normal and tumor tissues
Generation of human PLCδ4 overexpressing MCF-7 cells
Since the murine PLCδ4 has been found to be expressed predominantly in nuclei , nuclear fraction from a PLCδ4 overexpressing clone (Sample#2, Figure 3, panel B) was prepared to examine the distribution of PLC activity in nuclear vs whole cell extracts. The nuclear extract was found to contain about 1/3 of the total PLC activity when compared to whole cell extract, suggesting that, unlike murine PLCδ4  but similar to rat PLCδ4 , human PLCδ4 is mainly found in cytoplasmic fractions. The structure of the PH domain is the major factor that determines the subcellular localization of PLC [18, 19]. Consistent with cytoplasmic location based on cell fractionation data, fusion protein with green fluorescent protein (GFP) linked to the PH domain of PLCδ4 overexpressed in cells are associated with cytoplasmic membrane structures (Figure 3, panel C). GFP fusion constructs for PH domain of PLCβ1 have also been found in cytoplasmic membranes , whereas that of PLCδ1 have been found mainly in plasma membranes . Including conservative amino acid changes, the matches between the PH domains of PLCδ1 and PLCδ4 exceed 80%, suggesting subtle changes in the PH domain can affect protein localization.
PLCδ4 selectively activates protein kinase C-φ in MCF-7 cells
Constitutive activation of extracellular signal-regulated kinase (ERK) pathway in PLCδ4 overexpressing cells
Upregulation of EGFR/ErbB1 and HER2/ErbB2 in PLCδ4 overexpressing cells
EGFR can also form heterodimers with other members of erbB receptors such as the ligand-less erbB2 to promote tumor progression . ErbB2/HER2 is overexpressed in 20%–30% of breast and ovarian cancer patients and is associated with aggressive tumor characteristics and poor prognosis. Analysis of erbB2 total protein level with anti-erbB2 showed erbB2 protein levels were also elevated in all four independent isolated lines of PLCδ4 overexpressing cells, whereas little ErbB2 was detected in all five independent isolated lines of vector control cells and in untransfected MCF-7 cells.
PLCδ4 overexpression enhances MCF-7 cells proliferation in low serum, colonies formation in soft agar, and spheroid density on hydrogel
The induction of growth signaling response is not reversible by siRNA specific for PLCδ4
The expression of the rodent phosphoinositide-specific phospholipase C δ-4 (PLCδ4) has been found to be elevated upon serum stimulation in Swiss 3T3 cells and Northern blot analysis have linked the upregulation of PLCδ4 expression with rapid proliferation in hepatocytes and with oncogenic transformation in certain rodent cell lines , suggesting that PLCδ4 may be an enzyme target for oncogenic intervention. Accordingly, we have isolated the cDNA for human PLCδ4 to probe the expression of PLCδ4 using northern blot and cDNA expression panel derived from normal tissues (Figure 1) and Cancer Profiling Arrays (BD Clontech, Palo Alto, CA) derived from genetically matched cDNA pairs isolated from various tumor and normal tissue samples (Figure 2). The truncated splice variant of PLCδ4, PLCδ4b, was found to expressed in the same tissues where full length PLCδ4 could be detected (Figure 1, lower right panel), consistent with the recent discovery of widespread use of a mechanism that coupled alternative splicing with a premature stop codon for the downregulation of a transcript through nonsense-mediated mRNA decay . A somewhat analogous splice variant with premature stop codon has been isolated for rat PLCδ4 with dominant-negative activity ; however, coexpression of human PLCδ4 along with PLCδ4b has not led to significant reduction of PLC activity in MCF-7 cells in our hands (data not shown) possibly due to subtle differences between the human and the rat sequences. Though PLCδ4 expression was found to be downregulated in the majority of tumor tissues, it was found to be upregulated in a high percentage (>25%) of breast and testicular tumor tissues. The overexpression of PLCδ4 in testicular tumor is of interest, as the only observed phenotype in PLCδ4-/- mice is having defective sperms , suggesting PLCδ4 plays an important role in testis and that deregulation of PLCδ4 expression in testis can lead to oncogenesis. With the exceptions of a few universal tumor antigens such as telomerase, survivin, mdm2, cytochrome P450 1B1  and sphingosine kinase 1  that are overexpressed in most tumor tissues, the differential overexpression of PLCδ4 in selected tumor tissues is consistent with the expression pattern of most individual oncogenes, where overexpression is only found in restricted tissues as opposed to all tumor tissues, despite the fact that various tumor tissues do share a common cluster of overexpressed genes related to cell cycling and DNA replication . Hence experiments in addition to expression profiling are required to determine whether PLCδ4 is a gene with oncogenic potential.
We have studied the effects of overexpression of human PLCδ4 on cell signaling and proliferation in this study. MCF-7, a breast cancer cell line of low tumorigenicity, was chosen for ectopic expression of PLCδ4, since profiling of tissue arrays showed PLCδ4 can be overexpressed in breast tumor tissues (Figure 2). Constitutive overexpression of PLCδ4 selectively activates protein kinase C-φ (Figure 4, left panel), whereas the activation states of all other isoforms of PKC studied are not affected by PLCδ4 overexpression based on using phospho-specific PKC isoform-specific antibodies. The data is consistent with results from another phospho-specific antibody against the consensus motif of PKC substrates, showing slight enhancement of phosphorylation of PKC substrates with PLCδ4 overexpression (Figure 4, right panel).
Using antibodies that target signaling pathways for cell proliferation, we have found that PLCδ4 overexpression leads to constitutive activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathway in MCF-7 cells (Figure 5) and of kinases both upstream and downstream of the Erk signaling pathway such as Erk kinases (MEK1/2) and the 90 kDa ribosomal S6 kinase (p90RSK), whereas key proteins involved in two alternative MAPK pathways and the PI3K pathway, such as SAPK/JNK, p38 MAPK, and Akt, respectively, showed no change in activation in PLCδ4 overexpressing cells. PLCδ4 overexpression also upregulates the expression of epidermal growth factor receptors EGFR/erbB1 and HER2/erbB2 in MCF-7 cells (Figure 6). Of the four ErbB isoforms, cells co-expressing ErbB1 and ErbB2 have been found to be the most aggressive in forming tumors in mice . Though upregulation of EGFR expression is often associated with the downregulation of estrogen receptor (ERα) expression in breast cancer cells [20, 21], no significant change was found in ERα expression in PLCδ4 overexpressing MCF-7 cells (Figure 6). Since both EGFR transcription [21, 30] and ErbB2 expression  have been found to be upregulated by phorbol ester, it is possible that the DAG generated from PLCδ4 overexpression may activate selective PKCs such as PKC-φ, leading to enhancement of EGFR and ErbB2 gene transcription and activation of the Erk signaling pathway.
We have examined several cell growth parameters to determine whether enhancement of ErbB1/2 expression and activation of Erk pathway in PLCδ4 overexpressing MCF-7 cells can be translated into a more proliferative state of cell growth. MCF-7 cells stably expressing PLCδ4 demonstrate several phenotypes of transformation; namely, enhanced rate of proliferation and saturating cell density in low serum (Figure 7), improved efficiency in forming colonies in soft agar (Figure 8), and capacity to form more densely packed spheroids in low-attachment plates (Figure 9). These data suggest that PLCδ4 overexpression may contribute to a transformation phenotype by promoting both anchorage dependent and independent cell growth.
A criterion for validation and selection of new drug targets is to determine whether there is evidence of the reversal of the transformation phenotype using genetic means or drug leads. We have been able to knock down the expression of PLCδ4 in PLCδ4 overexpressing cells by transfecting siRNAs specific for PLCδ4 sequences (Figure 10); however, no reduction in ErbB1 expression level and Erk1/2 phosphorylation in PLCδ4 overexpressing cells in the presence of siRNA could be detected. The growth signaling responses induced by PLCδ4 are hence not reversible by siRNA. The inability for siRNAs for PLCδ4 to knock down growth signaling responses suggests PLCδ4 may play a role in the initiation of carcinogenesis by inducing other genetic changes, as a neoplastic state can sometimes be irreversible by becoming independent of the initiating oncogenic event through induction of genome destabilization. For instance, even transient induction of MYC activity was able to sustain tumorigenesis in certain cell types . Overexpression of ErbB2, a receptor induced by PLCδ4 (Figure 6), has also been associated with genetic instability in cells . These results suggest that PLCδ4 per se may be a difficult target to be manipulated with drugs, and the sites for intervention may lie instead on other enzyme targets in pathways activated by PLCδ4, such as the ErbB receptors and the Ras/Raf/MAP kinase cascade. The validation of some these targets has been confirmed with two approved drugs, Herceptin® specific for ErbB2 and Iressa® for EGFR tyrosine kinase, with numerous inhibitors for the ErbB receptor family in clinical trials , and with the studies of Raf inhibitor BAY 37-9751 and MEK inhibitor CI-1040 along the ERK pathway in clinical trials .
As activation of ErbB can activate another isoform of PLC, PLCγ , it is also possible that activation PLCγ may compensate for the knockdown of PLCδ4 by siRNA and that compounds capable of inhibiting multiple isoforms of PLC may be more suitable for oncogenic intervention. A black-box approach can also be used to screen for compounds by high throughput screening that would inhibit growth signaling responses induced by PLCδ4 with differential cytotoxcity in PLCδ4 overexpressing MCF-7 cells versus matched vector control cells. The use of human engineered tumor cells has been successful in identifying new as well as classical genotype-selective anti-tumor compounds .
Overexpression or dysregulated expression of PLCδ4 may initiate oncogenesis in certain tissues through upregulation of ErbB expression and activation of ERK pathway. PLCδ4 can therefore be a useful tumor marker for breast or testicular cancer tissues. Since the growth signaling responses induced by PLCδ4 are not reversible by siRNA, a surrogate for a PLCδ4-specific inhibitor, PLCδ4 itself is probably not a suitable drug target for development, but enzymes in pathways activated by PLCδ4 are potential therapeutic targets for oncogenic intervention. Alternatively, a black-box approach can be used to screen compounds with differential cytotoxcity in PLCδ4 overexpressing MCF-7 cells.
Isolation of human PLCδ4 cDNA
Search of the Genbank database  of expressed sequence tag (dbEST) using the rat PLCδ4 protein sequences  as probe identified an I.M.A.G.E. Consortium [LLNL] cDNA clone (GenBank# AI366170), pBSK-D4, isolated from a human oligodendroglioma library containing an insert of ~2500 bp with three separate stretches of sequences containing extensive homology to the rat PLCδ4 coding sequence. These three coding regions do not share a common reading frame and is most likely derived from a spurious splice variant of human PLCδ4 with exons deletion and intron insertion in two separate areas. This cDNA clone thus codes for a truncated PLCδ4 containing the first 258 amino acids of full-length PLCδ4 with another 16 amino acids and a termination codon added at the C-terminal end as a result of exons deletion.
To isolate a human PLCδ4 cDNA encoding an active PLC enzyme, PCR primers, oD4-2f, 5'-AGACACGTCC CAGTCTGGAA CC-3', and oD4-2r, 5'-CTGCTTCCTC TTCCTCATAT TC-3' (Qiagen, Alameda, CA) with sequences, respectively, corresponding to the coding strand upstream of a Eco RI site and to the complementary strand downstream of a unique Hpa I site were used to screen cDNAs derived from various human cell lines to identify PCR products that are longer (due to insertion of additional exons) than the product (expected to be ~300 bp) when using pBSK-D4 as the template. Templates from cDNAs derived from CA-HPV-10, Capan-1, Hs.7661, MiaPaCa, A549, MDA-MB-231, Panc-1, NCI-H460, LnCaP, MCF-7, and HNMEC cells all gave PCR products of ~800 bp upon amplification. The human normal mammary epithelial cells (HNMEC) was fron Clonetics (Walkersville, MD), whereas the rest of the cell lines were from American Type Culture Collection (Manassas, VA). The ~800 bp PCR product was then cleaved with Eco RI and Hpa I to generate a ~730 bp fragment for ligating into the vector pBSK-D4 that had been cleaved with Eco RI and Hpa I for the generation of the plasmid pBSK-D43X. DNA sequence analysis shows the cDNA insert in pBSK-D43X contains an additional ~500 bp region with extensive homology to the rat PLCδ4 sequence not found within the insert in pBSK-D4. pBSK-D43X lacked the 5'-end region of PLCδ4 cDNA upstream of Ecor RI, as the ~840 bp Eco RI fragment that spans the Eco RI site found in the 5'-cDNA adaptor sequence and the internal Eco RI site found in the PLCδ4 coding region was removed during preparation of the Eco RI/Hpa I pBSK-D4 vector.
Separately, PCR primers with sequences corresponding to the coding strand upstream of Hpa I of pBSK-D4, oD4-3f, 5'-CTGGTGAAGG GGAAGAAGTT A-3', and to the complementary strand downstream of Bgl II, oD4-3r, 5'-TGTCTAGACG AACGCCAAAG AT-3' were used to screen cDNAs derived from human cell lines to identify PCR products that are ~300 bp shorter (due to removal of a putative intron sequence) than the product (~1050 bp) when using pBSK-D4 as the template. A PCR product of ~700 bp was detected from cDNA template derived from CA-HPV-10 cells. The ~700 bp PCR product was then cleaved with Hpa I and Bgl II to generate a ~670 bp fragment for ligating into the vector pBSK-D43X that had been cleaved with Hpa I and Bgl II for the generation of the plasmid pBSK-D43X1. DNA sequence analysis shows the cDNA insert in pBSK-D43X contains extensive homology to the rat PLCδ4 sequence.
Construction of vectors for expression of human hPLCδ4 in mammalian cells
To assemble a full-length human PLCδ4a cDNA for expression study, a ~600 bp Nhe I - Eco RI fragment was isolated from digestion with Nhe I + Eco RI of the ~2200 bp PCR product generated by amplification from the template pBSK-D4 using the primers oD4-1f, 5'-GTGATCTGGT GCTAGCTGGT GGAAC-3' and oD4-1r, 5'-ACACCAATGC ATTCCCGTGA AATGCCCACC-3'; and a ~1700 bp Eco RI - Nsi I fragment isolated from digestion with Eco RI + Nsi I of the ~1770 bp PCR product generated by amplification from the template pBSK-D43X1 using the primers oD4-2f and oD4-1r were combined and inserted into the pCivIrPu vector that had been cleaved with Nhe I and Nsi I via a three-part ligation for the generation of the plasmid pPuroD4F. pCivIrPu is derived from the plasmids pIRESpuro (Clonetech, Palo Alto, CA) and pCIneo (Promega, Madison, WI). Specifically, the 980 bp Spe I - Not I fragment from pCIneo was inserted into the cloning vector pSL1180 (Amersham Pharmacia, Piscataway, NJ) between the restriction sites Spe I and Not I to generate pSLCI. The 1000 bp Spe I - Nsi I fragment from pSLCI was then isolated for insertion into the vector pIRESpuro between the restriction sites Spe I and Nsi I to generate pCivIrPu.
To determine whether the human hPLCδ4a cDNA sequence encodes a protein with phospholipase c (PLC) activity, the plasmid pPuroD4F was stably transfected into MCF-7 cells (American Type Culture Collection, Manassas, VA) to generate puromycin-resistant clones. Specifically, pPuroD4F was digested with BspH I before electroporating into these cell lines with a Cell-Porator™ (Life Technologies, Gaithersburg, MD) using conditions described previously . After adherence of the transfected cells 48 hours later, the cells were grown in the presence of 1.0 μg/ml puromycin to select for cells that had incorporated the plasmid.
Construction of vectors for expression of GFP-PH/PLCδ4 fusion proteins in mammalian cells
Coding region corresponding to the PH domain of PLCδ4 splice variant were amplified with PCR primers oPHd4_1f, 5'-GCGCAGCGAA TTCATGGCGT CCCTGCTGCA AGAC-3' and o oPHd4_1r, 5'-GATCTAGTGT CGACCCAAGA TCCACCAACA GCTGGAG-3' to generate a 400 bp fragment. The fragment was then cleaved with EcoR I and Sal I to generate a 390 bp fragment for insertion between the EcoR I and Sal I sites of pEGFP-n1 (Clontech, Palo Alto, CA) for generation of pPHd4GFP, an expression plasmid for fusion protein containing the PH domain of PLCδ4 upstream of GFP.
Cell culture reagents were from Invitrogen (Carlsbad, CA). Human tumor cell lines were from the American Type Culture Collection (Manassas, VA). MCF-7, MCF-7 cells stably overexpressing PLCδ4, or MCF-7 cells transfected with control vector were grown in RPMI supplemented with 10% FBS. Spheroids were grown on Costar® Ultra-Low Cluster Plates (Corning, Acton, MA) in RPMI supplemented with 10% FBS. Cell proliferation assays were performed using the CellTiter 96® AQueous One Solution (Promega, Madison, WI). For colonies grow in soft agar, cells in soft agar colony assay were cultured in 6-well plates containing 104 or 105 cells/well in RPMI 1640 with 0.35% agar, 5% FBS, 10% tryptose phosphate broth, 100 nM 17-β-estradiol. The plates were incubated for 10 days, after which the cultures were inspected and photographed.
Phospho-PKC (pan), phospho-PKCα/βII (Thr638/641), phospho-PKCφ (Thr538), phosphoPKCζ/λ (Thr410/403), phospho-MEK1/2 (Ser217/221), phospho-Erk1/2 (Thr202/183+Tyr204/185), total Erk1/2, phospho-p90RSK (Thr359+Ser363), phospho-SAPK p54/p46 (Thr183+Tyr185), phospho-p38 MAPK (Thr180+Tyr182), phospho-Akt (Ser473), total ER-α, phospho-ER-α (Ser167), total EGFR, phospho-EGFR (Tyr992), phospho-EGFR (Tyr1068), phospho-(Ser) PKC substrate and Lamin A/C antibodies were from Cell Signaling Technology (Beverly, MA). Hsp90 antibodies were from Biosource International (Camarillo, CA). HER-2 antibodies were from Upstate Cell Signaling Solutions (Charlottsville, VA). β-actin antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal antibodies raised in rabbits immunized with a synthetic peptide WEQQQTMARHLTEI corresponding to amino acids 390–402 of human PLCδ4 were obtained from Covance Research Products (Berkeley, CA). Western blot analyses were performed using the ECL Plus™ detection reagents from Amersham Biosciences (Piscataway, NJ).
Assay of phospholipase C activity in PLCδ4 overexpressing cells
Puromycin-resistant clones were randomly selected to check for PLC activity in cell extracts using a assay that measures the conversion of radiolabeled PIP2 or PI to DAG and IP3 or inositol-1-phosphate (Ins(1)P). To prepare cell extracts for assay, cells from plates were scraped into PBS + protease inhibitors. These cells were sonicated 3 times for 20 seconds using a probe sonicator. Protein concentrations were determined and the lysates were used for the assays. The assay of hydrolysis of PIP2 or PI was based on methods described previously [39, 40]. Specifically, each PI hydrolysis assay was in a total volumn of 200 μl composed of 250 μM PI (50 nmol), 20,000 cpm (0.030 μCi) of [3H]PI, 100 mM NaCl, 1 mM EGTA, 4 mM CaCl2, 0.1 mM DTT, 50 mM HEPES buffer, pH 7.0, 1 mg/ml sodium deoxycholate. A substrate solution enough for 50 assays was prepared by drying 5 ml of 500 μM PI + 1.5 μCi [3H]PI under N2 before resuspending in 5 ml of 2 mg/ml sodium deoxycholate by sonication. A buffer solution (10×) was made up of 250 mM HEPES pH 7.0, 1 M NaCl, 1 mM DTT. A Ca2+/EGTA solution (4×) was made up of 4 mM EGTA, 12 mM CaCl2, 100 mM HEPES pH 7.0 (free 3 mM Ca2+ in assay). After mixing 20 μl buffer solution, 50 μl Ca2+/EGTA, and 30 μl enzyme extract, the reaction was started by addition of 100 μl substrate solution. After incubating for 10 min at 37°C, the reaction was terminated with 0.75 ml chloroform/methanol/HCl (100:200:0.6, by volume), followed by 0.25 ml of chloroform (water saturated) and then 0.25 ml 1 N HCL containing 5 mM EGTA. After centrifugation to separate the phases, 300 μl of aqueous phase was then counted in scintillation counter.
For the assay of PLC activity using PIP2 as the substrate, each PI hydrolysis assay was in a total volumn of 200 μl composed of 40 μM PI (8 nmol), 200 μM PE (40 nmol), 20,000 cpm (0.030 μCi) of [3H]PIP2, 3 mM MgCl2, 100 mM NaCl, 1 mM EGTA, 1 mM CaCl2 (free 45 μM Ca2+ in assay), 0.1 mM DTT, 50 mM HEPES buffer, pH 7.0, 1 mg/ml sodium deoxycholate. A substrate solution enough for 50 assays was prepared by drying 80 μM PIP2 (5 ml) + 400 μM PE + 1.5 μCi [3H]PIP2 under N2 before resuspending in 5 ml of 65 mM HEPES pH 7.0, 100 mM NaCl, 2 mg/ml sodium deoxycholate by sonication. A MgCl2 solution was made up of 12 mM MgCl2, 50 mM HEPES pH 7.0, 200 mM NaCl, 0.4 mM DTT. A Ca2+/EGTA solution was made up of 10 mM EGTA, 10 mM CaCl2, 50 mM HEPES pH 7.0. After mixing 50 μl MgCl2 solution, 20 μl Ca2+/EGTA, and 30 μl enzyme extract, the reaction was started by addition of 100 μl substrate solution. After incubating for 10 min at 37°C, the reaction was terminated with 0.75 ml chloroform/methanol/HCl (100:200:0.6, by volume), followed by 0.25 ml of chloroform (water saturated) and then 0.25 ml 1 N HCL containing 5 mM EGTA. After centrifugation to separate phases, 300 μl of aqueous phase was then counted in scintillation counter.
Preparation of nuclear extracts
MCF-7 cells were harvested with 0.2% trypsin and washed two times with cold PBS. The cells were suspended in buffer A (10 mM Tris/HCl, pH 7.8), 1% Nonidet P-40, 10 mM β-mercaptoethanol, 0.5 mM PMSF, 1 μg/ml aprotinin and leupeptin) for 2 min on ice. An equal volume of distilled H2O was added, and the cells were allowed to swell for 2 min. The cells were sheared by ten passages through a 22-gauge needle. The nuclei were recovered by centrifugation at 400 × g for 6 min and washed once with buffer B (10 mM Tris/HCl (pH 7.4), 2 mM MgCl2, 0.5 mM PMSF, 1 μg/ml aprotinin and leupeptin). These nuclei were sonicated 3 times for 20 seconds at an output of 1 using a probe sonicator prior to PI hydrolysis assay.
Northern blot, PCR and tissue array analysis
Northern hybridization was carried out to detect the expression of PLCδ4 mRNA in different normal tissues. Specifically, nylon membranes containing mRNA isolated from various human tissues and tumor cell lines (Clontech, Palo Alto, CA) and cDNAs derived from various matched tumor and normal tissues on Cancer Profiling Array I and II (Clontech, Palo Alto, CA) were hybridized in ULTRAhyb™ (Ambion, Austin, TX) with PLCδ4 (2000 bp Nhe I-Bgl II fragment from pPuroD4F) coding region cDNA fragment labeled with [α-32P]dCTP using a Strip-Ez labeling kit (Ambion, Austin, TX). To evaluate the relative abundance of full length PLCδ4 and splice variant PLCδ4b, primers, oD4_2f, 5'-AGACACGTCC CAGTCTGGAA CC-3', and oD4_2r, 5'-CTGCTTCCTC TTCCTCATAT TC-3', located upstream and downstream of the coding region for exons 7, 8, and 9 were used to detect PCR products in a Rapid-Scan™ Gene Expression Panel (OriGene, Rockville, MD) generated from cDNA samples derived from various normal human tissues. The full length PLCδ4 would generate a 800 bp product; whereas the splice variant PLCδ4b with exons 7, 8, and 9 deleted, a 300 bp product.
Preparation of siRNAs and transfections
Several siRNAs targeting PLC-δ4 (accession number BC006355) were synthesized by Dharmacon Research (Lafayette, CO). siRNAs were annealed essentially as described . The sense strands for each siRNA duplex were as follows. PLC-δ4 specific siRNA #1 beginning at nt 1612, 5'-GAGCAGAACCTTCAGAATAdTdT-3', #2 beginning at nt 457, 5'-GAGCAGGGCTTCACCATTGdTdT-3', #3 beginning at nt, 765, 5'-GGAAGGAGAAGAATTCGTAdTdT-3', #4 beginning at nt 1752, 5'-GATATCATCTTTCTCTGAAdTdT-3' and "Smart Pool" siRNAs which combined the above PLC-δ4 siRNAs #1–4. The following siRNAs were obtained from Dharmacon Research (sense strands); Lamin A/C 5'-CUGGACUUCCAGAAGAACAdTdT-3' and Non-specific Control I 5'-NNATGAACGTGAATTGCTCAA-3'. Human MCF7-PLCd4c16 breast cancer cells were grown in RPMI1640, supplemented with 10% fetal bovine serum. Cells were seeded at 60,000 cells/well in a six-well dish. These seed density resulted in 15% confluency and was optimal for transfection efficiencies and growth by 72 hrs. Cells were transfected with 27 nM siRNA in serum free Opti-MEM I using Oligofectamine (Invitrogen, Carlsbad, California). Following 4 hr incubation with siRNAs, fetal bovine serum to 2% v/v final was added. Cells were harvested and extracts prepared for Western analysis 72 hr after transfection.
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