1997;50:57C71. integrins showed no effect. With use of highly sensitive time-lapse videomicroscopy and computer-assisted cell tracking techniques, promigratory functions of CD44 were excluded. 1) Addition of HA did not increase the migratory cell population or its migration velocity, 2) blocking of the HA-binding Hermes-1 epitope did not affect migration, and 3) impaired migration after blocking or activation of 1 1 integrins was not restored via CD44. Because 21-mediated migration was neither synergized nor replaced by CD44CHA interactions, we conclude that the biophysical properties of 3-D multicomponent ECM impose more restricted molecular functions of adhesion receptors, thereby differing from haptokinetic migration across surfaces. INTRODUCTION Tumor cell invasion and migration are supported by different adhesion receptor systems, including integrins and CD44 (Stetler-Stevenson TCS 4D, Bensheim, Germany) was performed as previously described (Friedl TCS-4D software. In some experiments, time-series of migrating cells after prestaining with primary mAb and secondary Fab fragments were obtained as simultaneous two-channel scans, as described (Friedl 0.001, ***? 0.0001). mAbs: 2 (1), 6F1; 2 (2), P1E6; 3, P1B5; 5, SAM-1; 6, GoH3; v, 17E6; 1, 4B4.? MV3 Cell Migration Is Dependent on 21 but Not on Other Integrins To identify integrin chains involved in MV3 migration and associated matrix remodeling, blocking mAb against 2, 3, 5, 6, and v integrins were used alone and in combination. Blocking anti-2 integrin mAbs 6F1 and P1E6 significantly reduced the percentage of cells locomoting as well as percentage of time Varenicline locomoting (Table ?(Table1)1) but not, however, migration velocity of residually migrating cells. The inhibitory effect of mAb 6F1 was comparable with anti-1 integrin mAb 4B4, whereas mAb P1E6 was less efficient. Blocking of other -chains, including 3, 5, 6, and v, did not inhibit migration or increase the effect of blocking anti-2 mAb. HA Promotes Varenicline MV3 Cell Migration on 2-D Surface but Not in 3-D Collagen Lattices The CD44 ligand HA is ubiquitously distributed throughout interstitial tissues, forming soft interconnected lattices (Scott, 1995 ). With respect to HA binding, CD44 can exist in three functional states: nonactivable, activable (e.g., by substrate, mAb, or cytoplasmic serine phosphorylation), and constitutively active (Peck and Isacke, 1998 ). If seeded onto HA-coated coverglass, 95% of MV3 cells attached and spread on the substrate (Figure ?(Figure4A)4A) in a time-dependent manner, suggesting that CD44 was either constitutively active MSH4 or inducible in MV3 cells. Subsequently, time- and dose-dependent induction of haptokinetic migration was seen on HA-coated substrate, as compared with cells on uncoated substrate (Figure ?(Figure4,4, A and B). Both the number and time of cells locomoting and their velocities were increased, resulting in increased migration speed (Figure ?(Figure4C).4C). Haptokinetic migration on HA, but not on uncoated glass, was significantly reduced, although not completely blocked by anti-CD44 mAb Hermes-1, which is consistent with previously published data (Thomas em et al. /em , 1992 ; Goebeler Varenicline em et al. /em , 1996 ). Open in a separate window Figure 4 HA-induced migration on 2-D substrate is inhibited by mAb Hermes-1. Shown are individual cell paths at their actual position (A, n = 40 randomly selected cells), time-dependent migration speed (B), and migratory variation within the populations (C) in the presence or absence of mAb Hermes-1 (10 g/ml). One representative out of at least two experiments is shown. In vitro, HA spontaneously aggregates, forming multimeric branched strands (Scott em et al. /em , 1991 ), binds to fibrillar collagen (Turley em et al. /em , 1985 Varenicline ), and is further multimerized and stabilized by cross-linking CS, resulting in increased biomechanical resistance of the lattice (Nishimura em et al. /em , 1998 ). For constitution of multicomponent lattices, HA and CS were used in concentrations found in interstitial tissues (see MATERIALS AND METHODS). In Varenicline contrast to migration on 2-D HA-coated surfaces, no alteration in spontaneous MV3 cell locomotion was observed in 3-D collagen matrices supplemented with HA in the absence or presence of CS, as determined by the number of locomoting cells (Figure ?(Figure5A)5A) and their velocities (Figure ?(Figure5B).5B). The lack of HA-induced migration was present in highly purified HA as well as in high molecular weight HA (3.5C5 106 Da; Figure ?Figure5,5, C and D) for a.

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