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Home , Extracellular matrix, Keratin 18, Keratin 8

Proc. Natl. Acad. Sci. USA Vol. 90, pp. 4261-4265, May 1993 Expression of complete filaments in mouse L cells augments and YI-WEN CHU*, RAYMOND B. RUNYANt, ROBERT G. OSHIMAt, AND MARY J. C. HENDRIX§¶ * Biology Program and §Department of , College of Medicine, University of Arizona, Tucson, AZ 85724; tDepartment of Anatomy, University of Iowa, Iowa City, IA 52242; and tCancer Research Center, La Jolla Cancer Research Foundation, La Jolla, CA 92037 Communicated by Herbert E. Carter, February 1, 1993 (receivedfor review September 24, 1992)

ABSTRACT Intermediate fiament have been are usually expressed in epithelia; is used to diagnose the origin of specific cells. Classically, vimen- found in and endothelial cells whereas and tin is found in mesenchymal cells, and keratins are present in are markers for muscle and neuronal , epithelial cells. However, recent evidence suggests that the respectively (1). Although this cell lineage- and differentia- coexpression of these phenotype-specific proteins augments tion state-specific expression of IFs may be used in the tumor cell motility, and hence, . In the present study, diagnosis of tumors (11), noteworthy exceptions still exist in we used the mouse L-cell model to determine if a direct some tumor species and disease states. Work by Trask et al. correlation exists between the expression of additional keratins (12) showed that there are different sets ofkeratins expressed in these cells, which normally express only vimentin, and their in normal vs. tumor-derived mammary epithelial cells. The migratory ability. Mouse L cells were transfected with human peculiar switch of keratin expression during neoplastic trans- keratins 8, 18, and both 8 and 18. The results indicate that the formation may be a significant phenomenon associated with cells expressing complete keratin riaments have a higher tumor progression. Another interesting observation is the migratory and invasive ability (through extracellular - unusual coexpression of vimentin and keratin in various coated filters) compared with the parental and control- tumor cells, which in a few documented cases correlates with transfected clones. Furthermore, there is an enrichment of metastatic potential. Some examples are as follows: a non- keratin-positive cells from a heterogeneous population of L metastatic rat pancreas adenocarcinoma expresses only vi- clones selected over serial migrations. This migratory activity mentin, whereas a metastatic variant contains a high amount was directly correlated with the spreading ability ofthe cells on ofkeratins in addition to vimentin (13). In a study with murine Matrigel matrix, in which the keratin-positive transfectants sarcoma cells, the ascites from the sarcoma were found to maintain a round morphology for a longer duration, compared coexpress keratin and vimentin, whereas the solid tumor with the other L-cell populations. Collectively, these data from the same sarcoma expresses only vimentin (14). In suggest that keratins may play an important role(s) in migra- several human cell lines, vimentin, in addition tion, through a special interaction with the extracellular envi- to keratins, is expressed only in highly invasive cell lines but ronment, thereby influencing cell shape. not in cell lines of low metastatic potential (15). Last, human , which expresses vimentin as its classical IF Keratins make up the largest group in the intermediate marker, coexpresses keratins in recurrent and metastatic filament (IF) multigene family. There are at least 19 different states (16-18). Collectively, these data suggest that there is keratin proteins, which can be further subdivided into two an additional type of IF expression during the process of groups: type I (acidic) and type II (neutral-basic) keratins tumor metastasis that contributes to migratory and aggres- based on their sizes and isoelectric point (1). One major sive behavior in some manner. difference associated with keratins that distinguishes them To further elucidate if there is a direct correlation between from other IFs, such as vimentin and desmin, is that keratins the expression of additional keratins in cells and their migra- are expressed as heteropolymer pairs consisting of specific tory ability, we have used the mouse L-cell model. Mouse L type I and type II proteins. Their expression is highly cells are fibroblasts that express vimentin. For this study, regulated during embryonic development and cellular differ- these cells have been transfected with human and 18 entiation (2, 3). DNAs (19), and the formation of keratin filaments was The functional role(s) of IFs is still unclear (for review, see visualized by using indirect immunofluorescence staining. refs. 4-6). In , the keratin filaments are found Invasion analysis showed that the cells with complete keratin attached to the cell-cell adherence junctions, known as filaments have higher invasive ability and deformability ca- desmosomes (7), as well as to the cell-extracellular matrix pability to penetrate polycarbonate filters (containing 10-,um The pore size) than control cells, in addition to differential spread- (ECM) adhesion sites, known as (8). ing ability on an ECM. This study suggests that keratins may function ofthis interaction is thought to provide a mechanical a role in influencing cell scaffold for the epithelial sheet to maintain its integrity and play migration, by shape. structure. This hypothesis has been strengthened by recent MATERIALS AND METHODS investigations showing that transgenic mice expressing a Cells and . Mouse L cells transfected with mutant have a phenotype resembling a human different keratin DNAs have been described (19, 20) and for genetic disease known as simplex, our studies were cultured in the appropriate selection media. which is characterized by blistering of the skin due to the Immunofluorescence Staining. Cells were seeded onto glass cytolysis of the basal epidermal cells (9, 10). To our knowl- coverslips to 70% confluence, washed three times with edge, this is the first example demonstrating a relationship phosphate-buffered saline, fixed in cold methanol for 7 min, between abnormal keratin proteins and a human disease. Abbreviations: ECM, extracellular matrix; IF, intermediate fila- The publication costs of this article were defrayed in part by page charge ment; MICS, membrane invasion culture system; DMS, dynamic payment. This article must therefore be hereby marked "advertisement" morphology system. in accordance with 18 U.S.C. §1734 solely to indicate this fact. STo whom reprint requests should be addressed.

4261 Downloaded by guest on September 29, 2021 4262 : Chu et al. Proc. Natl. Acad. Sci. USA 90 (1993) and air dried. Indirect immunofluorescence staining was used RESULTS for staining keratin IFs, using mouse anti-human Keratin Filament Formation in Mouse L Cells. Although (CK5 ; ICN) followed by a rhodamine-conjugated keratins are the type of IFs present in epithelial cells, they secondary antibody (Organon Teknika-Cappel). All fluores- have been expressed in several nonepithelial cell lines by cence staining was observed with a Zeiss standard 18 fluo- using DNA transfection techniques (19, 26). Previously, we rescence microscope equipped with automatic rhodamine have transfected mouse L-cell fibroblasts with human simple and fluorescein filter sets. epithelial keratin 8, 18, or both 8 and 18 DNAs and obtained Invasion and Transfilter Migration Assays. The membrane several different stably transfected lines (19). They are invasion culture system (MICS; refs. 21 and 22) was used to termed LK8 cells, which are L fibroblasts that received the measure cell invasion. A polycarbonate membrane contain- plasmid (LK442-K8) containing keratin 8 cDNA driven by a ing 10-,um size pores (Poretics) was coated with a reconsti- human f3- promoter; LK18, which are L cells that were tuted gel (Matrigel; Collaborative Re- cotransfected with vectors containing a neomycin-resistance search) and placed between the upper and lower well plates marker and a keratin 18 gene; and LK18+K8, which repre- of the MICS chamber. Subsequently, 5 x 104 cells were sent LK18 transfected again with the LK442-K8 plasmid. The resuspended in 10% NuSerum (Collaborative Research) and fate and expression of these transfected keratin DNAs have seeded into the upper wells. After incubation for 72 h at 37°C, been analyzed in detail (19), including RNA and cells that had invaded the Matrigel-coated membranes were analysis (19). Fig. 1 shows the results of the immunofluores- harvested from the lower wells, stained, and counted. Inva- cence staining of these cell lines with an anti-human keratin sion potential was calculated as the percentage of cells that antibody that recognizes simple epithelial keratins: the pa- invaded the Matrigel compared to the total number of cells rental L cells show negative staining as expected; LK8 and seeded (after correction for proliferation). LK18 also display no staining for keratin filaments, indicating The method for measuring transfilter migratory ability was a single type of keratin alone cannot form filaments in the modified from the procedure of McCarthy et al. (23). Instead cells. The LK18+K8 (clone 7) cells have a normal and of Matrigel coating, the polycarbonate membranes were complete filamentous keratin network that extends from the soaked in a solution (0.1 mg of gelatin per ml in 0.02 nuclear membrane to the plasma membrane. Furthermore, M glacial acetic acid) and placed in the MICS chamber with the keratin filament formation in LK18+K8 cells does not cells for 6 h at 37°C. Subsequently, those cells that had affect the endogeneous vimentin IF network normally pro- migrated through the pores were harvested and counted to duced by the mouse fibroblasts (data not shown). calculate the migratory rate. Invasive and Migratory Ability of the Transfected L Cells. Time-Lapse Cinematography. Aliquots of cells were ap- Does coexpression of vimentin and keratins 8 and 18 con- plied to Matrigel-coated dishes at a 1:3 dilution of the original tribute to the invasive ability of the LK18+K8 cells, as we volume. After 2 h of incubation (5% C02/95% air, 37°C), the had observed previously in melanoma (18)? To address this question, we decided to compare the LK18+K8 cells, which cells were videotaped for a 3-h interval using an Olympus contain full-length keratin IFs in addition to , with IMT-2 inverted microscope equipped with optics from Hoff- L cells and LK8 and LK18 clones, which do not form man Optics, Inc. (Hyde Park, NY) and an environmental chamber designed by the microscope's manufacturer. Sub- sequently, videotapes were analyzed on the dynamic mor- phology system (DMS) of the Cell Motility Center of the University of Iowa (24). Each tape was analyzed by tracing the cell outlines of all cells in a particular field (7-27 cells) at 10-min intervals for 2 or 3 h. Analysis by the DMS system included parameters of centroid movement (,tm/h) and cell shape (percent roundness). Data reported are the average of measurements made over the entire recording period. The scale was determined from the included image of a stage micrometer on the videotape filmed at the time of migration. Selection of Migratory Cells. To select subpopulations from a heterogeneous population of LK18+K8 cells, as well as from the keratin-negative parental L cells, a modified MICS chamber (MegaMICS; ref. 25) was used. Cells (1.5 x 106) were seeded in MegaMICS, and the migration assays were performed as described above. Cells were then collected from the bottom wells and plated either on glass coverslips for immunofluorescence staining or in culture flasks to ex- pand for second-round selection. This procedure was re- peated for third-round selection. The number of keratin filament-positive cells was determined by immunofluores- cence staining with the CK5 antibody. The migratory abilities ofall the selected populations were measured simultaneously in parallel experiments. Cell Spreading. To determine the time required for the cells FIG. 1. Immunofluorescence localization of keratins 8 and 18. to spread on different matrices, cells were plated on 24-well Cells were grown on glass coverslips for 24 h, fixed with methanol, tissue culture dishes coated with Matrigel, , col- and stained with mouse anti-human simple epithelial keratin antibody followed by rhodamine-conjugated goat anti-mouse IgG. The cell lagen I, or IV (Collaborative Research). Cell shapes lines are designated as follows: L, parental L cells; LK8, L cells and morphology were monitored periodically using a Wild transfected with keratin 8 cDNA; LK18, L cells transfected with M-40 inverted light microscope, and pictures were taken keratin 18 DNA; LK18&K8, L cells transfected with keratin 18 DNA using Ilford PANF 135 film. and keratin 8 cDNA. (xllOo.) Downloaded by guest on September 29, 2021 Cell Biology: Chu et al. Proc. Natl. Acad. Sci. USA 90 (1993) 4263 additional keratin filaments. The invasive potential of these is probably due to lower levels of expression of keratins in cell lines was determined in an in vitro invasion chamber over these cells, which provided an additional parameter to test in a 72-h period. The ability of the cells to invade Matrigel- the selection experiments. The cells that migrated (over 6 h) coated filters is presented as a series of bar graphs showing through the filters were collected, expanded, and selected percent invasion ± SE (Fig. 2). Cells transfected with K8 or sequentially through MegaMICS for a total of three rounds. K18 alone have similar invasive ability as parental L cells. The number of keratin-positive cells associated with each Interestingly, LK18+K8 cells have the highest invasive round was visually assessed with a fluorescence microscope, ability, which is -3.5-fold greater than that of L cells. and their migratory ability was measured simultaneously. One of the major mechanisms involved in cell invasion is Interestingly, a starting population containing 44.2% keratin- motility (27); therefore, cells demonstrating higher invasive positive clone 7 cells was enriched to 58.3% after the first activity usually show higher migratory ability. To test if this round of selection (clone 7-1) and enhanced again to 68.0% was the case with the transfected L cells, cell movement was and 74.8%, respectively, for clones 7-2 and 7-3. The migra- initially measured as the ability to move in a three- tion analysis of these cells showed there is an increase in dimensional manner from the upper wells of the MICS migration rate that is directly proportional to the percentage chamber through a gelatin-coated filter (containing 10-gm of keratin positivity in each cell population (Fig. 3A). Simi- size pores) over 6 h (Fig. 2). The data reveal that the larly, keratin-positive cells in LK18+K8 clone 5 were en- LK18+K8 showed the highest ability to deform and move riched from 33.9% to 40.0% after the first-round selection through the pores (which are much smaller than the cell size), (clone 5-1) and then increased to 46.5% (clone 5-2) and 57.4% compared to the keratin-negative L cells, as well as the LK8 (clone 5-3) after further selections. Their migration rate also and the LK18 cells. However, no significant difference gradually increased with the enhancement of the number of between L and LK18+K8 cell movement was measured (by keratin-positive cells (Fig. 3B). We have also performed time-lapse cinematography) in a lateral, two-dimensional control selection experiments using keratin-negative L cells. direction after 2 or 24 h on Matrigel matrix (data not shown). The results showed no increase in the migratory ability of Selection of Keratin-Positive Cells from a Heterogeneous migration-selected L cells from the MegaMICS, compared LK18+K8 Population. Not all LK18+K8 cells or other dou- with the parental L cells (data not shown). ble-transfected fibroblasts contain immunofluorescence- Cell Attachment and Spreading in Response to Basement detectable keratin filaments even after subcloning (19). The Membrane Substrate. The invasion of cells through ECM heterogeneity of the population of a particular clone is likely substrates requires several sequential steps: cell attachment due to rearrangement and loss of one of either of the keratin to the ECM, cell spreading via cell matrix-specific receptors, vectors. However, the heterogeneity of the LK18+K8 cells and cell migration through degraded matrix (27). We tested permitted a test of our hypothesis that keratin-positive trans- the ability of the transfected cells to attach to a Matrigel- fected L cells are more invasive and migratory. We per- coated membrane using [35S]methionine-labeled cells. In a formed selection assays using heterogeneous LK18+K8 pop- 1-h assay, L, LK8, LK18, and LK18+K8 all showed -60% ulations (clone 7 and an additional clone 5) that contained of the cells attached to the matrix, with no significant only a certain percentage of keratin filament-positive cells, difference among the four cell lines (data not shown). How- which were seeded into a MegaMICS chamber for migration ever, observations of cell spreading indicated there is a great analyses. Interestingly, the majority of the clone 5 transfec- difference between L and LK18+K8 with regard to the time tants displayed "incomplete" keratin-positive filaments (i.e., required for the cells to spread on the Matrigel matrix. Fig. the filaments were more concentrated in the perinuclear area 4 shows the phase-contrast pictures taken at with fragmented extensions throughout the ). This several time points after the cells were seeded on Matrigel- coated dishes. At 12 h, both cell lines still maintained a rounded morphological shape; after 24 h, L cells started to extend their ; and by 36 h, they had formed a L webnet type of organization. On the other hand, LK18+K8 cells did not spread as well after 36 h. However, the mor- phology and spreading area of both cell lines are similar after 72 h, indicating they both have the ability to interact with Matrigel components for spreading, but it takes much longer LK8 for the LK18+K8 cells to approximate a flattened morphol- ogy. The spreading data were further quantified by comput- erized digital analysis of the photomicrographs shown in Fig. 4. After 24 h on Matrigel, the LK18+K8 cells are -80% If-- ... LK1 8 round in morphology, while the L cells are -50% round. By 36 h, the roundness factor has diminished to 54.2% for LK18+K8 cells and 43.1% for L cells; by 72 h, little differ- ence in shape exists (18.2% for L cells vs. 26.2% for LK18+K8 cells). LK8 and LK18 showed the same pattern of LK18+K8 rapid spreading as L cells (data not shown). We have also used different substrates to test this difference in spreading phenomenon. Both cell lines (L and LK18+K8) respond very 40 30 20 10 0 5 10 15 20 25 quickly and similarly to type I and IV , as well as Percent Invasion Percent Migration fibronectin (data not shown), suggesting that the spreading response on the more complex Matrigel matrix is unique. FIG. 2. Invasive and migratory ability of L cells. Hatched bars depict the percent invasion of L (9.28% ± 1.77%), LK8 (13.94% ± DISCUSSION 3.60%), LK18 (10.61% ± 1.86%), and LK18+K8 (32.54% ± 2.80%) Investigation of the functional role(s) of IFs poses a challenge cells measured over 72 h using Matrigel-coated filters. Cross-hatched as well as a dilemma. Recent breakthroughs, however, for bars show the percent migration of L (5.47% ± 0.15%), LK8 (5.98% keratin studies have advanced our observational ± 1.37%), LK18 (6.26% ± 0.41%), and LK18+K8 (18.39% ± 1.98%) opportunities: cells measured over 6 h using gelatin-coated filters. Standard error specifically, a transgenic mouse model containing a in bars represent data generated from three experiments (n = 4). keratin gene 14, which results in the development of the human Downloaded by guest on September 29, 2021 4264 Cell Biology: Chu et al. Proc. Natl. Acad. Sci. USA 90 (1993)

A B 10 0: Clone 5 0: Clone 5-1 A: Clone 5-2 R- A: Clone 5-3

z 0 0

c- 0

40 45 50 55 60 65 70 75 80 35 40 45 50

% KERATIN POSITIVE CELLS % KERATIN POSITIVE CELLS

FIG. 3. Selection of keratin-positive cells and their migratory abilities. The migratory cells from two heterogeneous LK18+K8 clones, clone 7 (A) and clone 5 (B), were selected by their ability to move through gelatin-coated polycarbonate filter(s) one, two, or three times.

genetic disease epidermolysis bullosa simplex (9), and the we transfected a deleted keratin cDNA into the highly keratin knockout approach, which allows us to study the metastatic melanoma cells and showed (i) the disruption of requirement of keratins in (28). endogenous keratin filament organization and (ii) a dramatic For the present study, we employed gene transfection decrease in invasive and metastatic ability (29). These data technology to address a fundamental question regarding the suggested that the additional keratin filament expression in role(s) of keratin filaments in migration and invasion. Inter- advanced stages of melanoma may be necessary for their estingly, the present study is an extension of an observation aggressive behavior. Recently, we have successfully trans- made in our laboratory with human melanoma tumor cells in fected a human melanoma cell line, A375P, of low invasive/ which we have reported the unusual coexpression of two metastatic ability (which normally expresses only vimentin) different types of IFs-vimentin (which is normally ex- with keratin 8 and 18 DNAs and tested its ability to migrate. pressed) and keratins 8 and 18 in highly invasive and meta- These experiments have shown a 3- to 4-fold increase in the static melanoma (18). We hypothesized that additional IF ability of the transfected cells to migrate compared to the expression may offer a selective advantage for cells to be controls transfected with a neomycin-resistance marker, more migratory. To test this hypothesis, in a previous study which provides additional confirmatory evidence for the role of keratins in augmenting motility (Y.-W.C., R.G.O., E. Ruoslahti, and M.J.C.H., unpublished data). Hence, we have set forth to further test our hypothesis in I the L-cell model and found many similarities in the data generated from this study and the previous melanoma results. Transfection of L cells (which normally express only vimen- tin) with a single type ofkeratin did not result in the formation of keratin filaments in either the LK8 or LK18 stable trans- fectants (19, 20), as expected. However, L cells transfected with both keratins 8 and 18 (LK18+K8) demonstrated a complete keratin filamentous network with immunofluores- cence staining. We next discerned the ability of the L-cell 24 lines to invade Matrigel-coated filters over a 72-h period, as well as to migrate through gelatin-coated filters over 6 h. The results showed the invasive ability of the cells more or less coincided with the transfiter migration rates. To further delineate potential differences in migration across the surface of the Matrigel matrix, we measured the rate of lateral migration (gm/h) ofL vs. LK18+K8 cells at 2 and 24 h, using 36 microcinematography. We found no significant difference between the cell lines; however, this measurement of lateral migration is different from transfilter migration and invasion in that the latter two events encompass deformability in a three-dimensional manner through 10-,um size pores. Re- cently, Robey et al. (30) have also shown that the presence of keratins 18 and 19 in human retinal pigment epithelial cells .;i~ ~ plays an important role in the active migration ability of the 72 S _ .*>tu !,fl~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~cells using a similar type of transfilter migration assay pro- tocol. Interestingly, a correlation between cell deformability *i0*#.:.fiSs _, and metastatic potential has been proposed (31, 32), which indicates that more metastatic cells have a greater ability to *2i_jsnifr5: , _; .F _'t deform and migrate. The invasion and transfilter migration rates of LK18+K8 of L cells on Phase-contrast mi- FIG. 4. Morphology Matrigel. were the highest among all the transfected cells, which crographs ofparental L cells and LK18+K8 cells after 12, 24, 36, and melanoma are Percent supports our hypothesis proposed from the study. 72 h of attachment and spreading on Matrigel compared. is the roundness from an average of 7-27 cells was measured at each time In addition, the most striking result in the present study point and parameter with DMS. Standard error for each value is enrichment of keratin-positive populations by selection via 'l10%o of the calculated percent roundness value. transfilter migration. The heterogeneity of keratin filament Downloaded by guest on September 29, 2021 Cell Biology: Chu et al. Proc. Natl. Acad. Sci. USA 90 (1993) 4265 expression in LK18+K8 cells is likely due to the recombi- facilitate movement; or (ii) in a "negative" model, additional nation and segregation of the many copies of the vectors that IFs may interfere with the binding ofreceptors to the cytoskel- are integrated. However, the number of keratin-positive cells eton, thus preventing them from retarding the mobility of the can be enhanced by several rounds of selection from the cell. Hence, our report has provided an additional approach in migratory cells. Moreover, the population with the higher studying the function ofIFs and has made a simple observation percentage of keratin-positive cells has a higher migratory that enrichment for cells with enhanced ability to migrate rate; in fact, there is a linear correlation between the per- through small pores also selects for an increased frequency of centage of keratin positivity and the migration rate. A control keratin filament expression. Obviously, these cytoskeletal experiment using migration-selected L cells showed that proteins have more complicated roles than just simply pro- there is no increase in the migratory ability of the selected L viding structural support in cells. They may, in fact, be a cells. This indicates that the observed increase in migration critical determinant in motility, affecting both normal and rate is not a general phenomenon associated with the selec- abnormal cell migration. tion procedure. On the other hand, it is uniquely associated with the number of keratin-positive cells in the population. We gratefully acknowledge the scientific contributions of Dr. David Soll and the DMS of the Cell Motility Center at the University These results indicate that indeed keratins are associated of Iowa. We also thank Dr. Richard E. B. Seftor and Mr. Hanifa A. with the increased migratory behavior of the cells. Our data Jones for the preparation of the illustrations. This work was sup- strongly suggest that cells with both vimentin and "com- ported by Public Health Service Grants CA-54984 and CA-59702 plete" keratin IFs can move and invade faster than the ones (from the National Cancer Institute to M.J.C.H.) and CA-42302 with only one type of filament. (from the National Cancer Institute to R.G.O.). The mechanisms involved in the invasion process, proposed 1. Steinert, P. M. & Roop, D. R. (1988) Annu. Rev. Biochem. 57, 593-625. by Liotta (27), can be simplified to consist of (i) attachment to 2. Franke, W. W., Schmid, E., Schiller, D. L., Winter, S., Jarasch, E. D., an ECM, (ii) degradation of the ECM, and (iii) motility of cells Moll, R., Denk, H., Jackson, B. W. & Illmensee, K. (1981) Cold Spring through the degraded matrix. We, therefore, focused our Harbor Symp. Quant. Biol. 46, 431-453. 3. Fuchs, E. (1990) J. Cell Biol. 111, 2807-2814. attention on measuring potential differences in the degradative 4. Klymkowsky, M. W., Bachant, J. B. & Domingo, A. (1989) Cell Motil. ability of the cells that could account for their invasive 14, 309-331. behavior through Matrigel. However, measurement oftype IV 5. Oshima, R. G. (1992) Curr. Opin. Cell Biol. 4, 110-116. collagenase (72 kDa), mouse stromelysin, and in- 6. Skalli, 0. & Goldman, R. D. (1991) Cell Motil. Cytoskeleton 19, 67-79. 7. Jones, J. C. R. & Goldman, R. D. (1985) J. Cell Biol. 101, 506-517. dicated no differential expression of these between 8. Quaranta, V. & Jones, J. C. R. (1991) Trends Cell Biol. 1, 2-4. the parental L cells and the transfected clones. We then 9. Coulombe, P. A., Hutton, M. E., Letai, A., Hebert, A., Paller, A. S. & focused on measuring potential differences in attachment Fuchs, E. (1991) Cell 66, 1301-1311. ability among the cell lines. In these experiments, we found no 10. Vassar, R., Coulombe, P. A., Degenstein, L., Albers, K. & Fuchs, E. (1991) Cell 64, 365-380. differential ability for attachment to Matrigel. These findings 11. Osbom, M. & Weber, K. (1986) Trends Biochem. Sci. 11, 469-472. then prompted us to focus on the process of cell spreading, 12. Trask, D. K., Band, V., Zajchowski, D. A., Yaswen, P., Suh, T. & which involves the interaction of cell surface receptors with Sager, R. (1990) Proc. Natl. Acad. Sci. USA 87, 2319-2323. the ECM. It is also an important step in the process of tumor 13. Ben-Ze'ev, A., Zoller, M. & Raz, A. (1986) Cancer Res. 46, 785-790. 14. Gunther, A., Kinjo, M., Winter, H., Sonka, J. & Volm, M. (1984) Cancer cell metastasis in which cells oflow metastatic potential spread Res. 44, 2590-2594. and attach firmly to the matrix, whereas highly metastatic cells 15. Thompson, E. W., Paik, S., Brunner, N., Sommers, C. L., Zugmaier, do not spread as well, thus facilitating their ability to move G., Clarke, R., Shima, T. B., Torri, J., Donahue, S., Lippman, M. E., freely and detach more easily from the primary tumor mass. Martin, G. R. & Dickson, R. B. (1992) J. Cell. Physiol. 150, 534-544. 16. Trejdosiewicz, L. K., Southgate, J., Kemshead, J. T. & Hodges, G. M. Our data indicate that a longer time is required for the (1986) Exp. Cell Res. 164, 388-398. LK18+K8 cells to spread on a basement membrane matrix 17. Miettinen, M. & Franssila, K. (1989) Lab. Invest. 61, 623-628. composed of a complex meshwork of , type IV colla- 18. Hendrix, M. J. C., Seftor, E. A., Chu, Y.-W., Seftor, R. E. B., Nagle, gen, entactin, , and R. B., McDaniel, K. M., Leong, S. P. L., Yohem, K. H., Leibovitz, A. M., Meyskens, F. L., Conaway, D. H., Welch, D. R., Liotta, L. A. (33, 34) compared with the L cells. It is still unknown how & Stetler-Stevenson, W. (1992) J. Natl. Cancer Inst. 84, 165-174. keratins may affect the spreading process; nevertheless, the 19. Kulesh, D. A., Cecefia, G., Darmon, Y. M., Vasseur, M. & Oshima, retarded spreading of LK18+K8 correlates very well with R. G. (1989) Mol. Cell. Biol. 9, 1553-1565. their invasive ability. We speculate that the less-spread mor- 20. Kulesh, D. A. & Oshima, R. G. (1988) Mol. Ceal. Biol. 8, 1540-1550. 21. Hendrix, M. J. C., Seftor, E. A., Seftor, R. E. B. & Fidler, I. J. (1987) phology exhibited by LK18+K8 cells is more conducive for Cancer Lett. 38, 137-147. their migration through 10-,um pores. 22. Hendrix, M. J. C., Seftor, E. A., Seftor, R. E. B., Misiorowski, R. L., The occurrence of IF coexpression has been reported in Saba, P. Z., Sundareshan, P. & Welch, D. R. (1989) Invasion Metastasis several other species (35) and in specific cases of neoplastic 9, 278-297. 23. McCarthy, J. B., Palm, S. L. & Furcht, L. T. (1983) J. Cell Biol. 97, progression. Moreover, it is a prevailing phenomenon in early 772-777. embryogenesis. It has been suggested that the coexpression 24. Soll, D. R. (1988) Cell Motil. Cytoskeleton 10, 91-106. phenotype in parietal endoderm cells of the early mouse 25. Seftor, E. A., Seftor, R. E. B. & Hendrix, M. J. C. (1990) BioTech- embryo may be involved in the ability of cells to detach and niques 9, 324-331. 26. Domenjoud, L., Jorcano, J. L., Breuer, B. & Alonso, A. (1988) Exp. Cell migrate away from an epithelial sheet (36). The embryonic Res. 179, 352-361. cells are thought to be very motile and communicate differ- 27. Liotta, L. A. (1986) Cancer Res. 46, 1-7. ently with the ECM compared with differentiated cells. 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