Phospholipase C-δ1 Expression Is Linked to Proliferation, DNA Synthesis, and Cyclin E Levels
2008; Elsevier BV; Volume: 283; Issue: 20 Linguagem: Inglês
10.1074/jbc.m800752200
ISSN1083-351X
AutoresJonathan D. Stallings, Yue Zeng, Francisco Narvaez, Mario J. Rebecchi,
Tópico(s)Cell death mechanisms and regulation
ResumoWe previously reported that phospholipase C-δ1 (PLC-δ1) accumulates in the nucleus at the G1/S transition, which is largely dependent on its binding to phosphatidylinositol 4,5-bisphosphate (Stallings, J. D., Tall, E. G., Pentyala, S., and Rebecchi, M. J. (2005) J. Biol. Chem. 280, 22060-22069 ). Here, using small interfering RNA (siRNA) that specifically targets rat PLC-δ1, we investigated whether this enzyme plays a role in cell cycle control. Inhibiting expression of PLC-δ1 significantly decreased proliferation of rat C6 glioma cells and altered S phase progression. [3H]Thymidine labeling and fluorescence-activated cell sorting analysis indicated that the rates of G1/S transition and DNA synthesis were enhanced. On the other hand, knockdown cultures released from the G1/S boundary were slower to reach full G2/M DNA content, consistent with a delay in S phase. The levels of cyclin E, a key regulator of the G1/S transition and DNA synthesis, were elevated in asynchronous cultures as well as those blocked at the G1/S boundary. Epifluorescence imaging showed that transient expression of human phospholipase C-δ1, resistant to these siRNA, suppressed expression of cyclin E at the G1/S boundary despite treatment of cultures with rat-specific siRNA. Although whole cell levels of phosphatidylinositol 4,5-bisphosphate were unchanged, suppression of PLC-δ1 led to a significant rise in the nuclear levels of this phospholipid at the G1/S boundary. These results support a role for PLC-δ1 and nuclear phospholipid metabolism in regulating cell cycle progression. We previously reported that phospholipase C-δ1 (PLC-δ1) accumulates in the nucleus at the G1/S transition, which is largely dependent on its binding to phosphatidylinositol 4,5-bisphosphate (Stallings, J. D., Tall, E. G., Pentyala, S., and Rebecchi, M. J. (2005) J. Biol. Chem. 280, 22060-22069 ). Here, using small interfering RNA (siRNA) that specifically targets rat PLC-δ1, we investigated whether this enzyme plays a role in cell cycle control. Inhibiting expression of PLC-δ1 significantly decreased proliferation of rat C6 glioma cells and altered S phase progression. [3H]Thymidine labeling and fluorescence-activated cell sorting analysis indicated that the rates of G1/S transition and DNA synthesis were enhanced. On the other hand, knockdown cultures released from the G1/S boundary were slower to reach full G2/M DNA content, consistent with a delay in S phase. The levels of cyclin E, a key regulator of the G1/S transition and DNA synthesis, were elevated in asynchronous cultures as well as those blocked at the G1/S boundary. Epifluorescence imaging showed that transient expression of human phospholipase C-δ1, resistant to these siRNA, suppressed expression of cyclin E at the G1/S boundary despite treatment of cultures with rat-specific siRNA. Although whole cell levels of phosphatidylinositol 4,5-bisphosphate were unchanged, suppression of PLC-δ1 led to a significant rise in the nuclear levels of this phospholipid at the G1/S boundary. These results support a role for PLC-δ1 and nuclear phospholipid metabolism in regulating cell cycle progression. Phosphoinositides are metabolized by a multifaceted and highly regulated set of phosphoinositide-specific enzymes (2Katan M. Biochim. Biophys. Acta. 1998; 1436: 5-17Crossref PubMed Scopus (191) Google Scholar, 3Rebecchi M.J. Pentyala S.N. Physiol. Rev. 2000; 80: 1291-1335Crossref PubMed Scopus (817) Google Scholar, 4Rhee S.G. Annu. Rev. Biochem. 2001; 70: 281-312Crossref PubMed Scopus (1209) Google Scholar). Kinases sequentially phosphorylate the inositol head-group of phosphatidylinositol, and phosphatases reverse this process (5Downes C.P. Ross S. Maccario H. Perera N. Davidson L. Leslie N.R. Adv. Enzyme. 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Biochem. 1995; 64: 315-343Crossref PubMed Scopus (310) Google Scholar); both can generate phosphatidylinositol 4,5-bisphosphate (PIP2), 2The abbreviations used are: PIP2 or PI(4,5) P2, phosphatidylinositol 4,5-bisphosphate; XTT, sodium 3′-[1-[(phenylamine)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate; PLC, phospholipase C; PBS, phosphate-buffered saline; siRNA, small interfering RNA; EGFP, enhanced green fluorescent protein; TBS, Tris-buffered saline; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; FACS, fluorescence-activated cell sorting; PIP, phosphatidylinositol phosphate. 2The abbreviations used are: PIP2 or PI(4,5) P2, phosphatidylinositol 4,5-bisphosphate; XTT, sodium 3′-[1-[(phenylamine)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate; PLC, phospholipase C; PBS, phosphate-buffered saline; siRNA, small interfering RNA; EGFP, enhanced green fluorescent protein; TBS, Tris-buffered saline; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; FACS, fluorescence-activated cell sorting; PIP, phosphatidylinositol phosphate. the principle substrate of phospholipase C (PLC) (2Katan M. 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Although PLCδ1 is not essential, homozygous deletion in mice results in aberrant expression of terminal differentiation markers in several types of skin cells as well as the development of alopecia and spontaneous skin tumors (21Nakamura Y. Fukami K. Yu H. Takenaka K. Kataoka Y. Shirakata Y. Nishikawa S. Hashimoto K. Yoshida N. Takenawa T. EMBO J. 2003; 22: 2981-2991Crossref PubMed Scopus (87) Google Scholar). These effects appear to result from increased expression of proinflammatory cytokines (22Nakamura Y. Ichinohe M. Hirata M. Matsuura H. Fujiwara T. Igarashi T. Nakahara M. Yamaguchi H. Yasugi S. Takenawa T. Fukami K. FASEB J. 2008; 22: 841-849Crossref PubMed Scopus (49) Google Scholar). Mice that lack both PLCδ1 and PLCδ3, however, die between embryonic days 11.5 and 13.5 due to abnormal cellular proliferation and apoptosis in placental trophoblasts (23Nakamura Y. Hamada Y. Fujiwara T. Enomoto H. Hiroe T. Tanaka S. Nose M. Nakahara M. Yoshida N. Takenawa T. Fukami K. Mol. Cell. Biol. 2005; 25: 10979-10988Crossref PubMed Scopus (55) Google Scholar).Saccharomyces cerevisiae that lack PLC1-1, a homolog to mammalian PLCδ1, missegregate chromosomes (24Payne W.E. Fitzgerald-Hayes M. Mol. Cell. Biol. 1993; 13: 4351-4364Crossref PubMed Scopus (79) Google Scholar) and exhibit osmotic and temperature sensitivity and defects in metabolism and growth (25Flick J.S. Thorner J. Mol. Cell. Biol. 1993; 13: 5861-5876Crossref PubMed Scopus (168) Google Scholar, 26Yoko-o T. Matsui Y. Yagisawa H. Nojima H. Uno I. Toh-e A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1804-1808Crossref PubMed Scopus (136) Google Scholar). The extents to which these phenotypes are displayed depend on the genetic background of each yeast strain, suggesting that plc1-1 modulates complex multigene processes having significant redundancies. Transformation of these yeast mutants with rat PLCδ1 can rescue growth defects (26Yoko-o T. Matsui Y. Yagisawa H. Nojima H. Uno I. Toh-e A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1804-1808Crossref PubMed Scopus (136) Google Scholar), consistent with a high degree of functional conservation. Furthermore, overexpression of cyclin-dependent kinase inhibitors, SPL1 or SPL2, rescues these same defects (27Flick J.S. Thorner J. Genetics. 1998; 148: 33-47Crossref PubMed Google Scholar), suggesting that PLC1 is somehow linked to cell cycle regulation.Yagisawa et al. (28Yagisawa H. Sakuma K. Paterson H.F. Cheung R. Allen V. Hirata H. Watanabe Y. Hirata M. Williams R.L. Katan M. J. Biol. Chem. 1998; 273: 417-424Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) first reported that PLCδ1 harbored both nuclear export and import sequences that contribute to its shuttling between the cytoplasm and nucleus. We have demonstrated that PLCδ1 accumulates in the nucleus at the G1/S boundary in NIH-3T3 fibroblasts and C6 glioma (1Stallings J.D. Tall E.G. Pentyala S. Rebecchi M.J. J. Biol. Chem. 2005; 280: 22060-22069Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), and many of these observations have been confirmed (29Yagisawa H. J. Cell Biochem. 2006; 97: 233-243Crossref PubMed Scopus (27) Google Scholar). Here we set out to determine whether this protein plays a role in the cell cycle. We find that suppression of PLCδ1 increases cyclin E levels, alters S phase progression, and inhibits cell proliferation.EXPERIMENTAL PROCEDURESSynchrony, Flow Cytometry, and Cell Cycle Analysis—Rat C6 glioma cells (American Type Culture Collection) were maintained in RPMI 1640 (Invitrogen) supplemented with 7.5% fetal bovine serum and 1 mm penicillin and streptomycin (all supplements from Invitrogen) at 37 °C in a 5% CO2 humidified incubator. To synchronize cells to the G1/S boundary, adherent glioma cells were twice blocked with 2 mm thymidine (1Stallings J.D. Tall E.G. Pentyala S. Rebecchi M.J. J. Biol. Chem. 2005; 280: 22060-22069Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Briefly, cultures were washed with RPMI 1640 and incubated in growth medium supplemented with 2 mm thymidine for 12 h, washed three times with growth medium, and incubated for an additional 10 h without thymidine. Cultures were again blocked with 2 mm thymidine for an additional 12 h. To release them from G1/S block, cells were washed three times in RPMI 1640 and fed normal growth medium. To analyze DNA content, cells were harvested by treatment with trypsin/EDTA, washed with phosphate-buffered saline containing 1.0 mm calcium chloride and 2.0 mm magnesium chloride (PBS-CaMg), and fixed in 70% ethanol in phosphate-buffered saline (PBS) with 0.1% fetal bovine serum at 4 °C overnight (1Stallings J.D. Tall E.G. Pentyala S. Rebecchi M.J. J. Biol. Chem. 2005; 280: 22060-22069Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). To stain DNA, ethanolfixed glia were incubated in PBS with 50 mm citrate buffer, 50 μg/ml propidium iodide, and 50 μg/ml RNase A for 30 min at 37 °C (1Stallings J.D. Tall E.G. Pentyala S. Rebecchi M.J. J. Biol. Chem. 2005; 280: 22060-22069Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). After washing with PBS, cell cycle analysis was immediately performed using a fluorescence-activated cell sorter (FACScan; BD Biosciences). Histogram data were analyzed using the program Cychlred and the Origin 7.5 (OriginLab Corp.) peak fit module.siRNA, Expression Plasmids, and Cell Transfection—We targeted the mRNA sequence 151-172 bp (5′-ggA CCC Cag gCC gCU Cgg TT-3′) of rat PLCδ1 and designed and synthesized a corresponding duplexed siRNA (Proligo) based on previously described protocols (30Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Nature. 2001; 411: 494-498Crossref PubMed Scopus (8075) Google Scholar, 31Tuschl T. Hannon G. J RNAi: A Guide to Gene Silencing. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2003: 280-288Google Scholar). For these experiments, C6 glioma cells were plated on plastic tissue culture dishes or #1.0 borosilicate chambered glass coverslips (Nalge Nunc International) coated with poly-l-lysine (Sigma). Cells were transfected with PLCδ1-specific siRNA (δ1-siRNA) or a commercially available nonspecific control C-siRNA (Ambion), ranging from 0.01 to 320 nm, using FuGene6 (Roche Applied Science) or RNAiMax (Invitrogen) according to manufacturer's protocol. Three commercially available rat PLCδ1-specific siRNAs (Ambion numbers 200652, 49731, and 49541; designated here as siRNA1, siRNA2, and siRNA3, respectively) were also tested for their capacity to knock down expression, and the resulting phenotypes were also assessed. In some experiments, siRNA was delivered with human PLCδ1 fused to enhanced green fluorescence protein (EGFP), at ∼0.28 μg DNA/cm2, in the same FuGene6 transfection. These expression vectors have been previously described (1Stallings J.D. Tall E.G. Pentyala S. Rebecchi M.J. J. Biol. Chem. 2005; 280: 22060-22069Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar).siRNA transfection efficiency using the Fugene6 or RNAiMax reagents was found to be nearly 100% as assessed using a fluorescently labeled, short double-stranded RNA (Block-It, AlexaFluor Red; Invitrogen). On the other hand, plasmid transfection efficiency using either Fugene6 or FugeneHD (Roche Applied Science) rarely exceeded 25% whether using plasmids encoding the PLC-δ1 EGFP fusion protein or EGFP itself.Cell Proliferation and Viability Assays—To estimate the rate of growth in the presence or absence of δ1-siRNA, C6 glioma were plated at densities of 1 × 103 to 1.5 × 105 cells/2-cm2 well and allowed to grow for several days; cell numbers were determined every 24 h with a hemocytometer, and growth rate constants were estimated with the following equation, GR = (ln(nf/ni)/t), where ni = initial cell number, nf = final cell number, and t = time (h). Trypan blue (Invitrogen) was used to determine whether cell membrane integrity was compromised as a result of siRNA treatment. Viability was also assessed using XTT (Biological Industries), which is metabolized to the colored formazin product in the mitochondria (32Scudiero D.A. Shoemaker R.H. Paull K.D. Monks A. Tierney S. Nofziger T.H. Currens M.J. Seniff D. Boyd M.R. Cancer Res. 1988; 48: 4827-4833PubMed Google Scholar). During these procedures, C6 glioma cells were cultured in an equivalent medium lacking phenol red. XTT (1 mg/ml) was prepared in serum-free medium containing phenazine methylsulfate (1.53 mg/ml), which stimulates mitochondrial metabolism of XTT (32Scudiero D.A. Shoemaker R.H. Paull K.D. Monks A. Tierney S. Nofziger T.H. Currens M.J. Seniff D. Boyd M.R. Cancer Res. 1988; 48: 4827-4833PubMed Google Scholar). 100 μl of XTT/phenazine methylsulfate reagent was transferred to each well containing 400 μl of medium and incubated for 1 h. The conditioned medium from each well was then collected, and the absorbance was measured at 475 nm.SDS-PAGE and Western Blotting—Each 35-mm dish of monolayer cells was washed twice, with 2 ml each of warm PBS-CaMg. Soluble cellular proteins were then extracted with 0.5 ml of ice-cold extraction buffer (200 mm NaCl, 0.2% Nonidet P-40, 20 mm Tris, pH 8, 1 mm dithiothreitol, 1 mm MgCl2, 1 mm EGTA, and 1% mammalian anti-protease mixture (Sigma)) and incubated for 5 min at 4 °C. The cells were gently scraped up with a rubber policeman and transferred to 1.7-ml Eppendorf tubes. Using this method, the nuclei remained intact, and little of the cellular DNA was extruded. These samples were then subjected to centrifugation at 1000 × g for 4 min at 4 °C, and the supernatant fluids were transferred to new tubes. A portion of each sample was removed for determination of protein concentration. Protein concentration was determined via Bradford assay (Bio-Rad) according to the manufacturer's protocol. An equal volume of acetone at -20 °C was then added to the remaining samples, which were then incubated at -20 °C for at least 30 min. Following centrifugation at 12,000 × g for 5 min, the pellets were washed once with -20 °C acetone/water (1:1, v/v) and dried under vacuum. The dried samples were dissolved in SDS sample buffer to a concentration of 1-2 μg of protein/μl. Samples containing equal concentrations of total protein were separated in an 8% or 10% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membrane (Bio-Rad) using a Trans Blot semidry transfer apparatus (Bio-Rad) at 14 V for 1.5 h. The blot was then blocked with Tris-buffered saline (TBS) containing 5% nonfat dry milk and 0.1% Tween 20 for 60 min at room temperature. The membrane was incubated in solution containing anti-PLCδ1 S-11-2 monoclonal antibody (Upstate Biotechnology, Inc.) or anti-PLCδ1 polyclonal (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-cyclin E polyclonal, anti-cyclin A monoclonal, or anti-β-tubulin polyclonal antibody (Invitrogen). The membrane was incubated with secondary antibody solution containing either goat anti-mouse or anti-rabbit IgG (H + L)-horseradish peroxidase conjugate (Bio-Rad). Enhanced chemiluminescence (ECL Plus; Amersham Biosciences/GE HealthCare) was used to detect the binding of the secondary antibody following the manufacturer's protocol. The membranes were imaged using a CCD camera (Eastman Kodak Co.).[3H]Thymidine Incorporation Assay—C6 glioma cells were transfected with either 160 nm control C-siRNA or PLCδ1-specific siRNA and grown in 24-well plates. 48-72 h post-transfection, cultures were washed with PBS-CaMg and incubated in 0.5 ml of RPMI 1640 (7.5% fetal bovine serum, 1% phosphatidylserine) containing 1 μCi/ml [3H]thymidine for 2.5 h (a measure of the number of cells synthesizing nascent DNA, (33Nebigil C.G. Biochemistry. 1997; 36: 15949-15958Crossref PubMed Scopus (31) Google Scholar)). Alternatively, cultures were transfected and synchronized to the G1/S boundary as described above and then labeled with [3H]thymidine following their release from G1/S block (rate of thymidine incorporation). In each experiment, a portion of each culture was used to determine cell number. Incorporated [3H]thymidine was precipitated with 500 μl of 10% trichloroacetic acid on ice for 20 min. The precipitate was washed twice with 500 μl of each of 10% trichloroacetic acid. Finally, pellets were dissolved with 200 μl of 0.1 n NaOH for 15 min and transferred to a vial containing 4 ml of scintillation fluid and counted in a liquid scintillation spectrometer.Epifluorescence Microscopy and Indirect Immunofluorescence—Cell monolayers were rinsed once in PBS-CaMg and then fixed with freshly prepared 3.7% (w/v) formaldehyde solution (Fisher) in PBS for 10 min at room temperature. Samples were then washed three times in TBS for 5 min and permeabilized with 0.5% Nonidet P-40 (Sigma) in TBS for 5 min at room temperature. The detergent solution was replaced with blocking solution (TBS containing 5% goat serum (Pierce)) for 30 min at room temperature and then replaced with primary antibody solution (1:200; rabbit anti-cyclin E in TBS with 1% goat serum) overnight at 4 °C. Samples were washed three times in 1 ml of TBS for 7 min each and then incubated at 37 °C for 1 h in goat anti-rabbit IgG (H + L) conjugate Texas Red (Molecular Probes) diluted 1:3000 in TBS containing 1% goat serum. Each well was then washed three times in TBS for 7 min each. Indirect immunofluorescence of PLCδ1 was performed as previously described (1Stallings J.D. Tall E.G. Pentyala S. Rebecchi M.J. J. Biol. Chem. 2005; 280: 22060-22069Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). In some experiments, cells incubated with 4′,6-diamidino-2-phenylindole (5 μg/ml) for 5 min to assess the percentage of nuclei that appeared apoptotic. Images were captured with an AxioCam 330mA 12-bit CCD camera (Zeiss) and viewed with Carl Zeiss Axovision 3.1 software. Alternatively, fixed cells were visualized by epifluorescence microscopy (Olympus IMT-2 inverted microscope with a 100-watt mercury arc lamp), and images were taken with a Nikon Plan Fluor ×40 oil objective (numerical aperture 1.3) and Olympix AstroCam (LSR). These images were processed and analyzed with Esprit imaging software (LSR). To assess the fraction of cells (scored positive or negative) having nicked DNA as a result of siRNA treatments, an in situ TUNEL Assay Cell Death Detection kit (Roche Applied Science) was used according to the manufacturer's directions.Reverse Transcription-PCR—Total RNA was extracted from siRNA-treated C6 cultures using an RNeasy extraction kit (Qiagen) as per the manufacturer's instructions. Following purification, 0.1-1 μg of total RNA was first heated to 70 °C in the presence of random hexanucleotide primers. The RNA was then transferred to Ready-to-Go reverse transcription-PCR beads (GE Healthcare) and incubated at 42 °C for 30 min. Following the reverse transcription step, cyclin E primers (5′-GTGAAAAGCGAGGATAGCAG-3′;5′-TGTTGTGATGCCATGTAACG-3′) or glyceraldehyde-3-phosphate dehydrogenase primers were added, and the reactions were cDNA-amplified (18-26 cycles) in a Gene AMP PCR System 2000 thermocycler (PerkinElmer Life Sciences) with each cycle programmed for 95 °C melting for 0.5 min, 55 °C annealing for 0.5 min, and 72 °C extension for 1 min. The reaction products were separated on a 2% agarose gel that was subsequently stained with SYBR Green I (Molecular Probes, Inc., Eugene, OR). Images were recorded using a Kodak Gel Imager system, and the fluorescent bands were quantified using the Kodak gel analysis software. The cyclin E mRNA levels were normalized to expression of the glyceraldehyde-3-phosphate dehydrogenase amplicon in each sample.Subcellular Fractionation and Lipid Analysis—Nuclei were purified as previously described (1Stallings J.D. Tall E.G. Pentyala S. Rebecchi M.J. J. Biol. Chem. 2005; 280: 22060-22069Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Following siRNA treatment for 24 h, cultures were synchronized to the G1/S boundary and labeled with 10 μCi/ml [3H]myoinositol for at least 24 h. In some cases, cultures were released from G1/S block for 3 h prior to lipid extraction. Labeled cultures were rinsed with ice-cold PBS-CaMg and then treated briefly with PBS containing 1 mm EDTA to release them from the plastic dishes. The released cells were then subjected to centrifugation at 600 × g for 5 min at 4 °C. Cells were promptly resuspended in 500 μl of prechilled hypotonic resuspension buffer (RSB; 10 mm NaCl, 1.5 mm MgCl2, 10 mm Tris-HCl, pH 7.4) on ice for 7 min. Swollen cells were then transferred to a Dounce homogenizer and lysed by 20 strokes of the glass pestle. Nuclei and debris were then layered onto a sucrose cushion (320 mm sucrose, 7.7 mm MgCl2, 2.1 mm EGTA, and 0.1 mm phenylmethylsulfonyl fluoride) and centrifuged at 300 × g for 3 min at 4 °C. The pellet was washed twice with 0.5 ml of RSB and resuspended in 750 μl of methanol, 0.1 m HCl (v/v, 1:1), placed in silicanized borosilicate glass tubes, and mixed vigorously for 30 s. The lipids were subsequently extracted as previously described (1Stallings J.D. Tall E.G. Pentyala S. Rebecchi M.J. J. Biol. Chem. 2005; 280: 22060-22069Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Lipid extracts were applied to prescored Linear KD silica plates (Whatman) that had been pretreated with 40% methanol, 1% potassium oxalate, and 1 mm EGTA in water and heat-activated. The solvent system used was chloroform/methanol/water/concentrated ammonium hydroxide (v/v/v/v; 60:47:11.3:2) (1Stallings J.D. Tall E.G. Pentyala S. Rebecchi M.J. J. Biol. Chem. 2005; 280: 22060-22069Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Areas corresponding to migration of phosphoinositide, phosphatidylinositol 4-phosphate, and PI(4,5) P2 standards were scraped into vials containing 100 μl of a mixture of methanol and 10% Nonidet P-40 in water (v/v, 1:1). 4 ml of scintillation fluid (EcoLite) was added to each vial and mixed, and the vials were counted in a liquid scintillation spectrometer. Counts/min values were normalized to total lipid phase-extractable phosphorus.Statistical Analysis—All statistical analyses were preformed in GraphPad Prism. To determine the significance of the differences between mean values, one-way analysis of variance with Newman-Keul's post-test or Student's t test was used where appropriate (***, p < 0.001; **, p <0.01; *, p < 0.05). Fisher's exact test was used to analyze the frequency data obtained from indirect immunofluorescence images.RESULTSsiRNA-mediated Knockdown of PLCδ1 in Rat C6 Glioma Inhibits Proliferation—RNA interference is a sequence-specific post-transcriptional gene silencing mechanism that suppresses synthesis of a specific protein by degrading the mRNA encoding a target protein (30Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Nature. 2001; 411: 494-498Crossref PubMed Scopus (8075) Google Scholar, 31Tuschl T. Hannon G. J RNAi: A Guide to Gene Silencing. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2003: 280-288Google Scholar, 34Huppi K. Martin S.E. Caplen N.J. Mol. Cell. 2005; 17: 1-10Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). We identified a candidate site within the rat PLCδ1 mRNA coding sequence (residues 152-172) and designed a specific RNA duplex (δ1-siRNA). In addition, we purchased three unique siRNA duplexes predicted to suppress expression of rat PLCδ1.Treatment of C6 glioma cultures with δ1-siRNA reduced the levels of PLCδ1 by >80% by 72 h compared with control siRNA or untreated cultures (Fig. 1A). This was associated with reduced cell numbers. Under our transfection conditions, the significant changes in the apparent growth rates (Fig. 1B, inset) were well correlated with reduced expression of PLCδ1 (see Fig. 1A (δ1-siRNA1), and see supplemental Fig. 1A). Commercially available siRNA1 and siRNA3, greatly reduced expression of rat PLCδ1, whereas siRNA2 was less effective (Fig. 1C). Suppression of cell proliferation mirrored their effects on PLCδ1 levels (Fig. 1D; see supplemental Fig. 1A), supporting the idea that slower growth is a specific effect of PLCδ1 suppression. As the knockdown experiments progressed, it became evident that siRNA3 rapidly reduced PLCδ1 levels and profoundly and rapidly reduced the fraction of cells synthesizing DNA by 48 h following treatment. In order to provide a sufficient time window for measuring the relevant cell cycle variables, we compared δ1-siRNA and siRNA1 in further studies, since these siRNAs took longer to suppress PLC-δ1 expression.To address the possibility that the apparent decrease in proliferation was due to an increased rate of cell death, we investigated the cytotoxicity of the various siRNA and whether treatment with these reagents induced apoptosis. Trypan blue staining revealed no significant differences between control and δ1-siRNA treatments up to 72 h post-transfection (Fig. 2A). The ability of treated C6 cell mitochondria to metabolize XTT, another indicator of cell viability
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