Actin in human colon adenocarcinoma cells with differentmetastatic potential*Dorota Nowak1, Agnieszka Krawczenko2, Danuta Duœ2andMaria Malicka-B³aszkiewicz1½1Department of Cell Pathology, Institute of Biochemistry and Molecular Biology, University of Wroc³aw,Wroc³aw, Poland;2Institute of Immunology and Experimental Therapy, Polish Academy of Sciences,Wroc³aw, PolandReceived: 20 October, 2002; revised: 04 November, 2002; accepted: 12 November, 2002Key words: actin, state of actin polymerization, colon adenocarcinoma cell linesFour human colon adenocarcinoma cell line variants with different metastatic po-tential were used to examine whether a correlation exists between actin level, state ofactin polymerization and invasiveness of tumour cells.Monomeric (G), total (T) and filamentous (F) actin were determined in the cytosolicfraction of these cells. A statistically significant decrease in G actin level and increasein the state of actin polymerization (measured by F:G actin ratio) were found in thecytosol of three cell variants with higher metastatic potential and invasiveness (EB3,3LNLN, 5W) compared with the parental cell line (LS180).Our experimental data lead to the conclusion that there is a correlation between themetastatic capacity of human colon adenocarcinoma cells and the state of actin poly-merization.Actin is the most abundant and highly con-served protein occurring in all eukaryoticcells. Actin microfilaments are one of thethree major components of the cytoskeleton,the structure involved in many cellular func-tions, including cell motility, contractile ringformation during cytokinesis, maintenance ofcell shape, and signal transduction. The struc-tural and motile functions of actin are possi-ble through a dynamic conversion betweenmonomeric actin (G actin) and filamentousactin (F actin), a linear polymer of G actin sub-units (Sheterline et al., 1998).Vol. 49 No. 4/2002823–828QUARTERLY*The results work were presented in part at the 38th Meeting of the Polish Biochemical Society inWroc³aw, September, 2002.½To whom correspondence should be addressed: Maria Malicka-B³aszkiewicz, Department of Cell Pathol-ogy, Institute of Biochemistry and Molecular Biology, University of Wroc³aw, S. Przybyszewskiego 63,51-148 Wroc³aw, Poland; tel.: (48 71) 375 6205; fax: (48 71) 375 6234; e-mail: [email protected]: DNase I, deoxyribonuclease I from bovine pancreas (EC 3.1.21.1); DTT, dithiothreitol;i.i., intrailleal; i.s., intrasplenic; i.v., intravenous; PBS, phosphate-buffered saline.Adhesion and locomotion are one of the keycapabilities of cells in tumour growth and me-tastasis formation. They depend on the archi-tecture of cytoskeletal elements and especiallyon the actin microfilaments system. The alter-ations in actin organization are known to ac-company malignant transformation of manycell types (Ben-Ze’ev, 1985; Hansell et al.,1995; Button et al., 1995). The disruption ofactin stress fibers, as observed in many can-cer cells, may result from the changes in actincontent and its ability to polymerize and alsofrom a lack of physiological equilibrium be-tween actin assembly and disassembly, so im-portant for its functions.Interesting data have accumulated on the re-lation between actin organization, changes inactin isoforms expression and the ability ofcancer cells to form metastases (Pokorna etal., 1994; Janmey & Chaponnier, 1995; Nowak& Malicka-B³aszkiewicz, 1999). Differences inactin distribution were observed in popula-tions of sarcoma cells with different meta-static potential, in MDCK (Madine Darby Ca-nine Kidney) cells transformed with Moloneysarcoma virus (Le et al., 1998) and in humansalivary gland adenocarcinoma (Suzuki et al.,1998). The invasive variants of these cells con-tain no stress fibers in contrast to theirnoninvasive counterparts, which show highorganization of the actin cytoskeleton. Cyto-plasmic b actin was identified to appear at thetips of pseudopodia of invasive cells but not innoninvasive cells (Le et al., 1998; Shestakovaet al., 1999).Our former observation of actin duringhepatoma Morris 5123 tumour growthshowed that a remarkable increase in totaland polymerized actin level in tumour cells co-incides with the first metastasis formation tothe lungs of tumour bearing rats (Malicka-B³aszkiewicz et al., 1995; Nowak et al., 1995).In this paper four phenotypically well-chara-cterized human colon adenocarcinoma LS180cell line variants — including those with in-creased motility and metastatic potential —showing different organ localization of metas-tasis were used to examine whether a correla-tion exists between actin level, state of actinpolymerization and invasiveness of tumourcells.MATERIALS AND METHODSCells. The human colon adenocarcinoma cellline LS180 was obtained from DeutscheKrebsforschungzentrum (Heidelberg, Ger-many). Endothelial cell line HPLNEC.B3 —human microvascular endothelial cells from aperipheral lymph node of a patient with Hodg-kin’s lymphoma — was isolated and character-ized by Kieda et al. (2002). Cell lines and fur-ther selected variant sublines were propa-gated in OptiMEM medium supplementedwith 3% fetal bovine serum (Gibco, Grand Is-land, NY, U.S.A.). Cells were cultured in 25cm2tissue culture flasks at 37°Cin5%CO2/95% humidified air (Falcon or Costar) andpassaged weekly, using 0.25% trypsin/0.05%EDTA solution (Gibco, Grand Island, NY,U.S.A.).In vitro selection of LS180 cells with in-creased affinity for human HPLNEC.B3endothelial cells. HPLNEC.B3 cells wereplated onto the bottom of a 24-well TC plate.The colon carcinoma LS180 cells (1 ´ 105cells/100 ml) were superposed onto the uppersurface of an 8 mm, Matrigel-coated nucleo-pore Transwell filter (Costar, France). After48–72 h, LS180 cells which had migrated tothe lower surface of the filter towards the en-dothelial cell monolayer were removed by abrief 0.25% trypsin/0.05% EDTA treatment,washed and propagated in vitro. The manipu-lation was repeated three times. The obtainedcolon cancer subline was named LS180EB3(EB3).In vivo selection of colon adenocarci-noma cells. The in vitro selected EB3 cellswere passaged in vivo by various routes of in-oculation, to select metastatic variants. Foranimal experiments, athymic NCr nu/nu malemice, obtained from the National Cancer In-824 D. Nowak and others 2002stitute, Frederic Cancer Research and Devel-opmental Center (Frederick, MD, U.S.A.)were used. Animal experiments were per-formed according to the