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[CANCER RESEARCH 47. 3802-3807, July 15, 1987] Effect of Adhesion Factors , , and Type IV on Spreading and Growth of Transformed and Control Liver Epithelial Cells James L. Junker and Ursula I. Heine1

Ultrastructural Studies Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute, Frederick Cancer Research Facility, Frederick, Maryland 21701

ABSTRACT cell proliferation and movement (31,32) as, for example, during (33, 34) and embryonal development (35). To examine the specific effects of individual The cells used in this study are from a liver-derived epithelial components on the behavior of transformed cells of epithelial origin, cell line which underwent neoplastic transformation following ethionine-transformed cells and control cells at low and high passage exposure in vitro to the epigenetic carcinogen DL-ethionine (36); levels were seeded on glass that had been coated with fibronectin, laminili, or type IV collagen. The cells used were sublines of the liver-derived the corresponding control cells of high passage level, however, TRL 1215 epithelial cell line, a line in which transformation has been remained nontransformed. In this model system, transforma shown to be accompanied by increased cell-substrate adhesion and cell tion expressed by the capability of the cells to form soft agar spreading. Cell spreading on the different basement membrane compo colonies and become tumorigenic in syngeneic is accom nents was determined by morphometry, and growth (proliferation) was panied by an increase in cell-substrate adhesion as shown by measured by and DNA analyses. Laminin increased spreading increased cell spreading and increased numbers of actin stress and growth in transformed and control sublines. Laminin also induced libers (37). Cellular fibronectin also increases, and there is a changes in cell shape that were indicative of increased cell motility. For focal loss of the laminin reticulum (38). In this study, in order the control cells, fibronectin and also type IV collagen were less effective specifically to test the hypothesis that the increased level of than laminiti in stimulating cell spreading and growth. However, for the ethionine-transformed cells, fibronectin was as effective as laminin in fibronectin or the decreased level of laminin in the matrices of stimulating cell spreading. With the exception of the spreading response the transformed cells was related to their increased proliferative to fibronectin, the ethionine-transformed cells were less sensitive to the capabilities, and, more generally, to determine the effects of defined substrata than were the control sublines. Moreover, only the basement membrane components on cell spreading and growth ethionine-transformed cells were able to proliferate in serum-free me in this cell line, transformed and control cells were seeded on dium. Thus, greater autonomy is characteristic of transformation for defined substrata of fibronectin, laminin, or type IV collagen. these epithelial cells and is exemplified by the reduced influence of and Laminin is shown to be the most potent stimulator of growth. dependence on exogenous factors, both substrate-bound and soluble, for The results also provide evidence of increased sensitivity to spreading and growth. fibronectin by the transformed cells but more strongly demon strate the autonomy of the transformed cells relative to the INTRODUCTION controls. The extracellular has been shown to influence the behavior of cells (1) not only in terms of cell shape (2-4) but MATERIALS AND METHODS also in terms of cell proliferation (5, 6), differentiation (7), protein synthesis (8-10), and, ultimately, gene expression (11- . The TRL 1215 cell line used in this study (36) is an epithelial cell line derived from rat liver and it consists of three popu 14). The interactions of a cell with the substratum are of lations (sublines) defined as: (a) ETC2) of approximate passage level particular interest in carcinogenesis because in the processes of P60; (b) untreated HPC at the same passage level as the ETC; and (c) invasion and metastasis, the normal configuration of the base a control for the effects of passage level, untreated LPC, P15-P20. Cells ment membrane is disrupted (15, 16) and, in the case of were propagated in Williams' Medium D containing 10% fetal bovine metastasis, the vascular basal laminae must also be traversed serum (Gibco, Grand Island, NY) as previously described (35, 39). (17, 18). For the spreading and growth experiments, the serum used in the One approach to the examination of cell-matrix interactions medium was depleted of fibronectin by using -linked Sepharose is to study the modulation of cell behavior by individual com (Pharmacia, Uppsala, Sweden) (40). ponents of the basement membrane (19-22). In this study we Preparation of Defined Substrata. For cell spreading assays, square glass coverslips (9-mm) were cleaned with hot acid (concentrated sul have used type IV collagen, laminin, and fibronectin. Type IV furie acidxoncent rated nitric acid 4:1, 11OX"), neutralized with potas collagen provides the scaffolding for laminin and other com ponents of the basement membrane (23), and increased colla- sium carbonate, rinsed with distilled water, dried, autoclaved, and placed in 16-mm-diameter wells of 24-well culture dishes (Costar, genäseactivity correlates positively with invasion and metas Cambridge, MA). All substrate-containing solutions (except for type tasis (24, 25). Laminin is involved in epithelial cell attachment IV collagen) were diluted from frozen aliquots of stock solutions and (26), and cell surface laminin and plasmalemmal laminin recep sterile filtered (Centrex centrifugal microfilters, blue; Schleicher and tors appear to play a role in the adherence of a metastatic cell Schuell, Keene, NH) immediately prior to use. Type IV collagen was to the vascular basement membrane (27-29). Fibronectin is diluted prior to being aliquoted for freezing. The concentrations of another adhesion factor (30) and is involved primarily in mes- basement membrane components that were selected were those at which enchymal cell attachment. Fibronectin is reduced on or absent cell attachment reached its maximum in the supplier's assay system. from the surfaces of many transformed cells, and its addition For laminin the adequacy of this concentration was confirmed by the to culture medium can alter the phenotype of transformed cells observation that the extract, which is mostly laminin and toward a more normal morphology. Fibronectin also fosters which was used at a higher concentration, gave results that were similar to those of purified laminin. The bovine serum albumin concentration Received 10/30/86; revised 4/8/87; accepted 4/16/87. was chosen to be comparable to that of the basement membrane The costs of publication of this article were defrayed in pan by the payment of page charges. This article must therefore be hereby marked advertisement in 3The abbreviations used are: ETC, ethionine-transformed cells; LPC, low accordance with 18 U.S.C. Section 1734 solely to indicate this fact. passage (TRL 1215) cells; HPC, high passage cells; BSA, bovine serum albumin; 1To whom requests for reprints should be addressed. CAPS, cyclohexylaminopropanesulfonic acid. 3802

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1987 American Association for Cancer Research. LAMININ ENHANCEMENT OF CELL GROWTH components and is well below the level reported to inhibit attachment of established cell lines (2, 41). Substrates were applied to wells as follows: (a) control, no substrates added; (b) BSA, S ¿tgprotein/well, diluted with phosphate buffered saline; (c) laminin (Bethesda Research Laboratories, Gaithersburg, MD), 2.5 Mg/well,diluted with 10 mM CAPS-2 Murea, pH 11.0(CAPS- urea buffer); (

Holm-Swarm tumor (42), a gift from Dr. Hynda Kleinman, National D Institute of Dental Research, Bethesda, MD], 25 ¿tg/well,diluted with CAPS-urea buffer (e) plasma fibronectin (Bethesda Research Labora tories), 2.5 ^g/well diluted with CAPS-urea buffer; and (/) type IV collagen (Bethesda Research Laboratories), 5.0 ^g/well, diluted with 0.5 M acetic acid. Williams' Medium D (0.5 ml) was then added to each well and the dishes were allowed to sit at room temperature for 3-4 h. An exception was type IV collagen in the cell spreading experi ments for which 0.5 ml 0.1 M acetic acid was added to each well and the wells were allowed to air dry in a sterile environment. Prior to seeding, all wells were washed three times with Williams' Medium D. [V^l £222 IKX1 Cells were seeded at 2.5 x IO4cells/well for ETC and HPC and at 5.0 Fig. 1. Cell spreading on defined substrata 2 days after seeding. Substrata x IO4cells/well for LPC. were uncoated glass control (C), BSA, fibronectin f/TV), type IV collagen (CIV), laminin (/..If), or basal lamina extract (HI.). Columns, mean projected cell area of Cell Spreading. Two days after seeding, cells were fixed with 2.5% at least 100 cells. Bars, SE *, values that are significantly different from control glutaraldehyde, buffered with 0.1 M sodium cacodylate for 30 min at room temperature, and stained with 0.1% Coomassie blue. Projected cell areas of randomly selected cells were measured using a Zeiss (Fig. 2, b and c), the clusters were loose and many cells showed Videoplan image analysis system. ruffled membranes (Fig. 2, e and /). At confluence (Fig. 3), Cell Growth. DNA and protein analyses, performed in triplicate, were used to study cell growth. Cells were grown on 21-mm glass although the differences in cell shape were no longer present, coverslips in 35-mm-diameter wells. Preparation of the substrate was cells grown on laminin or basal lamina extract still had a larger done as above except that the amount of each substrate was increased projected cell area. 4-fold. Cells were seeded at 10" cells/well (ETC and HPC) or 2 x IO4 Fibronectin was as effective as laminin or basal lamina extract cells/well (LPC). At designated time points, following the procedure of only for ETC (Fig. 1). For the control cells (LPC and HPC), Shiu and Paterson (43), cells were extracted with perchloric acid, the although spreading on fibronectin was significantly increased supernatants were analyzed for DNA by the method of Burton (44), relative to BSA-coated or uncoated glass, it was also signifi and the resolubilized pellets were analyzed for protein by the method cantly less than with laminin or basal lamina extract (P < 0.01). of Bradford (45). Scanning Electron Microscopy. Cells on 9-mm2 cover glasses were The effect of type IV collagen was similar to that of fibronectin for LPC and HPC and similar to that of BSA-coated or un fixed by adding an equal volume of 6% glutaraldehyde-0. l M sodium cacodylate directly to the medium. After fixation for 1.5 h at room coated glass for ETC (P < 0.01). temperature, cells were dehydrated through alcohol to Freon 113, Cell Growth. Measurements of total protein and DNA were critical-point dried with Freon 13, and coated with carbon and gold- made 4 and 6 days after seeding. The overall pattern indicated palladium. that the best growth was found with basal lamina extract and Statistics. Following analysis of variance, statistical significance was laminin, followed by type IV collagen and fibronectin, followed determined by the Student-Newman-Keuls multiple comparison test by BSA-coated and uncoated glass. This pattern was most (46). clearly seen with LPC (Figs. 4 and 5). By day 4, cover glasses with basal lamina extract, laminin, type IV collagen, and fibro nectin had higher DNA levels than was found with BSA-coated RESULTS or uncoated glass, but these differences were not statistically Cell Spreading and Morphology. In pilot experiments it was significant. Protein values for laminin, basal lamina extract, found that differences in cell spreading were greater when glass and type IV collagen were significantly higher (P < 0.05) than was used than when tissue culture was used, although those for BSA-coated or uncoated glass. At day 6, when LPC the absolute values were greater on plastic. As shown in Fig. 1, were still subconfluent, DNA values for basal lamina extract, in subconfluent cultures, mean projected cell areas on uncoated laminin, type IV collagen, and fibronectin were significantly glass were 760 /an2 for LPC, 870 ?m2 for HPC, and 1610 /urn2 higher (P < 0.05) than for BSA-coated or uncoated glass. For for ETC. The difference between the HPC and ETC contrasts protein, laminin and basal lamina extract values were signifi with our previously published results (37). Among the changes cantly higher (P < 0.05) than were values for type IV collagen, in experimental protocol which might have contributed to re fibronectin, and BSA-coated or uncoated glass. duced spreading by HPC was the use in this study of fibronectin- The same pattern held true for HPC at day 4. The differences depleted serum in the medium. were not statistically significant for DNA, but for protein, the All cell sublines spread best on a substratum of laminin or laminin value was significantly higher than were the values for basal lamina extract (P < 0.01). The projected cell area was fibronectin, type IV collagen, and BSA-coated or uncoated glass maximally increased 89% for LPC, 105% for HPC, and 34% (P < 0.05). The protein value for basal lamina extract was for ETC when compared to uncoated or BSA-coated cover intermediate and was not statistically distinguishable from glasses. The increased spreading was accompanied by altera either the low group or the laminin value. As the HPC ap tions in morphology (Fig. 2). Cells grown on laminin or basal proached confluence at day 6, all values tended to converge, lamina extract were not only better spread and flatter but also except for the type IV collagen values which were significantly were more irregularly shaped and gave indication of increased higher (P < 0.01). motility. These changes were most striking for HPC and ETC. For ETC the pattern breaks down further, but even in these Rather than appearing as discrete clusters of polygonal cells cultures the laminin protein value was significantly higher (P < 3803 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1987 American Association for Cancer Research. LAMININ ENHANCEMENT OF CELL GROWTH

Fig. 2. Scanning electron microscopy of cells grown 2 days on uncoated (a-r) or lami- nin-coated (

Fig. 3. Scanning electron microscopy of confluent cells grown on uncoated (or) or laminin-coated (d-f) cover glasses. LPC (a,

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ation in a manner similar to that observed in . If this were true, an analogy could be drawn to proposed autocrine mechanisms of transformed cell growth (47, 48). In the present study all of the basement membrane compo nents fostered increased adhesion as judged by cell spreading measurements. Furthermore, with the nontumorigenic LPC and HPC in subconfluent cultures (i.e., LPC at day 6, HPC at day 4), substrates that induced the greatest spreading (laminin and basal lamina extract that is largely laminin) also supported the greatest cell growth. Cells grown on fibronectin and type IV collagen spread less well and grew less well but did spread better and generally grew better than did cells grown on BSA- coated or uncoated glass. For fibronectin this lower growth level was unexpected given that an extensive fibronectin matrix correlates with maximal spreading and growth under normal ITTI e IV^l BSA g?73 FN E353 OH SX3Q LU EäSäBL culture conditions (37). The data are consistent with those of fig. 4. Protein content of cells grown on defined substrata for 4 and 6 days. Hirata et al. (21), who showed laminin to be more effective Maximum protein values are usually seen with laminin or basal lamina extract. than type IV collagen or fibronectin in fostering the attachment Bars, SE, n = 3. *, values that are significantly different from control (P < 0.05). and growth of fetal liver cells in primary culture. This ranking of the substrata is clearest with LPC; it is less striking with the HPC, which we consider to be preneoplastic, and it breaks down further with the tumorigenic ETC. With respect to cell spreading, ETC show a maximal response on both laminin and fibronectin, the characteristic epithelial and mesenchymal adhesion . However, relative to LPC and HPC, the percentage of increase in spreading on laminin and basal lamina extract was much lower. Laminin had a positive effect on cell growth in subconfluent cultures of ETC, and this was statistically significant in the protein assay. The fact that significant differences in spreading and growth existed between experimental and control samples emphasizes the sensitivity of the cells to defined substrata. The specific effects of preadsorbed substrata were not obscured by the ability of the cells to produce their own basement membrane compo nents (38,49) or by the presence of growth and adhesion factors,

ITTI c IVsl BSA U77X FN Rx?S civ IXX1 LM ESS &L fibronectin excepted, from the serum in the medium. Serum Fig. 5. DNA content of cells grown on defined substrata for 4 and 6 days. albumin absorbed onto the cover glass from the medium may Absorbance at 600 nm. Maximum DNA values are usually seen with laminin or basal lamina extract. Burs, SE, n = 3. *, values that are significantly different have contributed to the effectiveness of the preadsorbed sub from control (P < 0.05). strata by saturating protein binding sites on the glass, thereby inhibiting the subsequent absorption to the glass of secreted 0.05) than the others at day 4. By day 6, as ETC also approached attachment factors (2, 20). confluence, all values were statistically equivalent. In their positive response to laminin in spreading, and prob Growth in Serum-free Medium. The autonomy of the ETC ably also growth, the ETC behaved in a manner that would be was demonstrated by their ability to grow without any exoge expected of epithelial cells (50, 51). In contrast to HPC and nous factors from serum. LPC, HPC, and ETC were grown 6 LPC, their spreading response to fibronectin was similar to that days on tissue culture plastic in serum-free medium. LPC did for laminin, providing evidence that changes in sensitivity to not survive. Some HPC survived, but replication was inhibited. basement membrane components accompany transformation Some ETC survived and were also able to replicate, albeit at a in this cell line. rate much less than in serum-containing medium. By day 6, The behavior of the cells on laminin and basal lamina extract parallel cultures grown in medium containing 1% or 10% serum is also noteworthy. In addition to increased spreading and had become confluent. growth, the morphology of the cells in subconfluent cultures was altered markedly, indicating greater motility, a haptotactic response to the substrate (52, 53). Irregularly shaped cells with DISCUSSION leading and trailing edges are usually found only in cultures of Because transformation by DL-ethionine is accompanied by ETC, but on laminin substrates such cells were also seen com increased cell spreading and adhesion in the TRL 1215 cell monly in cultures of HPC and LPC. line, this line provides an interesting model for studying in cells The lower proportional increases in spreading and growth on of epithelial origin the relationship of adhesion and other cell- laminin and basal lamina extract shown by ETC relative to substrate interactions to tumorigenesis and to the invasiveness HPC and LPC, rather than indicating "poor" growth on the that characterizes malignant tumors. In nontransformed cells, added substrates, indicate the strong growth of ETC on BSA- cell-substrate adhesion has been linked to DNA replication and coated or uncoated surfaces. Thus, the ETC were less affected thus to log phase growth (6). Earlier we had posed the question by material adsorbed to their growth surface, which serves to (37) of whether the increased fibronectin deposition on the emphasize that the concept of the increased autonomy of trans substrate exhibited by the ETC acted to stimulate cell prolifer- formed cells (54) encompasses not only reduced dependence on 3805 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1987 American Association for Cancer Research. LAMININ ENHANCEMENT OF CELL GROWTH

surface contact while nuclear events respond to cell shape in anchorage- soluble factors in medium or but also insol dependent . Cell, 21: 365-372, 1980. uble factors of the substrate or . Reiter, T., Penman, S., and Capeo, D. G. Shape-dependent regulation of Laminiti and, to a less extent, type IV collagen do continue cytoskeletal protein synthesis in anchorage-dependent and anchorage-inde to exert some controlling effect on the overgrowth of ETC, pendent cells. J. Cell Sci., 76: 17-33, 1985. 10. Ungar, F., Geiger, B., and Ben-Ze'ev, A. Cell contact- and shape-dependent perhaps by tying up receptors that would otherwise be involved regulation of vinculin synthesis in cultured fibroblasts. (Lond.), 319: in triggering the transition of the cell through phases of the cell 787-791, 1986. cycle (3, 55-57). However, this control is short lived, and ' Benecke, B.-J., Ben-Ze'ev, A., and Penman, S. The control of mRNA production, and turnover in suspended and reattached anchorage- multilayered rather than monolayered growth prevails. dependent fibroblasts. Cell, 14:931-939, 1978. Much attention has been focused on the association between '- Bissell, M. J., Hall, H. G., and Parry, G. How does the extracellular matrix loss of cell-substrate adhesion and transformation, given the ,, direct gene expression? J. Theor. Biol., 99: 31-68, 1982. Spiegelman, B. M., and Ginty, C. A. Fibronectin modulation of cell shape frequency of this correlation in fibroblastic models. However, and lipogenic gene expression in 3T3-. Cell, 35: 657-666, 1983. this correlation is not a fundamental principle of transformation 14 Lee, E. Y.-H. P., Lee, W. H., Kaetzel, C. S., Parry, G., and Bissell, M. J. Interaction of mouse mammary epithelial cells with collagen substrata: both from the perspective of the many exceptions to this phe regulation of casein gene expression and secretion. Proc. Nati. Acad. 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3807 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1987 American Association for Cancer Research. Effect of Adhesion Factors Fibronectin, Laminin, and Type IV Collagen on Spreading and Growth of Transformed and Control Rat Liver Epithelial Cells

James L. Junker and Ursula I. Heine

Cancer Res 1987;47:3802-3807.

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