~Copyright 1992by The Humana Press Inc. All rights of any nature whatsoeverreserved. 0163-4992/91/1802-0123504.20

Transglutaminase Stabilizes Melanoma Adhesion Under Laminar Flow D. G. MENTER, *'1 J. T. PATTON, 2 T. V. UPDYKE, 1 R. S. KERBEL, 3 M. MAAMER,4 L. V. MCINTIRE, 2 AND G. L. NICOLSON I

1Department of Tumor Biology, The University of Texas M. D. Anderson Cancer Center, Houston, TX; 2Cox Laboratory for Biomedical Engineering, Institute for Biosciences and Bioengineering, Rice University, Houston, TX; 3Cancer Research Division, Sunnybrook Health Science Centre, Toronto, Ontario, M4N 3M5 Canada; and 4lnnothera Recherche, AreweU, Cedex, France ABSTRACT To resist substantial wall shear stress (WSS) exerted by flowing blood, metastatic melanoma cells can form adhesive contacts with subendothelial extracellular matrix proteins, such as fibronectin (FN). Such contacts may be stabilized by transglutaminase catalyzed-crosslinkage of cell focal adhesion proteins. We analyzed human melanoma cell adhesion under flow by decreasing the flow (WSS) of melanoma cell suspensions and allowing them to adhere to immobilized wheat germ agglutinin or FN. At the wall shear adhesion threshold (WSAT), cell adherence was rapid with no rolling. Following cell adherence, we increased the flow and determined the wall shear detachment threshold (WSDeT). Cells spread and remained adherent on immobilized FN at high WSDeTs (___32.5 dynes/cm2). The high resistance of adherent cells to shear forces suggested that transglutarninase-mediated crosslinking might be involved. Transglutaminase inhibitors monodansylcadaverine and INO-3178 decreased WSAT, and at low concentrations completely inhibited tumor cell spreading and promoted detachment at low WSDeTs (0.67 dynes/cm2). In static adhesion assays, transglutaminase inhibitors decreased cell adhesion to immobilized-FN * Author to whom all correspondence and reprint requests should be addressed.

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in a dose-dependent manner and prevented the formation of crosslinked ~2SI-FN complex that failed to enter a SDS-polyacrylamide gradient gel. The data suggest that transglutaminase-catalyzed crosslinking, particularly in the presence of WSS, may be important in stabilizing cellular adhesive contacts during adhesion to immobilized-FN. Index Entries: Parallel plate flow chamber; wall shear stress; transglutaminase; tumor cell adhesion; cell adhesion threshold; cell detachment threshold; cell spreading; melanoma; detergent-insoluble protein complex; fibronectin; integrin; wheat germ agglutinin.

INTRODUCTION

Blood-borne tumor metastasis formation involves several sequential steps (for reviews, see 1-3). Once in the blood, large numbers of tumor cells may circulate, but only a small fraction of these are capable of successfully implanting in the microcirculation, invading at the secondary site, and establishing metastatic colonies (4,5). Tumor cell implantation in the microcirculation is thought to be a critical step in the metastatic sequence, and inhibiting the adhesive properties of malignant cells with drugs (6-8) or peptide inhibitors (9,10) can significantly reduce the formation of experimental blood-borne tumor metastases. In addition, highly metastatic animal tumor cell lines also show higher rates of adhesion than their poorly metastatic counterparts to microvessel endothelial cells (11,12) or their subendothelial matrix (13). The adhesion molecules involved in these interactions are only partially k n o w n (14). After adhering to endothelial cells in the microcirculation, tumor cells are capable of stimulating endothelial cell retraction and exposure of subendothelial matrix (15-17). The subendothelial matrix contains molecules, such as fibronectin (FN) and laminin, that stimulate tumor cells to adhere, spread, and stabilize adhesive contacts (13,18-20). The arrest and adhesion of tumor cells to vascular surfaces in vivo must occur u n d e r conditions involving fluid shear forces that are difficult to mimic in vitro. The use of a parallel plate flow chamber allows for evaluating the flow fields and assessing the wall shear stress (WSS) effects on cell adhesion (21-23). In our studies, we utilized a parallel plate flow chamber with well-defined shear flow to examine tumor cell adhesion to immobilized FN. We examined metastatic h u m a n MeWo melanoma cells and their wheat germ agglutinin-(WGA) resistant variants (70W, 3S5) for their adhesive behaviors under flow, focusing primarily on the stability of adhesion u n d e r WSS. The expression of WGA-binding sites on these selected m e l a n o m a lines correlates with their experimental metastatic potentials in n u d e mice (70W ~_MeWo > 3S5), but not with their ability to survive WGA-mediated cytotoxicity (24).

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The expression and distribution of cell surface receptors on metastatic cells may be important during adhesion to various substrates (7) since the efficiency with which such cells establish stable adhesive contacts may be related to their survival in the presence of shear forces in the circulation. Adhesion stabilization under shear requires a number of cellular properties, including the ability of cells to reorganize their cytoskeletal systems and spread. It may also be related to receptor stabilization by transglutaminase-mediated crosslinking. Transglutaminases catalyze the formation of E(v-glutamyl)lysine covalent crosslinks between substrates (25,26), and transglutaminase activity has been found in hepatocyte plasma membrane complexes (27-32). Since protein crosslinking of FN or fibrinogen into detergent-insoluble complexes was associated with hepatocyte cellcell adhesion or endothelial cell membrane-fibrinogen crosslinking (2934), we examined the effect of the competitive transglutaminase inhibitor monodanslycadaverine (MDC; 35, 36) and the irreversible transglutaminase inhibitor INO-3178 (3178 AQ), 2-[3-(diallylamino)proprionyl] benzothiophene (37-39) on irreversible tumor cell adhesion to and spreading on immobilized FN. We also examined 125I-FN crosslinking to tumor cell surfaces and the formation of high-Mr glycoprotein complexes following adhesion and spreading of melanoma cells. Our data suggest that cell surface glycoprotein and transglutaminase expression may be important during MeWo cell adhesion and stabilization under shear flow conditions.

METHODS Transglutaminase Inhibitors MDC was purchased from Sigma (St. Louis, MO). INO-3178 (3178AQ; 2-[3-(diallylamino)proprionyl] benzothiophene was synthesized as previously described (37,38).

Tumor Cell Lines The human MeWo melanoma parental cell line was propagated from a lymph node metastasis originating in a 78-year-old male Caucasian in 1974 by Koder and Bean (40). WGA was utilized to select MeWo variant cell lines in tissue culture (24). The 3S5 variant arose after 3 wk in tissue culture during exposure to graded concentrations of 30, 40, and 50/~g/mL WGA. The 70W variant arose during multiple tissue culture passages over a 20-wk period in graded concentrations of WGA (30, 40, 50, and 70 #g/mL. Tumor cells were passaged prior to reaching confluence using Ca 2§ Mg2§ HEPES (N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid) buffered saline (CMFH: 129 mM NaCI, 5 mM KC1, 0.3 mM Na2HPO4.7 H20, I mM NaHCOa, 5 mM Glc, 25 mM HEPES, pH 7.4, (41)

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containing 2 mM EDTA. Cells were cultured in a humidified 5% CO2 atmosphere in DMEM/F12 (1:1) containing 5% fetal bovine serum (FBS) without antibiotics.

Growth Analysis Cell growth responses to transglutaminase inhibitor treatment were analyzed as previously described (42). Briefly, subconfluent tumor cells grown in 100-ram tissue culture plates (Corning, Houston, TX) were washed with serum-free DMEM, pH 7.4, containing 25 mM HEPES and 25 mM D-Glc. Tumor cells were washed once with CMFH and removed from tissue culture plates using CMFH containing 2 mM EDTA, washed once with DMEM (pH 7.4), containing 25 mM HEPES, 25 mM D-Glc and 5% FBS, and resuspended at a concentration of 2.0 x 10S cells/mL. Transglutaminase inhibitors MDC and INO-3178 were prepared at twice the final concentration in serum-free DMEM and mixed 1:1 with twice the final concentration of tumor cells in serum-containing medium. Tumor cells were preincubated in drug or diluent containing medium for 30 min in a 50 mL conical tissue culture tube. Each growth assay was performed by the addition of 200/~L of a cell suspension (2.0 x 104 cells) to each well of a 96-well tissue culture plate in the presence or absence of the appropriate concentration of transglutaminase inhibitor. The plates were incubated at 37~ in a humidified 95% air/5% CO2 atmosphere for 2, 4, and 6 d. A single microtiter plate represented a single time-point using the various drug dilutions, and the incubation was terminated by vigorous washing with Delbucco's phosphate-buffered saline (DPBS). Each plate was immediately fixed by the addition of 0.1% gluteraldehyde in DPBS. Plates were washed twice with DPBS and incubated at 25~ with 0.1% crystal violet in DPBS for 30 min. Plates were then washed extensively with DPBS and the cells were solubilized with 1% SDS in 5 mM HEPES buffer, pH 7.0. Absorbance was determined at 590 nm on a Titertek Multiskan Model 310 C (Flow Laboratories, McLean, VA). Initial experiments were performed to assure a linear correlation between crystal violet dye absorbance equivalents and cell number using a Model ZBI Coulter Counter (Coulter Electronics, Hialeah, FL) (43).

Protein-Coated Glass Slides Human plasma FN (Collaborative Research, Bedford, MA) containing 140 mM CAPS [3-(cyclohexylamino)-propanesulfonicacid], 200 mM NaC1, and 2 mM CaC12 was reconstituted at a concentration of I mg/mL with 50 mM NaC1, 25 mM HEPES, pH 7.5. FN (3 mg) was concentrated and desalted using Amicon CM-30 Centricon to a dead stop volume of 50/~L, and was reconstituted in 2 mL of buffer containing 50 mM NaC1, 25 rnM HEPES, pH 7.5, and then diluted to 100/~g/mL with H20. Glass slides (75x38 ram, Curtis Mathis) were cleaned using RBS-detergent (Pierce

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Chemical Co., Rockford, IL), rinsed with H20, followed by 95% ethanol, placed in Petri dishes, and allowed to dry in a laminar flow hood. FN (3/~g/cm 2) was applied at a concentration of 100 #g/mL and allowed to dry in a 3.5 cm 2 area approximating the flow area of the parallel plate flow chamber on RBS-cleaned, ethanol-rinsed glass slides. Aseptic procedures were utilized during the coating process, and the slides were used within 1-2 h.

ADHESION ANALYSIS Parallel Plate Flow System A parallel plate flow chamber was constructed to form a uniform channel with a small gap-to-width ratio in order to ensure a geometry approximating two infinite parallel plates (21,23). Briefly, a polycarbonate shear deck supports a glass slide held in place by a vacuum applied through holes in a silastic support gasket. Fluid flow is through a slotted manifold to maintain fully uniform laminar flow over the entire chamber. The velocity profile is parabolic under laminar flow in this system (Reynolds number 32.48 dynes/cm 2. Varying cell concentrations between 1-5 x 10s cells/mL did not affect WSAT determinations.

Microtiter Adhesion Assay Untreated flat-bottomed, 96-well microtiter plates (Flow Laboratories Inc., McLean, VA) were coated with FN at a concentration of 2.5 #g/cm 2. Plates were washed twice with H20 and then incubated for I h with DMEM containing 1% BSA. Preconfluent tumor cells were prepared as described for the growth assays, except that they were resuspended in 2 mg/mL saltand fatty acid-free BSA at a concentration of 6.4x 105 cells/mL. Transglutaminase inhibitors MDC and INO-3178 were prepared at twice their final concentration in CMFH and mixed 1:1 with twice the final concentration of tumor cells in BSA-containing medium. Tumor cells were preincubated for 30-90 min in a 50 mL conical tissue culture tube, and the adhesion assay was performed by the addition of 100 #L of a cell suspension at a concentration of 5.0xl05/cm 2 to each well in the presence or absence of the appropriate concentration of transglutaminase inhibitor. Plates were incubated at 37~ in a humidified CO2 atmosphere for 5, 15, 30, 60, and 120 min.

Statistical Analysis Statistical analyses included: Fisher PLSD, Scheffe F-test, Dunnett-t, and matrix correlation coefficients; these were calculated using Statview 512 + on a Macintosh computer. Nonparametric data were analyzed for significance using Kruskal-Wallis analysis of variance and the H statistic from each test was evaluated using standardized tables containing critical values for Kruskal-Wallis H distribution.

lodination of FN Three milligrams of FN was reconstituted with H20, concentrated and desalted using Amicon CM-30 Centricon to a dead stop volume of 50 #L, and reconstituted in 250 /~L of buffer containing 50 mM NaC1, 25 mM HEPES, pH 7.5. Carrier-free [12sI] NaI (1 mCi//dVl, ICN) was added Cell Biophysics

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to the FN solution, followed immediately by the addition of 10/~L of 20 mM Chloramine-T (Aldrich, Milwaukee, WI). Labeling continued for 60 s and the reaction was terminated by the addition of 5 /~L of 20 mM sodium metabisulfite. Because of the inherent nonspecific adhesive properties of FN, substantial losses occurred on PD-10 cleanup columns, even when preblocked with 1% BSA or 2% polyvinylpyrolidone. Therefore, the radioactive FN was adsorbed to six well plates overnight and the unbound radioactivity was removed by washing vigorously six times with DPBS. The specific trichloroacetic acid-insoluble radioactivity activity of FN was 2.14+0.15 x 106 cpm//~g protein.

Cell Adhesion to 1251-FN Multiwell plastic tissue culture plates (6 wells/plate; Coming, Houston, TX) were coated with 2.5/~g/cm 2 of 12SI-FN and allowed to dry overnight. Plates were washed three times with 2 mL H20 and preincubated with DMEM, pH 7.4, containing 25 mM HEPES, 25 mM D-GLC, and I mg/mL salt- and fatty acid-free BSA. Preconfluent tumor cells were treated as described for static adhesion assays. The adhesion assay was performed by the addition of 2 mL of cell suspension to each well in the presence or absence of the appropriate concentration of transglutaminase inhibitor. Plates were incubated at 37~ in a humidified 95% air, 5% CO2 atmosphere, and cells were removed by the addition of 8 mM EDTA to each well. The tumor cells were removed by aspiration, and each well was washed twice with CMFH. The tumor cell suspension and washes were combined in 13 mL conical tissue culture tubes, cooled in wet ice, and centrifuged at 350g for 5 min. Cells were washed twice with 6 mL of CMFH by centrifugation at 200g at 4~ and the final pellet was resuspended in I mL of CMFH at 4~ and transferred to a I mL microfuge tube. Samples were centrifuged for 10 s in an Eppendorf microfuge (model 5414, Brinkmann Instruments, Westbury, NY). The resulting pellet was solubilized in 10 mM CHAPS, 147.6 mM Tris, 66.32 mM HC1, pH 8.22. This buffer also contained 10% glycerol, 50 mM NaC1, 40 ~ leupeptin, 2 mM PMSF, 10 mM dithiothreitol, 2 mM iodoacetamide, and 2 mM EDTA. SDS was added to a final concentration of 2% and denatured at 95 ~ for 5 min just prior to performing electrophoresis.

Gradient-SDS-PAGE The gel electrophoresis system consisted of a continuous, linear pH and buffer strength gradients without added SDS (44) and were designed to improve stacking and resolution of SDS-proteins. For polyacrylamide gradient gels, 6-12% acrylamide, 4% N, N'-methylene-bis-acrylamide were cast in a modified cassette size (15x16x0.1 cm). In addition to a polyacrylamide gradient, a moving boundary velocity gradient was formed by both buffer and pH gradients to improve stacking and resolution. The gradients consisted of a stacking leading phase buffer (369 mM Tris base, Cell Biophysics

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166 mM HC1, pH25o= 8.29) and a resolving leading phase buffer (08.22M Tris base, 0.068M HC1, pH2so=9.21). The anolyte buffer consisted of 20 mM Tris base, 10 mM HC1, pH25o= 8.07, and the catholyte buffer consisted of 20 mM glycine, 10 mM Tris base, 0.03% SDS, pH2so= 9.18. The Mr standards consisted of 50 ng each of [14C]-mol wt standards (BRL). GradientSDS-PAGE was run at 50 V until the dye-fronts entered the continuous gels, and they were run at 30 mA/gel until the dye-fronts were 5 m m from the bottom of the gel.

Silver Staining of Gels The gels were fixed in 200 mL of 45% ethanol, 10% acetic acid for 2x 30 min. The gels were neutralized and dehydrated with 50% ethanol for 3 x I h with quick water rinses. An ammoniacal silver solution was prepared from solution A (0.8 g silver nitrate dissolved in 4 mL H20), solution B (21 mL of 0.36% of NaOH and 1.4 mL of ammonium hydroxide reagent). Solution C was prepared by the slow addition of solution A to solution B with constant stirring, and then solution C was brought to 100 mL with H20. The gels were incubated in solution C for 15 min with constant aggitation. The gels were briefly washed three times with H20 followed by three times for 20 min with 200 mL of 20% ethanol. Fresh developer solution was added that contained 1.5 mL of 1% citric acid and 0.15 mL of 38% formaldehyde reagent in 300 mL of 20% ethanol. Staining was stopped with 3 mL of glacial acetic acid for 5 rain, followed by 6 mL of ammonium hydroxide reagent (28%) for 10 rain. The gels were rinsed four times with H20, stored in 300 mL of 20% ethanol at 4~ and then photographed. Dot Blot Analysis of SDS-lnsoluble Cell Lysates SDS-insoluble cell material was isolated by centrifuging at 10,000g and washing the SDS-insoluble material two times with CHAPS solubilizat-ion buffer containing 2% SDS as described in the preceeding section. SDSinsoluble material was then blotted to 0.45/~m Immobilon-P (Mlllipore, Bedford, MA) in a 96-well dot blot apparatus (Bio-Rad, Richmond, CA). Autoradiography of Dot Blots or Electrophoresis Gels Prior to exposing Kodak XAR 5 film, silver stained gels were incubated in 20% ethanol for I h and dried, or dot blots were rinsed with H20 and dried. After the appropriate exposure time, film was developed in an AFP Imaging, 14XL X-Ray Film Processor.

RESULTS

Determination of WSAT and WSDeT The adhesive potential of flowing MeWo cells was determined by beginning WSS at 1.79-3.68 dynes/cm2 and gradually decreasing the flow/wall shear rate until cells began to adhere (Table 1). The point at CellBiophysics

Table 1 Wall Shear Stress Threshold, Spreading, and Release Values of H u m a n Melanoma Cells Cell line adhesion parameter

Monodansylcadaverine Untreated

100~

200~

400~

INO-3178 10~

50~

100p.M

MeWo WSAT

0.67, 0.34 0.17, 0.34 0.34

WSDeT

5 x (_>32.48)

0.34 0.34

0.34 0.34

0.17 0.17

0.34 0.34

0.17 0.34

0.17 0.17

->32.48 _>32.48

3.58 3.58

1.79 1.79

6.83 13.88

3.58 3.58

3.58 3.58

% Cells spread

100, 94.4 77.0, 76.6 85.0

36.4 54.8

0 10.0

0 0

0 0

0 0

0 0

% Cells released

0, 10.0 11.0, 10.5 5.0

72.7 65.4

50.0 74.8

92.3 88.6

100.0 100.0

100.0 100.0

100.0 100.0

3S5 WSAT

0.34, 0.34 0.67, 0.34 0.34

WSDeT

5 x (___32.48)

% Cells spread

% Cells released

94.4, 93.8 100, 79.0 83.0 0, 0, 0, 26.7, 18.9

0.34 0.34

0.34 0.34

0.34 0.17

0.34 0.34

0.34 0.34

0.17 0.34

_>32.48 _>32.48

1.79 3.58

1.79 1.79

3.58 6.83

0.67 1.79

0.67 0.67

71.4 68.9

20.0 0

0 0

0 0

0 0

0 0

42.8 60.0

78.0 50.0

82.4 86.8

100.0 92.0

100.0 100.0

100.0 100.0

70W WSAT

WSDeT

% Cells spread

% Cells released

0.67, 0.67 0.67, 0.67 0.67, 0.67 0.67, 0.67, 0.67 9 x (-> 32.48) 91.4, 90.0, 100.0 92.3, 100 80.0, 90.5 90.6, 92.1 8.3,0,0, 15.3, 0 10.5, 0

0.67 0.67 0.67

0.67 0.67 0.67

0.34 0.34 0.34

0.67 0.67 0.67

0.67 0.67 0.67

0.34 0.34 0.34

>__32.48 _>32.48 ___32.48 ->32.48 _>32.48 _>32.48 90.0 91.7 90.9 92.8 92.0 93.3

3.58 3.58 1.79 80.0 85.0 79.7

13.88 ->32.48 13.88 0 0 0

6.83 6.83 3.58 0 0 0

3.58 3.58 0.67 0 0 0

14.3 9.0 10.3

8.3 0 13.3

15.0 10.0 16.5

28.6 25.9 23.5

23.3 53.3 25.0

100.0 87.5 100.0

6.5, 0 Adhesion studies were performed using human MeWo, 3S5, or 70W melanoma cells under flow in the presence of WSS defined using a parallel plate flow chamber. The WSS levels were calculated by varying the flow rates. The threshold WSS at which cells either adhered to or detached from proteincoated glass slides were defined as WSAT and WSDeT, respectively. FN or WGA were coated on glass slides at a concentration of - 2.5 gg/cm2. The percentage of cells that spread and the percentage of cells released as WSS was increased were determined using videomicroscopy. MeWo, 3S5, or 70W tumor cells were pretreated for 15-30 min with the appropriate concentration of transglutaminase inhibitor, and cell adhesion studies were performed in the presence of defined WSS. The percentage of cells that had spread and the percentage of cells released as WSS was increased were determined using videomicroscopy. The data are represented as individual determinations.

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Table 2 Statistical Correlations Between Melanoma Cell WSAT Values to Immobilized WGA and FN, Metastatic Potential and WGA Affinity

FN WSAT WGA WSAT WGA affinity Lung colonies

FN WSAT

WGA WSAT

WGA affinity

Lung colonies

1 0.786 0.652 0.643

1 0.941 0.849

1 0.94

1

Comparison of WGA binding affinity and lung colonizing ability were calculated. Matrix correlation coefficients were generated using raw data and a statistics program (see Materials and Methods). Data on number of lung nodules formed by the MeWo, 3S5, and 70W cell lines were from ref. 24. WGA affinity data was entered as the maximum bound CHAPS solubilized trans[3sS]-amino acid metabolically labeled MeWo, 3S5, and 70W cell lysates to a WGA affinity column in three different experiments.

which adhesion began was defined as the wall shear adhesion threshold (WSAT). Adhesion of untreated cells to immobilized WGA or FN was essentially instantaneous. At shear rates low e n o u g h to establish adhesive contacts, we observed no saltatory cell rolling. Based on correlation coefficient analysis, adhesion of the 70W, MeWo parental, and 3S5 cells to immobilized WGA in the presence of WSS correlated with their metastatic potential in n u d e mice (70W > MeWo > 3S5; Table 2). The MeWo parental cell line and the poorly metastatic 3S5 cell line exhibited a lower WSAT on immobilized-WGA than the high metastatic 70W cell line. The intermediate metastatic potential parental MeWo and poorly metastatic 3S5 cell lines exhibited WSATs on immobilized WGA of 1.2+0.6 d y n e s / c m 2 and 0.98+0.8 dynes/cm 2, respectively; this was approximately one-half the WSAT observed for the highly metastatic 70W cell line (2.38+0.8 dynes/cm2). Similarly, for adhesion of MeWo parental, 3S5, and 70W cells to immobilized-FN, we found WSATs of 0.37+0.18, 0.41+0.15, 0.67_+0 d y n e s / c m 2, respectively. The b i n d i n g of CHAPS lysates to immobilized W G A affinity columns (data not shown) and the WSAT observed for WGA correlated better with lung-colonizing potential than the WSAT of the cell lines to immobilized-FN (Table 2). Wall shear d e t a c h m e n t threshold (WSDeT) is important in maintaining adhesive interactions once they formed in the presence of substantial WSS. WSDeT was determined by increasing flow from 0.15 d y n e s / c m 2 to _>32.48 d y n e s / c m 2 in an attempt to dislodge the melanoma cells. The m e l a n o m a cells were able to withstand very high WSS levels on immobilized FN; b e t w e e n 82-94% of the cells examined remained a d h e r e n t to immobilized FN at WSS of _>32.48 dynes/cm 2, the highest WSS that could be maintained in our instrument. The ability of the t u m o r cells to maintain adhesion to immobilized FN in the presence of substantial WSS could be related to their ability to spread on this substrate. H u m a n m e l a n o m a cells spread within 15 min after adhesion to immobilized FN in the presence of defined WSS. The Cell Biophysics

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percentage of spread melanoma cells was inversely related to the number of cells released (Table 1). The greater the extent of cell spreading, the less detachment occurred with increasing WSS.

Tr a n s g l u t a m ina se Inhibitors Since the WSS required to remove MeWo melanoma cells adherent to immobilized FN was very high, strong molecular forces must be involved in the adhesion process. Thus, we investigated the possibility that covalent linkages catalyzed by transglutaminase might be involved in stabilizing the adhesive process. We examined tumor cell adhesion and the potential involvement of transglutaminases by treating cells with MDC (a competitive inhibitor) or INO-3178 (an irreversible inhibitor). Differences in WSAT levels were only observed following treatment of melanoma cells with concentrations ~ 400 ~ MDC or 100 ~ INO-3178. At these inhibitor concentrations, the WSAT levels of MeWo and 3S5 tumor cells were decreased from 0.34 dynes/cm2 to 0.17 dynes/cm2. In comparison, WSAT levels of the highly metastatic 70W tumor cells were decreased from 0.67 dynes/cm2 to 0.34 dynes/cm2. In general, the detachment threshold values were significantly different by comparison to WSAT values as a function of transglutaminase inhibitor concentration. At a concentration of 100 ~ MDC, a high WSDeT (___32.48 dynes/cm2) was necessary to remove the adherent cells, but at 200 ~ MDC, the MeWo and 3S5 cells had WSDeT values that were substantially lower than the highly metastatic 70W cells. When treated with 400 ~ MDC, all three cell lines exhibited WSDET values of ___3.58 dynes/cm2 (Table 1). In general, tumor cells treated with 100 ~M INO-3178 were dislodged from immobilized FN at a significantly lower WSDeT than those treated with an equimolar dose of MDC. The poorly metastatic 3S5 cells were removed from immobilized FN at lower WSDeT levels than either the MeWo or 70W cells (Table 1). In comparison with the MeWo and 3S5 cells, highly metastatic 70W cells were more capable of spreading in the presence of MDC (Table 1). INO-3178 was extremely effective at preventing melanoma cell-spreading. Even at the lowest concentration (10 /dV/) of INO-3178 examined, cell spreading was not observed. Interestingly, the highly metastatic 70W cells did not visibly spread on immobilized FN, yet only 23.5-25.0% of the cells were dislodged when they were treated with 10-50 ~ INO-3178. Under these conditions, all of the melanoma cells treated with 100 INO-3178 were dislodged from immobilized FN.

T r a n s g l u t a m ina se Inhibitor Effects on Cell Viability and Growth Transglutaminase inhibitors had little effect on cell viability over the course of the adhesion experiments. Greater than 95% of transglutaminase inhibitor-treated tumor cells remained phase bright and were capable of Cell Biophysics

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vital dye exclusion over the 4-8 h course of the experiments. In comparison, long-term growth studies revealed a dose-dependent effect of transglutaminase inhibitors on cell division rates over a 6-d period (Fig. 1). All three melanoma cell types appeared, however, to be capable of adaptation or recovery from these drug treatments. MDC and INO-3178 appeared to have significant effects on cell growth at concentrations >_ 200 pdVi, but the cells appeared to begin growth recovery within 2-4 d.

Static Adhesion Assays We performed melanoma cell adhesion assays to immobilized FN under static conditions to examine transglutaminase inhibitor effects. MDC was ineffective at preventing adhesion at doses lower than 100/d~d, but we observed inhibition of adhesion at 200 ~ (Fig. 2). Inhibition increased substantially when the concentration of MDC was increased to 400 ~ (Fig. 2A). The irreversible inhibitor INO-3178 was substantially more effective than MDC at blocking MeWo melanoma cell adhesion over the entire concentration range tested (Fig. 2B).

Adherent Cell CHAPS-Insoluble Complex Formation with 12SI-FN Substrate To evaluate the total amount of FN associated with the cell surface during adhesion and spreading on immobilized FN, we performed a static adhesion immobilized 12H-FN. Cells were allowed to adhere, and the total amount of cell-bound FN was determined after removal of cells with 8 mM EDTA, followed by extensive cell washing. Packed tumor cell pellets were solubilized in 5 volumes of solubilization buffer B that contained 2 mM EDTA and 10 mM dithiothreitol as a reducing agent and 2 mM iodoacetamide to acetylate and irreversibly inactive transglutaminase. Treatment with 100 p.M MDC or 100 p.M INO-3178 caused a decrease in bound 12SI-FN. The formation of an SDS-insoluble multiprotein complex containing ~2SI-FNwas inhibited by 100 ~ MDC, and to a greater extent by 100 pdVlINO-3178 (Fig. 3A). Using gradient-SDS-PAGE, we analyzed for the appearance of transglutaminase inhibitor-dependent high-Mr CHAPS-insoluble complexes containing radiolabeled FN (Fig. 3B). These complexes failed to migrate into the gradient-SDS-PAGE system (Fig. 3C, arrows). The untreated cells of all three cell types bound the greatest amount of 12SI-FN. By both 12SI-FNbinding and association of ~2SI-FNwith the high-Mr CHAPS-insoluble complex seen in gradient-SDS-PAGE, there was nearly a 2-3-fold reduction in the amount of ~2SI-FN bound by the MeWo and 3S5 cells. Treatment of the 70W cells with 100 ~ INO3178 caused less than a twofold reduction in amount of 12SI-FN bound based on gradient-SDS-PAGE (Fig. 3).

Cell Biophysics

Stabilization of Melanoma Adhesion

.n,

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1061 0.0 ~M

lO5 *

3.1

6.3 laM

10 4 4

12.5 lxM 25.0 .aM

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50.0 .aM 100.0 ~M 200.0 .aM

10 1

400.0 .aM 10 ~

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Day 2

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Day 4

Day 6

10 6 -----

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1.6 .aM

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6.3

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12.5 .aM 25.0 .aM

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Day 4

Day 6

200.0 .aM

Fig. 1. Melanoma cellular growth responses to transglutaminase inhibitor treatment. Cellular growth was analyzed in microtiter plates in the presence or absence of the appropriate concentrations of transglutaminase inhibitor at 2, 4, and 6 d. Growth assays were terminated by vigorous cell washing, and the cells were immediately fixed and stained with 0.1% crystal violet. The plates were then washed and the cells were solubilized with HEPES-buffered SDS (see Materials and Methods for details). A linear correlation was found between crystal violet dye binding absorbance at 590 nm and cell number. Each time-point of a given experiment was calibrated based on dye-binding absorbance equivalents and comparative cell numbers. Data were then converted to cell number and statistically analyzed. These are data on MeWo cells and are representative of all three cell lines. A, MDC-treated cells, and B, INO-3178-treated cells.

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A

120

100 0.0 I.tM

.

80 i
_50 v.M INO-3178) were required to influence WSAT of human melanoma cells. Although at a concentration of 10 p.M INO-3178 melanoma cells attached for 1-5 s and then detached, the requirement of higher relative concentrations of drug to affect WSAT suggests that the cell surface receptors involved in the initial adhesive events function normally in the presence of transglutaminase inhibitors. Thus, subsequent intracellular or cell surface reactions are probably required to stabilize the adhesive interactions, and once they occur, these may involve transglutaminase-mediated crosslinks. The differences in cell spreading behavior of MeWo, 3S5, and 70W cells on immobilized FN in the presence of transglutaminase inhibitors also suggest that multiple factors are involved in stabilizing the adhesive interactions. At a concentration of 10/dVI, INO-3178 completely inhibited melanoma cell spreading on immobilized FN, and WSDeT was reduced from 32.48 dynes/cm 2 to between 3.58-13.88 dynes/cm 2, correlating with 92-100% cell detachment from the FN substrate. The amount of melanoma cell spreading was inversely proportional to the number of cells detached from immobilized FN, suggesting that cells that are rounded up in the presence of INO-3178 might present higher profiles to WSS than spread cells, possibly because they extend further into the laminar fluid flow. The cell spreading and the shear resistance profiles exhibited by rounded vs spread cells may not be the only factor associated with cell detachment. Another important consideration for stable adhesion is the assemblege or aggregation of multiple adhesion components into zones where multiple adhesive interactions can occur (49). A number of recent reports prompted us to examine the effects of transglutaminase inhibitors on the formation of crosslinked cell surface components during cell adhesion to immobilized FN and SDS-insoluble FN-containing complexes. Cornwell et al. (48) demonstrated the inhibition of Chinese hamster ovary cell adhesion to plastic in the presence of serum components by MDC. In other studies, Slife et al. (27,28,31,32) showed that hepatocyte transglutaminase catalyzed the formation of high-Mr detergent-insoluble complexes that are too large or complex to enter low percentage SDS-PAGE gels. When soluble 12SI-FN or 125I-fibrinogen binds to rabbit hepatocytes or human umbilical vein endothelial cells, it becomes crosslinked into an insoluble complex within 3 h (30,31, 50). Interestingly, these reports also demonstrated that transglutaminasemediated crosslinking of 125I-fibrinogen into SDS-insoluble complexes occurred within < 15 rain. We observed formation of an SDS-insoluble complex following adhesion of MeWo melanoma cells to immobilized-FN (Fig. 3A). The time required for 12sI-fibrinogen crosslinking was found by Martinez et al. (50) to be similar to the time observed for the formation of stable melanoma cell-adhesive interactions with immobilized FN in the presence of WSS. Using gradient-SDS-PAGE, we also observed substan-

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tial inhibition of immobilized FN association with melanoma cell surfaces after 3-4 h in the presence of transglutaminase inhibitors. Our results are consistent with previous reports using soluble 125I-FN, in that virtually all of the immobilized protein that associates with the cell surface is in an insoluble complex within 3-4 h (30,31,50). Transglutaminases are present in most tissues (25) and their covalent crosslinking activities should be important if they are involved in stabilizing the formation of adhesive complexes. We have demonstrated that transglutaminase inhibitors prevent cell spreading, the formation of stable adhesive contacts, and crosslinked membrane complexes between metastatic human melanoma cells and immobilized FN. Tissue transglutaminases possess a number of characteristics, including calcium-dependency, potential membrane association through lipid coupling (51), presence of potential N-glycosidation sites (52), and GTP-binding/hydrolyzing properties (53-55). Calcium and GTP have opposing effects in regulating transglutaminase activity (53,55). GTP binding inhibits tissue transglutaminase actvity, and subsequent GTP hydrolysis may reactivate the enzyme (53,55). Tissue transglutaminases are associated with cell membranes through lipid binding, and calcium activation and GTP binding/hydrolysis activities are suggestive of a potential role in G-proteinmediated signal transduction. Tissue transglutaminase may also be involved in G-protein-mediated signaling through GTP-regulated inhibition of crosslinking activity that affects cell shape change, adhesion, motility, and growth. Furthermore, calcium influx and calcium-dependent activation of transglutaminase may initiate crosslinking that stabilizes cytoskeletal and adhesive elements in focal adhesion sites. Thus, transglutaminasemediated crosslinking may have a profound influence on malignant cells, especially those properties, such as cell adhesion, that play important roles in the metastatic process.

SUMMARY Human metastatic melanoma parental MeWo and variants 3S5 and 70W cell lines form adhesive contacts with immobilized FN that resist substantial WSS and may be stabilized by transglutaminase catalyzedcrosslinkages. Analyzing cell adhesion under flow, we determined that cell adherence was rapid with no visible cell rolling. Under flow, the WSAT was greatest for the highly metastatic 70W cells compared to less metastatic MeWo and 3S5 cells. Following cell adherence, we determined the WSDeT of untreated cells was ~ 32.5 dynes/cm 2. Transglutaminase inhibitors, monodansylcadaverine and INO-3178: (1) decreased WSAT at high concentrations; (2) at low concentrations completely inhibited tumor cell spreading; and (3) promoted cell detachment at low WSDeTs (0.67 dynes/cm2). In static adhesion assays, transglutaminase inhibitors de-

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creased cell adhesion to immobilized FN and prevented the formation of crosslinked 12SI-FN complexes. The data suggests that cellular adhesive contacts with immobilized FN may be stabilized by transglutaminase and may be of particular importance in the presence of WSS.

ACKNOWLEDGMENTS The authors w o u l d like to thank Sridhar Rajagopalan for his contributions to these studies. S u p p o r t e d by N . I . H grant R35-CA44352 from the N.C.I. to G.L.N., N.I.H. grant RO1-HL-17432 to L.V.M., and N.I.H. training grant T32-CA-09299 to D.G.M.

REFERENCES 1. 2. 3. 4.

Weiss, L. (1985), Principles of Metastasis, Academic Press Inc~, London. Nicolson, G. L. (1988), Biochim. Biophys. Acta 984, 175-244. Nicolson, G. L. (1991), Curr. Opinion Oncol. 3, 75-92. Cole, W. H., Robert, S. S., Webb, R. S., Strehl, F. W., and Oates, G. D. (1965), Ann. Surg. 161, 753-768. 5. Salsbury, A. J. (1975), Cancer Treat. Rev. 2, 55-72. 6. Irimura, T., Gonzalez, L., and Nicolson, G. L. (1981), Cancer Res. 41, 3411-3418. 7. Nicolson, G. L., Fidler, I. J., and Poste, G. (1986), J. Natl. Cancer Inst. 76, 511-519. 8. Lichtner, R. B., Erkell, L. J., Schirrmacher, V., and Nicolson, G. L. (1989), Clin. Exp. Metastasis 7, 175-186. 9. Iwamoto, Y., Robey, F. A., Graf, I., Sasaki, M., Kleinman, H. K., Yamada, Y., and Martin, G. R. (1987), Science 238, 1132-1134. 10. Humphries, M. J., Yamada, K. M., and Olden, K. (1988), J. Clin. Invest. 81, 782-790. 11. Auerbach, R., Lu, W. C., Pardon, E., Gurnkowski, F., Kaminska, G., and Kaminski, M. (1987), Cancer Res. 47, 1492-1496. 12. Nicolson, G. L., Belloni, P. N., Tressler, R. J., Dulski, K., Inoue, T., and Cavanaugh, P. G. (1989), Invasion Metastasis 9, 102-116. 13. Lichtner, R. B., BeUoni, P. N., and Nicolson, G. L. (1989), Exp. Cell Biol. 57, 146-152. 14. Nicolson, G. L. (1991), Semin. Cancer Biol. 2, 143-154. 15. Krarner, R. H. and Nicolson, G. L. (1979), Proc. Natl. Acad. Sci. USA 76, 5704-5708. 16. Zamora, P. O., Danielson, K. G., and Hosick, H. L. (1980), Cancer Res. 40, 4631-4639. 17. Vlodavsky, I., Schirrmacher, V., Ariav, Y., and Fuks, Z. (1983), Invasion Metastasis 3, 81-97. 18. Bastida, E., AlmiraU, L., Bertomeu, M. C., and Ordinas, A. (1989), Int. J. Cancer 43, 1174-1178.

Cell Biophysics

142

Menter et al.

19. Murray, C. J., Liotta, L. A., Rennard, S. I., and Martin, G. R. (1980), Cancer Res. 40, 347-351. 20. Terranova, V. P., Liotta, L. A., Russo, R. G. and Martin, G. R. (1982), Cancer Res. 42, 2265-2269. 21. Lawrence, M. B., Smith, C. W., Eskin, S. G., and McIntire, L. V. (1990), Blood 75, 227-237. 22. Muggli, R. and Baumgartner, H. R. (1978), Proceedings of the First Munich Symposium on Biological Connective Tissue Gastpar, H., Kuhn, K., and Marx, R., eds., F. K. Schattauer, Verlag, pp. 289. 23. Hubbell, J. A., and McIntire, L. V. (1986), Biophys. J. 50, 937-945. 24. Ishikawa, M., Dennis, J. W., Man, S., and Kerbel, R. S. (1988), Cancer Res. 48, 665-670. 25. Folk, J. E. (1980), Ann. Rev. Biochem. 49, 517-531. 26. Lorand, L. and Conrad, S. M. (1984), Mol. Cell. Biochem. 58, 9-,35. 27. Slife, C. W., Dorsett, M. D., Bouquett, G. T., Regeister, A., Taylor, E., and Conroy, S. (1985), Arch. Biochem. Biophys. 241, 329-336. 28. Slife, C. W., Morris, G. S., and Snedeker, S. W. (1987), Arch. Biochem. Biophys. 257, 39--47. 29. Barsigian, C., Fellin, F. M., Basse, W., Schaeffer, A., Fish, S., and Martinez, J. (1987), J. Biol. Chem. 262, 3674-3679. 30. Barsigian, C., Fellin, F. M., Jain, A., and Martinez, J. (1988), J. Biol. Chem. 263, 14015-14022. 31. Fellin, F. M., Barsigian, C., Rich, E., and Martinez, J. (1988), J. Biol. Chem. 263, 1791-1797. 32. Tyrrell, D. J., Sale, W. S., and Slife, C. W. (1988), J. Biol. Chem. 263, 1946-1951. 33. Tyrrell, D. J., Sale, W. S., and Slife, C. W. (1986), J. Biol. Chem. 261, 14833-14836. 34. Tyrrell, D. J., Sale, W. S., and Slife, C. W. (1988), J. Biol. Chem. 263, 8464-8469. 35. Lorand, L., Ong, H. H., Lipinski, B., Rule, N. G., Downey, J., and Jacobsen, A. (1966), Biochem. Biophys. Res. Commun. 25, 629-637. 36. Lorand, L., Rule, N. G., Ong, H. H., Furlanetto, R., Jacobsen, A., Downey, J., Oner, N., and Burner-Lorand, J., (1968), Biochemistry 7, 1214-1223. 37. Samama, M., Sofia, J., Soria, C., Maamer, M., and Otera, A. (1979), Prog. Chem. Fibrinolysis Thrombolysis 4, 235-240. 38. Chignard, M., Maamer, M., Lalau Keraly, C., Greiss, M., Aurousseau, M., and Benveniste, J. (1986), Thromb. Res. 44, 445-454. 39. Lorand, L., Barnes, N., Bruner-Lorand, J. A., Hawkins, M., and Michalska, M. (1987), Biochemistry 26, 308-313. 40. Fogh, J., Wright, W. C., and Loveless, J. D. (1977), J. Natl. Cancer Inst. 58, 209-214. 41. Updyke, T. V. and Nicolson, G. L. (1986), Clin. Exp. Metastasis 4, 273-284. 42. Cavanaugh, P. G. and Nicolson, G. L. (1989), Cancer Res. 49, 3928-3933. 43. Cavanaugh, P. G. and Nicolson, G. L. (1990), J. Cell. Biochem. 43, 127-138. 44. Wyckoff, M., Rodbard, D., and Crambach, A. (1977), Anal. Biochem. 78, 459-482. 45. Atherton, A. and Born, G. V. R. (1973), J. Physiol. 233, 157-166.

CellBiophysics

Stabilization o f Melanoma A d h e s i o n

143

46. Fesus, L., Metsis, M., Muszbek, L. and Koteliansky, V. E. (1986), Eur. f. Biochem. 154, 371-374. 47. Wright, C. S. and Raikhel, N. (1989), J. Mol. Evol. 28, 327-336. 48. CornweU, M. M., Juliano, R. L., and Davies, P. J. A. (1983), Biochem. Biophys. Acta 762, 414-419. 49. Nicolson, G. L. (1973), Nature New Biol. 243, 218-220. 50. Martinez, J., Rich, E., and Barsigian, C. (1989), J. Biol. Chem. 264, 20502-20508. 51. Harsfalvi, J., Arato, G., and Fesus, L. (1987), Biochem. Biophys. Acta 923, 42-45. 52. Ikura, K., Nasu, T., Yokota, H., Tsuchiya, Y., Sasaki, R., and Chiba, H. (1988), Biochemistry 27, 2898-2905. 53. Achyuthan, K. E. and Greenberg, C. S. (1987), J. Biol. Chem. 262, 1901-1906. 54. Bergamini, C. M. (1988), Fed. Eur. Biochem. Soc. 2, 255-258. 55. Lee, K. N., Birkbichler, P. J., and Patterson, M. K., (1989), Biochem. Biophys. Res. Commun. 162, 1370-1375.

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