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• W2045707271 abstract "Nonmuscle myosin IIs play an essential role during cytokinesis. Here, we explore the function of an alternatively spliced isoform of nonmuscle myosin heavy chain (NMHC) II-C, called NMHC II-C1, in the A549 human lung tumor cell line during cytokinesis. NMHC II-C1 contains an insert of 8 amino acids in the head region of NMHC II-C. First, we show that there is a marked increase in both the mRNA encoding NMHC II-C1 and protein in tumor cell lines compared with nontumor cell lines derived from the same tissue. Quantification of the amount of myosin II isoforms in the A549 cells shows that the amounts of NMHC II-A and II-C1 protein are about equal and substantially greater than NMHC II-B. Using specific siRNAs to decrease NMHC II-C1 in cultured A549 cells resulted in a 5.5-fold decrease in the number of cells at 120 h, whereas decreasing NMHC II-A with siRNA does not affect cell proliferation. This decreased proliferation can be rescued by reintroducing NMHC II-C1 but not NMHC II-A or II-B into A549 cells, although noninserted NMHC II-C does rescue to a limited extent. Time lapse video microscopy revealed that loss of NMHC II-C1 leads to a delay in cytokinesis and prolongs it from 2 to 8-10 h. These findings are consistent with the localization of NMHC II-C1 to the intercellular bridge that attaches the two dividing cells during the late phases of cytokinesis. The results suggest a specific function for NMHC II-C1 in cytokinesis in the A549 tumor cell line. Nonmuscle myosin IIs play an essential role during cytokinesis. Here, we explore the function of an alternatively spliced isoform of nonmuscle myosin heavy chain (NMHC) II-C, called NMHC II-C1, in the A549 human lung tumor cell line during cytokinesis. NMHC II-C1 contains an insert of 8 amino acids in the head region of NMHC II-C. First, we show that there is a marked increase in both the mRNA encoding NMHC II-C1 and protein in tumor cell lines compared with nontumor cell lines derived from the same tissue. Quantification of the amount of myosin II isoforms in the A549 cells shows that the amounts of NMHC II-A and II-C1 protein are about equal and substantially greater than NMHC II-B. Using specific siRNAs to decrease NMHC II-C1 in cultured A549 cells resulted in a 5.5-fold decrease in the number of cells at 120 h, whereas decreasing NMHC II-A with siRNA does not affect cell proliferation. This decreased proliferation can be rescued by reintroducing NMHC II-C1 but not NMHC II-A or II-B into A549 cells, although noninserted NMHC II-C does rescue to a limited extent. Time lapse video microscopy revealed that loss of NMHC II-C1 leads to a delay in cytokinesis and prolongs it from 2 to 8-10 h. These findings are consistent with the localization of NMHC II-C1 to the intercellular bridge that attaches the two dividing cells during the late phases of cytokinesis. The results suggest a specific function for NMHC II-C1 in cytokinesis in the A549 tumor cell line. Nonmuscle myosin IIs belong to the conventional class II myosins, which form bipolar filaments at relatively low ionic strength and share a number of biological properties with skeletal, cardiac, and smooth muscle myosins (1Sellers J.R. Myosins. Oxford University Press, Oxford1999Google Scholar). Nonmuscle myosin IIs are expressed in both muscle and nonmuscle cells and are hexamers, consisting of a pair of heavy chains (200 kDa) and two pairs of light chains (20 and 17 kDa). They are one of the major motor proteins interacting with cytoskeletal actin and are involved in regulating cytokinesis, cell motility, and cell polarity in many eukaryotic cells (1Sellers J.R. Myosins. Oxford University Press, Oxford1999Google Scholar, 2Krendel M. Mooseker M.S. Physiology. 2005; 20: 239-251Crossref PubMed Scopus (285) Google Scholar). To date, three isoforms of nonmuscle myosin heavy chain (NMHC) 2The abbreviations used are: NMHC, nonmuscle myosin heavy chain; NMHC II-C0, noninserted isoform of NMHC II-C; NMHC II-C1, inserted isoform of NMHC II-C; siRNA, small interfering RNA; GFP, green fluorescent protein; DAPI, 4′,6′-diamidino-2-phenylindole; RT, reverse transcription. II, termed NMHC II-A, NMHC II-B, and NMHC II-C, have been identified in vertebrates (3Katsuragawa Y. Yanagisawa M. Inoue A. Masaki T. Eur. J. Biochem. 1989; 184: 611-616Crossref PubMed Scopus (115) Google Scholar, 4Shohet R.V. Conti M.A. Kawamoto S. Preston Y.A. Brill D.A. Adelstein R.S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7726-7730Crossref PubMed Scopus (102) Google Scholar, 5Takahashi M. Kawamoto S. Adelstein R.S. J. Biol. Chem. 1992; 267: 17864-17871Abstract Full Text PDF PubMed Google Scholar, 6Golomb E. Ma X. Jana S.S. Preston Y.A. Kawamoto S. Shoham N.G. Goldin E. Conti M.A. Sellers J.R. Adelstein R.S. J. Biol. Chem. 2004; 279: 2800-2808Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). In humans, the genes (MYH9, MYH10, and MYH14) encoding these myosin heavy chains are located on different chromosomes: 22, 17, and 19, respectively (7Simons M. Wang M. McBride O.W. Kawamoto S. Yamakawa K. Gdula D. Adelstein R.S. Weir L. Circ. Res. 1991; 69: 530-539Crossref PubMed Scopus (217) Google Scholar, 8Leal A. Endele S. Stengel C. Huehne K. Loetterle J. Barrantes R. Winterpacht A. Rautenstrauss B. Gene (Amst.). 2003; 312: 165-171Crossref PubMed Scopus (51) Google Scholar). All NMHC IIs are conserved with a 64-80% identity in amino acids among the various isoforms (6Golomb E. Ma X. Jana S.S. Preston Y.A. Kawamoto S. Shoham N.G. Goldin E. Conti M.A. Sellers J.R. Adelstein R.S. J. Biol. Chem. 2004; 279: 2800-2808Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar), suggesting that they might share some cellular functions, such as a role in cytokinesis and cell adhesion. For example, decreasing NMHC II-B in COS-7 cells results in multinucleation, a defect that can be rescued most efficiently by NMHC II-B but also to a significant extent by NMHC II-A and NMHC II-C (9Bao J. Jana S.S. Adelstein R.S. J. Biol. Chem. 2005; 280: 19594-19599Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). On the other hand, studies from a number of laboratories have also revealed that the different isoforms of non-muscle myosin II have distinct tissue and subcellular distributions and apparently play different roles, particularly during development (10Maupin P. Phillips C.L. Adelstein R.S. Pollard T.D. J. Cell Sci. 1994; 107: 3077-3090Crossref PubMed Google Scholar, 11Kelley C.A. Sellers J.R. Gard D.L. Bui D. Adelstein R.S. Baines I.C. J. Cell Biol. 1996; 134: 675-687Crossref PubMed Scopus (142) Google Scholar, 12Kolega J. Mol. Biol. Cell. 2003; 14: 4745-4757Crossref PubMed Scopus (101) Google Scholar, 13Kolega J. J. Cell Sci. 1998; 111: 2085-2095Crossref PubMed Google Scholar, 14Murakami N. Trenkner E. Elzinga M. Dev. Biol. 1993; 157: 19-27Crossref PubMed Scopus (38) Google Scholar, 15Conti M.A. Even-Ram S. Liu C. Yamada K.M. Adelstein R.S. J. Biol. Chem. 2004; 279: 41263-41266Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 16Uren D. Hwang H.K. Hara Y. Takeda K. Kawamoto S. Tullio A.N. Yu Z.X. Ferrans V.J. Tresser N. Grinberg A. Preston Y.A. Adelstein R.S. J. Clin. Invest. 2000; 105: 663-671Crossref PubMed Scopus (36) Google Scholar). Ablation of NMHC II-A in mice leads to a defect in cell adhesion during early development (15Conti M.A. Even-Ram S. Liu C. Yamada K.M. Adelstein R.S. J. Biol. Chem. 2004; 279: 41263-41266Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar), whereas ablation of NMHC II-B results in defects in the heart and brain, two organs known to be enriched for this isoform (16Uren D. Hwang H.K. Hara Y. Takeda K. Kawamoto S. Tullio A.N. Yu Z.X. Ferrans V.J. Tresser N. Grinberg A. Preston Y.A. Adelstein R.S. J. Clin. Invest. 2000; 105: 663-671Crossref PubMed Scopus (36) Google Scholar). Both NMHC II-B and II-C, but not NMHC II-A, undergo alternative splicing at homologous amino acids located in loop 1 and in loop 2 of their heavy chains. In the case of NMHC II-B, an exon encoding 10 amino acids is incorporated into loop 1 near the ATP binding region at amino acid 212 (NMHC II-B1), and an exon encoding 21 amino acids is inserted into loop 2 near the actin binding region at amino acid 622 (NMHC II-B2) (17Itoh K. Adelstein R.S. J. Biol. Chem. 1995; 270: 14533-14540Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). These inserted isoforms are only expressed in neuronal tissues, and recently, the results of ablating each of them in mice have been reported (18Ma X. Kawamoto S. Uribe J. Adelstein R.S. Mol. Biol. Cell. 2006; 17: 2138-2149Crossref PubMed Google Scholar). For NMHC II-C, an alternative exon encoding 8 amino acids is incorporated into loop 1 (NMHC II-C1), and another alternative exon encoding 41 amino acids is introduced into loop 2 (NMHC II-C2) at locations homologous to the NMHC II-B inserts. Whereas the tissue distribution of NMHC II-C2 is similar to that of II-B1 and II-B2 in being confined to neuronal tissues, 3S. S. Jana, unpublished observation. NMHC II-C1 is expressed in a variety of tissues, such as liver, kidney, testes, brain, and lung (6Golomb E. Ma X. Jana S.S. Preston Y.A. Kawamoto S. Shoham N.G. Goldin E. Conti M.A. Sellers J.R. Adelstein R.S. J. Biol. Chem. 2004; 279: 2800-2808Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). In a recent report, it was demonstrated that the presence of the C1 insert in NMHC II-C increases both the actin-activated MgATPase activity and the in vitro motility of heavy meromyosin derived from this isoform (19Kim K.Y. Kovacs M. Kawamoto S. Sellers J.R. Adelstein R.S. J. Biol. Chem. 2005; 280: 22769-22775Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). However, at present, little is known about the function of NMHC II-C1 at the cellular level. In this study, we report that NMHC II-C1 expression is markedly increased in two different tumor cell lines and is the only NMHC II-C isoform present in a number of tumor cell lines. Our studies show that NMHC II-A and II-C1 are the major isoforms in the A549 lung tumor cell line, each contributing ∼45% of the total nonmuscle myosin II at the protein level. Decreasing nonmuscle myosin II isoforms using siRNA had a different effect on cytokinesis. Whereas decreasing non-muscle myosin II-A had no significant effect on cell proliferation, lowering NMHC II-C1 decreased the number of cells 5.5-fold at 120 h by prolonging cytokinesis from 2 to 8-10 h. Decreasing NMHC II-C1 slowed the rate of cell proliferation by delaying the formation and retraction of the intercellular bridge that connects the two nascent daughter cells. Expression of exogenous NMHC II-C1-GFP, but not GFP-NMHC II-A or GFP-NMHC II-B, in the NMHC II-C1-depleted cells rescued the decrease in cell proliferation. These findings suggest a specific function for NMHC II-C1 in cytokinesis in the A549 tumor cell line. Plasmid Constructs and siRNAs—The cytomegalovirus promoter from pEGFP-C3 (Clontech, Palo Alto, CA) was inserted into the AseI/AgeI sites, replacing the pTRE promoter in the expression plasmids containing the GFP-tagged, full-length cDNAs of human NMHC II-A and NMHC II-B (9Bao J. Jana S.S. Adelstein R.S. J. Biol. Chem. 2005; 280: 19594-19599Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 20Wei Q. Adelstein R.S. Mol. Biol. Cell. 2000; 11: 3617-3627Crossref PubMed Scopus (153) Google Scholar). Mouse noninserted NMHC II-C0 and inserted NMHC II-C1 (6Golomb E. Ma X. Jana S.S. Preston Y.A. Kawamoto S. Shoham N.G. Goldin E. Conti M.A. Sellers J.R. Adelstein R.S. J. Biol. Chem. 2004; 279: 2800-2808Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar) were cloned into the SalI/BamHI sites of the pEGFP-N3 expression vector (Clontech). Expression of all four full-length cDNAs encoding NMHC IIs in cell lines was confirmed by immunoblot analysis and fluorescence microscopy. An siRNA (II-C1 siRNA) specific for the human C1 inserted sequence (6Golomb E. Ma X. Jana S.S. Preston Y.A. Kawamoto S. Shoham N.G. Goldin E. Conti M.A. Sellers J.R. Adelstein R.S. J. Biol. Chem. 2004; 279: 2800-2808Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar) and siRNA (II-C siRNA) specific to the 3′-untranslated region of NMHC II-C mRNA were chemically synthesized by Qiagen (Valencia, CA). A pool (SMARTpool) of siRNAs specific for human NMHC II-A (Ref Seq accession number NM_002473) or NMHC II-B (Ref Seq accession number NM_005964) was chemically synthesized by Dharmacon Research, Inc. (Lafayette, CO). Fluorescein-labeled nonspecific siRNA from Qiagen was used to determine the efficiency of transfection and as a non-specific control for siRNA experiments. Duplex siRNAs obtained from Qiagen were as follows: II-C1 siRNA duplex, sense strand (5′-r(CGUCAGCACCGUGUCUUAU)d(TT)-3′) and antisense strand (5′-r(AUAAGACACGGUGCUGACG)d(GA)-3′); II-C siRNA duplex, sense strand (5′-r(GGACUGGAGCUACCUUGCU)d(TT)-3′) and antisense strand (5′-r(AGCAAGGUAGCUCCAGUCC)d(TT)-3′); nonspecific siRNA duplex, sense strand (5′-r(UUCUCCGAACGUGUCACGU)d(TT)-3′) and antisense strand (5′-r(ACGUGACACGUUCGGAGAA)d(TT)-3′). Cell Lines—Human A549 (lung carcinoma), MCF-7 and HCC1569 (breast carcinoma), HepG2 (liver carcinoma), PC-3 (prostate carcinoma), PANC1 (pancreas carcinoma), NIH: OVCAR-3 (ovarian carcinoma), Beas-2B (nontumor lung), and MCF-10-2A (nontumor breast) were purchased from ATCC (Manassas, VA). All cell lines were maintained following standard ATCC protocols. For growth curves, cells were trypsinized and harvested at the indicated times, and the number of cells was counted using a hemocytometer. The (-)-enantiomer of blebbistatin was purchased from Sigma and dissolved at 10 mm in Me2SO. 50 μm blebbistatin was added after 72 h of NMHC II-C1 siRNA treatment. Within 8 min after the addition of blebbistatin, time lapse imaging was started. Transfection—1 μg of plasmid DNA/ml of culture medium and 200 nm siRNA were transfected using Effectene® and RNAiFect™ transfection reagents (Qiagen), respectively. Efficiency of siRNA transfection (as detected by the fluorescein signal) was >90%, and that for plasmid DNA (detected by the GFP signal) was 70-80%. Reverse Transcription (RT)-PCR and Real Time RT-PCR— Total RNA from cell lines was isolated using the RNeasy minikit (Qiagen). 1μg of total RNA was reverse-transcribed using random hexamers and the GeneAmp RNA PCR core kit (Applied Biosystems, Branchburg, NJ), and the resulting cDNA was amplified by PCR for individual gene products using specific primers. The primer sets were as follows: 5′-AAGACCGATCTCCTGTTGGA-3′ and 5′-ACCTTGCCGGCATAGTGGATA-3′ for human NMHC II-A; 5′-GCTGTTCAACCACACCATGT-3′ and 5′-ACAGTTCCGCTGCAAGACCTT-3′ for human NMHC II-B; 5′-ATGCTGCAGGATCGTGAGGACC-3′ and 5′-ATGAATTTGCCGAATCGGGAGG-3′ for human NMHC II-C; 5′-ATCTGGCACCACACCTTCTACAATGAGCTGCG-3′ and 5′-CGTCATACTCCTGCTTGCTGATCCACATCTGC-3′ for human β-actin. β-Actin was used to normalize the samples. PCR products were separated by 1.8% agarose gel electrophoresis and stained with ethidium bromide. For quantitation, real time RT-PCR was performed. The cDNA was amplified by PCR using a Quantitect SYBR green PCR kit (Qiagen) using the primers described above. The amount of cDNA from tumor and nontumor cell lines was normalized using β-actin. For each experiment, a standard curve was generated using 4-fold serial dilutions of plasmid containing each of the cDNAs. When PCR products fell within the range of the standard curve, the amount of cDNA of each gene was calculated relative to the standard curve using the Opticon Monitor program. Samples were run in triplicate using a PCR program with an initial cycle of 15 min at 95 °C, followed by 40 cycles of 15 s at 95 °C, 15 s at 54 °C (or 60 °C for II-C), and 1 min (or 30 s for II-C) at 72 °C. After each run, a melting curve was examined to ensure that no primer dimers or secondary products were formed. S.E. was calculated from three independent experiments. Immunoblot Analysis—Extracts of various cell lines were prepared as described previously (6Golomb E. Ma X. Jana S.S. Preston Y.A. Kawamoto S. Shoham N.G. Goldin E. Conti M.A. Sellers J.R. Adelstein R.S. J. Biol. Chem. 2004; 279: 2800-2808Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). Briefly, cells on tissue culture plates were washed twice with cold phosphate-buffered saline and directly lysed with Laemmli sample buffer. Proteins were separated by SDS-PAGE on 4-12% polyacrylamide gradient Tris-glycine gels or 6% polyacrylamide Tris-glycine gels (Cambrex (Rockland, ME) or Invitrogen), transferred to a polyvinylidene difluoride membrane (Invitrogen), and blocked in 5% nonfat milk in phosphate-buffered saline. The upper part of the blot was incubated with antibodies to the carboxyl terminus of NMHC II-C (1:10,000), the amino terminus of NMHC II-B (1:5,000 or 1:2,000), NMHC II-A (anti-platelet II-A, 1:100,000 or 1:50,000), or panmyosin (1:5,000; Covance Research Products, Inc., Berkeley, CA) at 4 °C overnight (6Golomb E. Ma X. Jana S.S. Preston Y.A. Kawamoto S. Shoham N.G. Goldin E. Conti M.A. Sellers J.R. Adelstein R.S. J. Biol. Chem. 2004; 279: 2800-2808Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 21Phillips C.L. Yamakawa K. Adelstein R.S. J. Muscle Res. Cell Motil. 1995; 16: 379-389Crossref PubMed Scopus (107) Google Scholar). The lower part of the blot was incubated with either anti-β-actin (1:10,000) (Sigma) or anti-α-tubulin (1:10,000) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) antibody. The blot was washed and then incubated with horseradish peroxidase-conjugated secondary antibodies (Pierce) at room temperature for 1 h. The blot was treated with SuperSignal West Pico or Femto luminal enhancer solution (Pierce). Luminescence signal was captured on Biomax MR film (Eastman Kodak Co.). Films were scanned using a densitometer (Molecular Devices, Sunnyvale, CA). Band intensity was calculated using ImageQuant software after normalizing with actin band intensity. Immunofluorescence Microscopy—A549 cells grown on chamber slides were rinsed with phosphate-buffered saline and fixed with 4% paraformaldehyde at room temperature for 30 min and permeabilized with 0.5% Triton X-100 for 10 min. For antibody staining, the samples were blocked with 0.1% bovine serum albumin and 10% normal goat serum in phosphate-buffered saline for 1 h at room temperature, incubated with affinity-purified polyclonal rabbit antibodies against NMHC II-C or NMHC II-A at 4 °C overnight. The secondary antibody, Alexa 594 goat anti-rabbit IgG (Molecular Probes, Inc., Eugene, OR) was incubated with cells at room temperature for 1 h. DAPI was used for nuclear staining. After washing, chamber slides were mounted using a Prolong antifade kit (Molecular Probes). The images were collected using a Leica SP1 confocal microscope (Deerfield, IL). Time Lapse Imaging—Time lapse imaging of cytokinesis was performed using an Olympus IX-70 microscope supported by the Metamorph program (Molecular Devices). Images were recorded every 5 min for 2 h for nonspecific siRNA or every 10 min for 10 h for NMHC II-C1 siRNA-treated A549 cells using a Photometric Cool Snap Camera. Images of blebbistatin-treated cells were recorded every 2 min using an Olympus IX-70 microscope supported by the Andor iQ program and Andor iXon DV887 camera (Andor Technology, Belfast, Northern Ireland). All time lapse imaging was performed under 5% CO2 and at 37 °C in a stage incubator. Statistical Analysis—Data were expressed as the means ± S.E. Statistical significance was tested with a two-way analysis of variance followed by the Bonferroni test. The differences were considered to be significant if p was <0.05. Expression of C1 Inserted mRNA in Human Epithelial Tumor Cell Lines—Golomb et al. (6Golomb E. Ma X. Jana S.S. Preston Y.A. Kawamoto S. Shoham N.G. Goldin E. Conti M.A. Sellers J.R. Adelstein R.S. J. Biol. Chem. 2004; 279: 2800-2808Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar) reported that the gene encoding NMHC II-C contains an alternatively spliced exon that inserts 24 nucleotides encoding 8 amino acids into loop 1 of NMHC II-C near the ATP binding region of the NMHC. Here, we investigated the expression of this alternatively spliced isoform of NMHC II-C, NMHC II-C1, in human tumor and related nontumor cell lines by RT-PCR, using primers flanking the inserted C1 exon. Fig. 1A shows that NMHC II-C1 is expressed in a number of different tumor cell lines, as shown by the generation of a nucleotide fragment of 269 bp instead of 245 bp (for the noninserted isoform). Note also that the noninserted isoform of NMHC II-C (Fig. 1A, II-C0) is not expressed in any of these cell lines at the mRNA level. In contrast to the lung and breast tumor cell lines (denoted as T in Fig. 1B), which express significant amounts of NMHC II-C1, Fig. 1B shows that a nontumor cell line derived from the same tissue, Beas-2B (lung) and MCF-10-2A (breast, denoted as N in Fig. 1B), express very little mRNA encoding either the NMHC II-C1 or noninserted NMHC II-C (Fig. 1B, top). We also determined the relative amounts of mRNA encoding NMHC II-A and II-B using isoform-specific primers. Fig. 1B shows that mRNAs encoding both isoforms are expressed in all four cell lines with no major difference in expression between the nontumor or tumor cell lines. These data were confirmed using real time PCR (see Table 1).TABLE 1Ratio of NMHC II isoforms in tumor (T) versus nontumor (N) cell lines for RNA and protein Results are expressed as mean ± S.E. from three independent experiments.IsoformLung (T:N)Breast (T:N)Real time PCR II-A0.69 ± 0.16:11.42 ± 0.29:1 II-B1.31 ± 0.01:10.66 ± 0.02:1 II-C130.8 ± 5.1:17.33 ± 1.22:1Immunoblot analysis II-A0.13 ± 0.01:10.33 ± 0.01:1 II-B0.03 ± 0.02:10.30 ± 0.01:1 II-C113.5 ± 0.8:14.16 ± 0.05:1 Open table in a new tab Immunoblot Analysis of NMH II-C in Tumor and Nontumor Cell Lines—We then quantified the relative difference in protein expression among the NMHC II isoforms in the lung and breast tumor and nontumor cell lines using antibodies specific for each of the isoforms. Fig. 2 shows immunoblots probed with antibodies to NMHC II-A, II-B, and II-C, and Table 1 quantifies the relative expression of each isoform, setting the quantity of NMHC II in the nontumor cell line as 1 for each isoform. We used a series of three different immunoblots and dilutions, one example of which is shown in Fig. 2. For NMHC II-C1 (third blot from the top), the protein expression analysis correlates with the increase in mRNA expression (Fig. 1B) and shows a 4-fold increase in the breast tumor cell line and a 13.5-fold increase in the lung tumor cell line over the respective nontumor cell lines. In contrast, the immunoblots detecting NMHC II-A and II-B show a marked reduction in each of the isoforms at the protein level in the tumor cell lines, differing from the mRNA results, which showed no significant change between the tumor and nontumor cell lines for both lung and breast (Fig. 1B and Table 1). These results suggest that expression of NMHC II-A and II-B protein was decreased at the translational level or due to protein instability, whereas NMHC II-C1 expression was increased transcriptionally or due to increased mRNA stability or both in the tumor epithelial cell lines. NMHC II-A and NMHC II-C1 Are the Major Isoforms in Lung Tumor A549 Cells—To compare the percentage of each NMHC II isoform at the protein level in the A549 lung tumor cell line, we made use of specific siRNAs and a panmyosin II antibody. As noted under “Experimental Procedures,” we lowered the mRNA encoding each NMHC II isoform using the appropriate siRNA and then analyzed the NMHC II protein levels using antibodies specific to each isoform as well as a pan-myosin antibody. We correlated the change in the immunoblot seen following siRNA treatment with that seen following detection with the panmyosin antibody. Fig. 3 shows that siRNA targeting NMHC II-A specifically lowered the II-A protein (II-A, compare control lanes 1 and 2 with lanes 3 and 4) and also significantly reduced the amount of total NMHC as detected in the panmyosin immunoblot (Pan-Myosin, lanes 3 and 4). On the other hand, lowering II-B protein with siRNA (II-B, lanes 5 and 6) had little effect on the panmyosin blot (Pan-Myosin, compare control lanes 1 and 2 with lanes 5 and 6). Lowering NMHC II-C1 (see Fig. 4A, II-C) also significantly lowered the total NMHC signal on the panmyosin blot (Fig. 3, lanes 9 and 10). Quantification of the panmyosin blot for three different immunoblots revealed that NMHC II-B is less than 8% of the total NMHC II, and NMHC II-A and II-C1 comprise about 45% each of the total NMHC II.FIGURE 4Decreasing the NMHC II-C1 isoform reduces cell proliferation. A, immunoblot confirming that siRNA specifically inhibits only NMHC II-C1 protein expression and not II-A or II-B. Two different amounts of sample were loaded on the gel. B, an equivalent number (2.5 × 104) of A549 cells were seeded and transfected with either nonspecific (NS) or NMHC II-C1 siRNA, and cell numbers were counted at 24-h time intervals until 120 h. Each assay was performed in triplicate, and the entire experiment was repeated three times. *, p < 0.01; **, p < 0.001 for siRNA II-C1 and II-A cell number versus nonspecific siRNA cell number. C, 0.75 × 104 cells were transfected with either nonspecific or NMHC II-C1 siRNA (passage 1). At 156 h, cells were replated at equal numbers, and cells were counted until 228 h (passage 2). D, quantification of NMHC II-C1 protein expression using immunoblots (not shown) from passage 1 and passage 2 cells (see C). Percentage of NMHC II-C1 protein expression was calculated with respect to the protein expression of NMHC II-C1 in nonspecific siRNA-treated cells. Results are expressed as mean ± S.E. for three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The Effect of Lowering NMHC II Isoforms on Cell Proliferation— Since myosin II-A and II-C1 are the major nonmuscle myosin II isoforms in the A549 lung tumor cell line, we were interested in exploring the effect of lowering each of these two isoforms on cell proliferation. We found that lowering NMHC II-A had only a small effect (although statistically significant) on cell proliferation compared with the effect of transfecting nonspecific siRNA (quantified in Fig. 4B, hatched bars compared with the black bars for nonspecific siRNA). In contrast, using siRNA directed specifically against the NMHC II-C1 mRNA lowered NMHC II-C1 protein (Fig. 4A) and markedly decreased cell proliferation (open bars) compared with the effect of nonspecific siRNA (Fig. 4B). At 120 h, there was almost a 5.5-fold difference in the number of NMHC II-C1 siRNA-treated cells compared with nonspecific siRNA-treated cells (Fig. 4B). When NMHC II-C1 siRNA-treated cells and nonspecific siRNA-treated cells were replated at 156 h after siRNA transfection and the cells were counted at 24, 48, and 72 h after replating, there was no longer a difference in the rate of cell proliferation between them (see Fig. 4C, Passage 2). This correlated with increased expression of NMHC II-C1 protein following replating, which was most likely due to the loss of siRNA. Fig. 4D quantifies the expression of NMHC II-C1 protein in the siRNA-treated cells compared with nonspecific siRNA-treated cells at the times indicated as determined by scanning immunoblots. It demonstrates that by 180 h after siRNA transfection, the effects of siRNA have decreased, resulting in increased expression of NMHC II-C1 to about 40% of control cells and restoring proliferation of NMHC II-C1 siRNA-treated cells to that found for nonspecific siRNA-treated cells. This shows that the effects of siRNA treatment are reversible and account for the decrease in cell proliferation. Of note is that there was no evidence for binucleation in the siRNA-treated cells. Rescuing the siRNA-induced Decrease in Cell Proliferation Using GFP NMHC IIs—We then assessed the ability of each NMHC II isoform to rescue the decrease in cell proliferation caused by down-regulation of NMHC II-C1. Each full-length NMHC II isoform tagged with GFP was introduced into the cells that had been previously treated with NMHC II-C siRNA for 24 h. For this experiment, siRNAs to lower NMHC II-C were targeted to the 3′-untranslated region of NMHC II-C mRNA to prevent the siRNA from lowering mRNA derived from the NMHC II-C1-GFP construct (see “Experimental Procedures”). We previously reported (9Bao J. Jana S.S. Adelstein R.S. J. Biol. Chem. 2005; 280: 19594-19599Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) that, unlike NMHC II-A and II-B, it was necessary to fuse GFP to the carboxyl terminus of the NMHC II-C molecule to prevent aggregation and for it to localize properly. Following transfection of the GFP-tagged NMHC II constructs into the NMHC II-C siRNA-treated cells, the cells were cultured for an additional 96 h, and the cell number was counted every 24 h after cDNA transfection. These experiments were carried out under conditions that normalized the transfection efficiency of all four GFP-tagged NMHC II constructs to 70% of the cells transfected, using the GFP signal viewed by fluorescence microscopy as an indicator. Fig. 5A shows a plot of cell numbers of the NMHC II-C siRNA-treated cells that were transfected with GFP-tagged NMHC II isoforms. Control cells transfected with nonspecific siRNA displayed the same rate of proliferation as did cells that were not transfected at all (data not shown). In contrast, NMHC II-C siRNA-treat" @default.
• W2045707271 created "2016-06-24" @default.
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• W2045707271 title "A Specific Isoform of Nonmuscle Myosin II-C Is Required for Cytokinesis in a Tumor Cell Line" @default.
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• W2045707271 cites W1981429363 @default.
• W2045707271 cites W1984953789 @default.
• W2045707271 cites W1987565809 @default.
• W2045707271 cites W2006667157 @default.
• W2045707271 cites W2009540102 @default.
• W2045707271 cites W2030255198 @default.
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• W2045707271 cites W2051165421 @default.
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