Assessing Cell Fusion and Cytokinesis Failure as Mechanisms of Clone 9 Hepatocyte Multinucleation In Vitro

Damir Simic1, Catherine Euler1, Christina Thurby1, Mike Peden1, Sarah Tannehill‐Gregg1, Todd Bunch1, Thomas Sanderson1, Terry Van Vleet1

1 Drug Safety Evaluation, Bristol‐Myers Squibb Co., Mount Vernon, Indiana
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 14.9
DOI:  10.1002/0471140856.tx1409s53
Online Posting Date:  August, 2012
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In this in vitro model of hepatocyte multinucleation, separate cultures of rat Clone 9 cells are labeled with either red or green cell tracker dyes (Red Cell Tracker CMPTX or Vybrant CFDA SE Cell Tracer), plated together in mixed‐color colonies, and treated with positive or negative control agents for 4 days. The fluorescent dyes become cell‐impermeant after entering cells and are not transferred to adjacent cells in a population, but are inherited by daughter cells after fusion. The mixed‐color cultures are then evaluated microscopically for multinucleation and analysis of the underlying mechanism (cell fusion/cytokinesis). Multinucleated cells containing only one dye have undergone cytokinesis failure, whereas dual‐labeled multinucleated cells have resulted from fusion. Curr. Protoc. Toxicol. 53:14.9.1‐14.9.17. © 2012 by John Wiley & Sons, Inc.

Keywords: multinucleation; hepatocyte; Clone 9; cytokinesis; cell fusion

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Table of Contents

  • Introduction
  • Basic Protocol 1: Hepatocyte Treatment Conditions and Culturing
  • Support Protocol 1: siRNA Knockdown of Genes to Refine Mechanism
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Hepatocyte Treatment Conditions and Culturing

  • Clone 9 cell medium: F‐12K medium (ATCC, cat. no. 30‐2004) containing 10% fetal bovine serum (FBS, e.g., Invitrogen, cat no. 16000‐044) and 1× penicillin/streptomycin (Invitrogen, cat. no. 15070‐063)
  • 75% (v/v) ethanol
  • Hepatocytes: Clone 9 cells (ATCC, cat. no. CRL‐1439)
  • 0.25% trypsin‐EDTA (Invitrogen, cat no. 25200‐056)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Vybrant CFDA SE Cell Tracer Kit—green cell marking (Invitrogen, cat. no.V12883)
  • Red Cell Tracker CMPTX—red cell marking (Invitrogen, cat. no. C34552)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Dimethylsulfoxide (DMSO; (e.g., Calbiochem, cat no. 317275)
  • 0.4% trypan blue (Sigma, cat no. T8154)
  • Drug treatments:
    • Rifabutin (Sigma, cat no. R3530)
    • Erythromycin stearate (Sigma, cat no. E9256)
    • Aurora kinase inhibitor (OM137, Sigma, cat no. O8140)
    • Rifampicin (Sigma, cat no. R3501)
    • Tamoxifen (Sigma, cat no. T5648)
    • Puromycin (Sigma, cat no. P7255)
    • siRNA (see protocol 2)
  • 4% (w/v) paraformaldehyde: dilute from 16% paraformaldehyde aqueous solution (Electron Microscopy Sciences, cat. no. 15710) as described under step 32, below
  • Antifade reagent with DAPI (Invitrogen, cat. no. S36938)
  • 4′,6‐diamidino‐2‐phenylindole (DAPI)
  • Clear nail polish
  • 25‐ and 75‐cm2 cell culture flasks with vented caps (e.g., BD Falcon)
  • Sterile, filtered pipet tips (2‐ to 1000‐µl)
  • 15‐ml conical tubes (e.g., BD Falcon)
  • Centrifuge
  • Upright cell culture microscope or alternative device for cell counting
  • Hemacytometer (e.g., Incyto C‐Chip disposable,
  • 6‐well dish
  • Microscope slides
  • Coverslips, sterile, to fit in wells of 6‐well dish
  • Confocal or fluorescent microscope
NOTE: All reagents and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.NOTE: All culture incubations are performed in a 37°C, 5% CO 2 humidified incubator unless otherwise indicated.

Support Protocol 1: siRNA Knockdown of Genes to Refine Mechanism

  • AurkB siRNA (Ambion, cat. no. AM16708; siRNA ID #201286; rat target gene symbol: Aurkb)
  • Control siRNA—non silencing (ABI/Ambion, cat no. 4390843)
  • Nuclease‐free H 2O (available from most molecular biology suppliers)
  • F‐12K medium (ATCC, cat. no. 30‐2004), serum free
  • Clone 9 cells (ATCC, cat. no. CRL‐1439) growing in culture (see protocol 1)
  • HiPerFect transfection reagent (Qiagen, cat. no. 301704)
  • Clone 9 cell medium: F‐12K medium (ATCC, cat. no. 30‐2004) containing 10% fetal bovine serum (FBS, e.g., Invitrogen, cat no. 16000‐044)
  • AurkB primer/probe set (ABI, TaqMan ID Rn01460656_m1)
  • Eukaryotic 18S rRNA endogenous control Primer/Probe set (ABI, cat no. 4333760F)
  • QScript cDNA Super Mix (Quanta, cat. no. 101414)
  • PerFecta qPCR Fast Mix ROX (Quanta cat. no. 101419)
  • RNA isolation kit: e.g., RNeasy kit (Qiagen) or Cells‐to‐Ct kit (Ambion)
  • RNaseZap (Ambion, cat no. AM9780; AM9782)
  • NanoDrop Spectrophotometer ND‐1000
  • BioRad iCycler or Applied Biosystems 7900 PCR system
  • Additional reagents and equipment for quantitation of RNA using NanoDrop spectrophotometer (Desjardins and Conklin, ) and RT‐PCR (Arany, )
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Literature Cited

Literature Cited
   Adams, R.R., Maiato, H., Earnshaw, W.C., and Carmena, M. 2001. Essential roles of Drosophila inner centromere protein (INCENP) and Aurora‐B in histone H3 phosphorylation, metaphase chromosome alignment, kinetochore disjunction, and chromosome segregation. J. Cell Biol. 153:865‐880.
   Arany, Z.P. 2008. High‐throughput quantitative real‐time PCR. Curr. Protoc. Hum. Genet. 58:11.10.1‐11.10.11.
   Auvigne, I., Pichard, V., Aubert, D., Robillard, N., and Ferry, N. 2002. In vivo cell lineage analysis in cypreterone acetate‐treated rat liver using genetic labeling of hepatocytes. Hepatology 35:281‐288.
   Brodbeck, W.G and Anderson, J.M. 2009. Giant cell formation and function. Curr. Opin. Hematol. 16:53‐57.
   Chung, D.H., Humphrey, M.B., Nakamura, M.C., Ginzinger, D.G., Seaman, W.E., and Daws, M.R. 2003. CMRF‐35‐like molecule‐1, a novel mouse myeloid receptor, can inhibit osteoclast formation. J. Immunol. 171:6541‐6548.
   Cui, W., Ke, J.Z., Zhang, Q., Ke, H.Z., Chalouni, C., and Vignery, A. 2006. The intracellular domain of CD44 promotes the fusion of macrophages. Blood 107:796‐805.
   Dadke, D., Jarnik, M., Pugacheva, E.N., Singh, M.K., and Golemis, E.A. 2006. Deregulation of HEF1 impairs M‐phase progression by disrupting the RhoA activation cycle. Mol. Biol. Cell. 17:1204‐1217.
   DeMoe, J.H., Santaguida, S., Daum, J.R., Musacchio, A., and Gorbsky, G.J. 2009. A high throughput, whole cell screen for small molecule inhibitors of the mitotic spindle checkpoint identifies OM137, a novel Aurora kinase inhibitor. Cancer Res. 69:1509‐1516.
   Desjardins, P.R. and Conklin, D.S. 2011. Microvolume quantitation of nucleic acids. Curr. Protoc. Mol. Biol. 93:A.3J.1‐A.3J.16
   Deward, A.D. and Alberts, A.S. 2009. Ubiquitin‐mediated degradation of the formin mDia2 upon completion of cell division. J. Biol. Chem. 284:20061‐20069.
   Dini, L., Pagliara, P., and Carla, E.C. 2002. Phagocytosis of apoptotic cells by liver: A morphological study. Microsc. Res. Tech. 57:530‐540.
   Duncan, A.W., Taylor, M.H., Hickey, R.D., Hanlon Newell, A.E., Lenzi, M.L., Olson, S.B., Finegold, M.J., and Grompe, M. 2010. The ploidy conveyor of mature hepatocytes as a source of genetic variation. Nature 467:707‐710.
   Fuji, E., Karasawa, Y., Kumano, E., Sakurai, T., Misawa, Y., Mori, T., Ito, T., Suzuki, M., and Sugimoto, T. 2004. Nuclearity and BrdU labeling of rat hepatocytes in cytocentrifuge preparations of freshly isolated hepatocytes with cumulative labeling of bromodeoxyuridine. J. Toxicol. Pathol. 17:43‐49.
   Frendo, J.L., Cronier, L., Bertin, G., Guibourdenche, J., Vidaud, M., Evain‐Brion, D., and Malassine, A. 2003. Involvement of connexin 43 in human trophoblast cell fusion and differentiation. J. Cell Sci. 116:3413‐3421.
   Gerets, H.H.J., Tilmant, K., Gerin, B., Chanteux, H., Depelchin, B.O., Dhalluin, S., and Atienzar, F.A. 2012. Characterization of primary human hepatocytes, HepG2 cells, and HepaRG cells at the mRNA level and CYP activity in response to inducers and their predictivity for the detection of human hepatotoxins. Cell Biol. Toxicol. 28:69‐87.
   Glotzer, M. 2005. The molecular requirements for cytokinesis. Science 307:1735‐1739.
   Gurtler, U., Tontsch‐Grunt, U., Jarvis, M., Zahn, S.K., Boehmelt, G., Quant, J., Adolf, G.R., and Solca, F. 2010. Effect of BI 811283, a novel inhibitor of Aurora B kinase, on tumor senescence and apoptosis. J. Clin. Oncol. 28:e13632.
   Hayashi, S., Fuji, E., Kato, A., Kimura, K., Mizoguchi, K., Suzuki, M., Sugimoto, T., Takanashi, H., Itoh, Z., Omura, S., and Wanibuchi, H. 2008. Characterization of multinuclear hepatocytes induced in rats by mitemcinal (GM‐611), an erythromycin derivative. Toxicol. Pathol. 36:858‐865.
   Helming, L. and Gordon, S. 2007. The molecular basis of macrophage fusion. Immunobiology 212:785‐793.
   Hollander, M.C., Sheikh, M.S., Bulavin, D.V., Lundgren, K., Augeri‐Henmueller, L., Shehee, R., Molinaro, T.A., Kim, K.E., Tolosa, E., Ashwell, J.D., Rosenberg, M.P., Zhan, Q., Fernandez‐Salguero, P.M., Morgan, W.F., Deng, C.X., and Fornace, A.J. Jr. 1999. Genomic instability in Gadd45a‐deficient mice. Nat. Genet. 23:176‐184.
   Hu, C., Ahmed, M., Melia, T.J., Sollner, T.H., Mayer, T., and Rothman, J.E. 2003. Fusion of cells by flipped SNAREs. Science 300:1745‐1749.
   Jin, J. and Woodgett, J.R. 2005. Chronic activation of protein kinase Bbeta/Akt2 leads to multinucleation and cell fusion in human epithelial kidney cells: Events associated with tumorigenesis. Oncogene 24:5459‐5470.
   Kim, J.R., Kee, H.J., Kim, J.Y., Joung, H., Nam, K.I., Eom, G.H., Choe, N., Kim, H.S., Kim, J.C., Kook, H., Seo, S.B., and Kook, H. 2009. Enhancer of polycomb1 acts on serum response factor to regulate skeletal muscle differentiation. J. Biol. Chem. 284:16308‐16316.
   Kodama, A., Takaishi, K., Nakano, K., Nishioka, H., and Takai, Y. 1999. Involvement of Cdc42 small G protein in cell‐cell adhesion, migration and morphology of MDCK cells. Oncogene 18:3996‐4006.
   Kostka, G., Palut, D., Kopec‐Szlezak, J., and Ludwicki, J.K. 2000. Early hepatic changes in rats induced by permethrin in comparison with DDT. Toxicology 142:135‐143.
   Kwong, J., Kulbe, H., Wong, D., Chakravarty, P., and Balkwill, F. 2009. An antagonist of the chemokine receptor CXCR4 induces mitotic catastrophe in ovarian cancer cells. Mol. Cancer Ther. 8:1893‐1905.
   Lin, J., Schyschka, L., Muhl‐Benninghaus, R., Neumann, J., Hao, L., Nussler, N., Dooley, S., Liu, L., Stuckle, U., Nussler, A.K., and Ehnert, S. 2012. Comparative analysis of phase I and II enzyme activities in 5 hepatic cell lines identifies Huh‐7 and HCC‐T cells with the highest potential to study drug metabolism. Arch. Toxicol. 86:87‐95.
   Liu, Z., Yue, S., Chen, X., Kubin, T., and Braun, T. 2010. Regulation of cardiomyocyte polyploidy and multinucleation by CyclinG1. Circ. Res. 106:1498‐1506.
   Lu, X. and Kang, Y. 2009. Cell fusion as a hidden force in tumor progression. Cancer Res. 69:8536‐8539.
   Lubberstedt, M., Müller‐Vieira, U., Mayer, M., Biemel, K.M., Knöspel, F., Knobeloch, D., Nüssler, A.K., Gerlach, J.C., and Zeilinger, K. 2011. HepaRG human hepatic cell line utility as a surrogate for primary human hepatocytes in drug metabolism assessment in vitro. J. Pharmcol. Toxicol. Methods 63:59‐68.
   Manning, K., Al‐Dhalimy, M., Finegold, M., and Grompe, M. 1999. In vivo suppressor mutations correct a murine model of hereditary tyrosinemia type I. Proc. Natl. Acad. Sci. U.S.A. 96:11928‐11933.
   Mbalaviele, G., Chen, H., Boyce, B.F., Mundy, G.R., and Yoneda, T. 1995. The role of cadherin in the generation of multinucleated osteoclasts from mononuclear precursors in murine marrow. J. Clin. Invest. 95:2757‐2765.
   McNally, A.K. and Anderson, J.M. 2005. Multinucleated giant cell formation exhibits features of phagocytosis with participation of the endoplasmic reticulum. Exp. Mol. Pathol. 79:126‐135.
   Ririe, K.M., Rasmussen, R.P., and Wittwer, C.T. 1997. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal. Biochem. 245:154‐160.
   Scampini, G., Nava, A., Newman, A.J., Della Torre, P., and Mazué, G. 1993. Multinucleated hepatocytes induced by rifabutin in rats. Toxicol. Pathol. 21:369‐376.
   Seglen, P.O. 1997. DNA ploidy and autophagic protein degradation as determinants of hepatocellular growth and survival. Cell Biol. Toxicol. 13:301‐315.
   Shen, E., Lei, Y., Liu, Q., Zheng, Y., Song, C., Marc, J., Wang, Y., Sun, L., and Liang, Q. 2009. Identification and characterization of INMAP, a novel interphase nucleus and mitotic apparatus protein that is involved in spindle formation and cell cycle progression. Exp. Cell Res. 315:1100‐1116.
   Singh, M., Cowell, L., Seo, S., O'Neill, G., and Golemis, E. 2007. Molecular basis for HEF1/NEDD9/Cas‐L action as a multifunctional co‐ordinator of invasion, apoptosis and cell cycle. Cell Biochem. Biophys. 48:54‐72.
   Sudo, H. and Maru, Y. 2007. LAPSER1 is a putative cytokinetic tumor suppressor that shows the same centrosome and midbody subcellular localization pattern as p80 katanin. FASEB J. 21:2086‐2100.
   Tsai, C.W., Liu, K.L., Lin, C.Y., Chen, H.W., and Lii, C.K. 2011. Structure and function relationship study of allium organosulfur compounds on upregulating the Pi class of glutathione S‐transferases. J. Agric. Food Chem. 59:3398‐3405.
   Yabuta, N., Okada, N., Ito, A., Hosomi, T., Nishihara, S., Sasayama, Y., Fujimori, A., Okuzaki, D., Zhao, H., Ikawa, M., Okabe, M., and Nojima, H. 2007. Lats2 is an essential mitotic regulator required for the coordination of cell division. J. Biol. Chem. 282:19259‐19271.
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