Membrane type I-matrix metalloproteinase-mediated degradation of type I collagen by oral squamous cell carcinoma cells.

Oral squamous cell carcinomas are highly invasive lesions that destroy adjacent tissues and invade bone and muscle, which is most likely the result of matrix metalloproteinase (MMP) activity. We examined three cell lines derived from squamous cell carcinoma of the tongue for their intrinsic capacities to degrade interstitial collagen with the goal of identifying the matrix-degrading enzymes. SCC-25 and SCC-15 cells degrade reconstituted fibrillar type I collagen in the absence of exogenous growth factors or cytokines when seeded as a colony on dried films. Degradation is confined to the subjacent matrix, is enhanced 2-3-fold by phorbol ester, and is strictly MMP-dependent, as it is blocked by BB-94 and tissue inhibitor of metalloproteinases-2 but not by inhibitors of serine and cysteine proteinases. Both cell lines express active (M(r) 57,000) membrane type I-MMP (MT1-MMP) on their surfaces, as detected by surface biotinylation and immunoprecipitation. Concomitantly, both cell lines activate endogenous MMP-2 when cultured on type I collagen films, as assessed by zymography. Phorbol ester treatment enhances collagen-induced MMP-2 activation, which is accompanied by the appearance of a surface-labeled M(r) 43,000 form of MT1-MMP. Treatment of cells with a synthetic furin inhibitor, which inhibits processing of the MT1-MMP zymogen, blocks collagen degradation. In contrast, CAL 27 cells do not degrade collagen under either basal or phorbol 12-myristate 13-acetate-stimulated conditions. Although proMT1-MMP (M(r) 63,000/65,000) is detectable in these cells by immunoblot analysis, they express greatly reduced levels of active MT1-MMP on their surfaces relative to SCC-25 and SCC-15 cells. Correspondingly, CAL 27 cells cultured on collagen express neither latent nor active gelatinases. Immunoblots of lysates and conditioned media revealed the constitutive expression of proMMP-1 and proMMP-13 in all three cell lines. We conclude that in the absence of exogenous growth factors or accessory stromal cells, degradation of interstitial collagen by oral squamous cell carcinoma cells requires a threshold level of active MT1-MMP on cell surfaces.

Induction of collagenase-3 (MMP-13) in rheumatoid arthritis synovial fibroblasts.

There is a growing body of evidence that implicates matrix metalloproteinases (MMPs) as major players in numerous diseased conditions. The articular cartilage degradation that is characteristic of rheumatoid arthritis (RA) is believed to be mediated by the collagenase subfamily of matrix metalloproteinases. The preference of collagenase-3 (CL-3) for collagen type II makes it a likely candidate in the turnover of articular cartilage and a potential target for drug development. In this study, RA synovial membrane tissue was shown to express CL-3 mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR) and protein by immunohistochemistry. Fibroblasts isolated and cultured from RA synovial membrane tissue were induced to express CL-3 mRNA. CL-3 mRNA was detected after PMA treatment in 16 of the 18 RA synovial membrane fibroblast cell lines established for this study. These fibroblasts also expressed mRNA for collagenase-1 (CL-1, MMP-1), membrane type-1 matrix metalloproteinase, gelatinase A, gelatinase B, stromelysin-1, stromelysin-2, TIMP-1, and TIMP-2. They were further shown to express CL-1 mRNA constitutively and CL-3 mRNA only after stimulation with PMA, IL-1, TGF-beta1, TNF-alpha, or IL-6 with IL-6sR. These fibroblasts also expressed after induction both CL-1 and CL-3 at the protein level as determined by Western blot analyses and immunofluorescence.

Stimulation of tumor cell motility linked to phosphodiesterase catalytic site of autotaxin.

A family of extracellular type I phosphodiesterases has recently been isolated by cDNA cloning, but a physiological function linked to the phosphodiesterase active site has remained unknown. We now present evidence that the phosphodiesterase catalytic site, 201YMRPVYPTKTFPN213, is essential for the motility stimulating activity of autotaxin (ATX), one member of the exophosphodiesterase family. Native ATX possesses phosphodiesterase activity at neutral and alkaline pH, binds ATP noncovalently, and undergoes threonine phosphorylation. Homogeneously purified recombinant ATX, based on the teratocarcinoma sequence, retains these same activities. A single amino acid in the phosphodiesterase catalytic site, Thr210, is found to be necessary for motility stimulation, phosphodiesterase activity, and phosphorylation. Two mutant recombinant proteins, Ala210- and Asp210-ATX, lack motility stimulation and lack both enzymatic activities; Ser210-ATX possesses intermediate activities. Another mutation, with the adjacent lysine (Lys209) changed to Leu209-ATX, possesses normal motility stimulation with sustained phosphodiesterase activity but exhibits no detectable phosphorylation. This mutation eliminates the phosphorylation reaction and indicates that the dephosphorylated state is an active motility-stimulating form of the ATX molecule. By demonstrating that the phosphodiesterase enzymatic site is linked to motility stimulation, these data reveal a novel role for this family of exo/ecto-enzymes and open up the possibility of extracellular enzymatic cascades as a regulatory mechanism for cellular motility.

Responses of tumor cell pseudopod protrusion to changes in medium osmolality.

The potential involvement of osmotically generated force in protrusion of tumor cell pseudopods was examined during a micropipette assay. Experiments were performed on single A2058 melanoma cells activated by a micropipette filled with soluble type IV collagen. Previous observations suggested that tumor cell pseudopod protrusion induced by type IV collagen took place in distinct, separable phases: an initial bleb (first phase) caused by localized Ca2+-activated actin filament severing resulting in an osmotic flux followed by an extension with an irregular shape (second phase) which required G protein-mediated actin polymerization (Dong et al., 1994, Microvasc. Res., 47:55-67). Presently we studied cell pseudopod protrusion in response to the changes in chemoattractant osmolality. Reduction of attractant osmolality by 20-25% from its baseline value (297 mmol/ kg) resulted in an increase in pseudopod length by 50% apparent in the initial phase. Increases in attractant osmolality by 25-30% from the baseline value arrested pseudopod protrusion significantly during both initial and later phases. Using a dual-pipette method, such osmotic influence on the cell pseudopod protrusion was shown to be only a local effect in a small region where the cell surface was stimulated by the micropipette. While forces derived from actin polymerization and osmotic pressure have been proposed to cause protrusion in general, our results suggested that osmotically generated force is more apparent in the initial phase of the pseudopod formation.

In vitro and in vivo growth inhibition of murine melanoma K-1735 cell by a dominant negative mutant alpha subunit of the Gi2 protein.

In murine and rat fibroblasts, activation of several G proteins (Gi2, Gq, G12) can stimulate cell growth or transformation, and can induce tumor formation in nude mice; contrastingly, inactivation of Gi2 inhibits fibroblast proliferation in vitro. We investigated whether it is possible to modulate malignant cell growth in vitro and in vivo through alteration of Gi2 protein function. To do so, we introduced mutated alpha subunits of Gi2 (alpha i2) in CL19 cells, a clone of the murine melanoma cell line K-1735. When we did this, a constitutively activated mutant (alpha i2-Q205L) and a dominant negative mutant (alpha i2-G204A) of alpha i2 were stably expressed in CL19 cells. We found that the in vitro motility of all alpha i2-transfected CL19 cells was increased; however, overexpression and alteration of the function of Gi2 did not increase metastasis formation by CL19 cells in nude mice. Expression of alpha i2-Q205L conferred a limited growth advantage to CL19 cells in vitro; in vivo, tumor formation and size, and overall survival of animals injected with CL19 cells expressing alpha i2-Q205L, were similar to controls. In contrast, expression of the inactive alpha i2-G204A mutant inhibited CL19 growth in vitro by at least 50% in all conditions tested, and mice injected with cells expressing the alpha i2-G204A mutant showed delayed tumor formation, reduced tumor size, and longer survival. We conclude that Gi2 proteins contribute to malignant cell growth, and more importantly, that inactivation of Gi2 proteins can inhibit proliferation of melanoma cells and possibly of other malignant cells both in vitro and in vivo.

Integrin alphavbeta3 mediates chemotactic and haptotactic motility in human melanoma cells through different signaling pathways.

Distinctions between chemotaxis and haptotaxis of cells to extracellular matrix proteins have not been defined in terms of mechanisms or signaling pathways. Migration of A2058 human melanoma cells to soluble (chemotaxis) and substratum-bound (haptotaxis) vitronectin, mediated by alphav beta3, provided a system with which to address these questions. Both chemotaxis and haptotaxis were completely inhibited by treatment with RGD-containing peptides. Chemotaxis was abolished by a blocking antibody to alphavbeta3 (LM609), whereas haptotaxis was inhibited only by approximately 50%, suggesting involvement of multiple receptors and/or signaling pathways. However, blocking antibodies to alphavbeta5, also present on A2058 cells, did not inhibit. Pertussis toxin treatment of cells inhibited chemotaxis by >80%, but did not inhibit haptotaxis. Adhesion and spreading over vitronectin induced the phosphorylation of paxillin on tyrosine. In cells migrating over substratum-bound vitronectin, tyrosine phosphorylation of paxillin increased 5-fold between 45 min and 5 h. Dilutions of anti- alphavbeta3 that inhibited haptotaxis also inhibited phosphorylation of paxillin (by approximately 50%) and modestly reduced cell spreading. In contrast, soluble vitronectin (50-100 microg/ml) did not induce tyrosine phosphorylation of paxillin. The data suggest that soluble vitronectin stimulates chemotaxis predominantly through a G protein-mediated pathway that is functionally linked to alphavbeta3. Haptotaxis is analogous to directional cell spreading and requires alphavbeta3-mediated tyrosine phosphorylation of paxillin.

Two phases of pseudopod protrusion in tumor cells revealed by a micropipette.

Pseudopod protrusion at the leading edge is a characteristic of migrating tumor cells as they traverse vascular subendothelial basement membranes to establish metastases. A micropipette system has been developed to study the dynamics of pseudopod protrusion in individual human melanoma cells in response to type IV collagen, a component of basement membranes. Soluble type IV collagen stimulates chemotaxis of A2058 melanoma cells through a G protein-coupled receptor, and induces an early burst of intracellular calcium (Savarese et al., 1992, J. Biol. Chem. 267, 21928-21935). A micropipette filled with type IV collagen solution (100 micrograms/ml) was positioned so that the tip was adjacent to a cell suspended in Dulbeceo's modified Eagle's medium. Within 10 min, tumor cells generated a pseudopod which entered the micropipette with an average velocity of 0.24 micron/min and proceeded to lengthen for 40 min. Pseudopods from individual cells ranged from 7.5-10 microns at this time and were characterized by an irregular shape which did not fill the lumen of the micropipette. Pretreatment of cells with pertussis toxin (0.5 microgram/ml), which inhibits cell migration by approximately 90% (but not the calcium burst), blocked formation of the irregular, extended pseudopod, while allowing a much smaller outpouching, or bleb, to form. Lengths of such blebs from individual PT-treated cells reached a plateau at approximately 20 min and ranged from 2.2-4.0 microns at the end point. Treatment of cells with bis-(amino-phenoxy)ethane tetraacetic acid (75 microM), an intracellular Ca2+ chelator, blocked initial bleb formation and prevented extension. From these observations we hypothesize that tumor cell pseudopod protrusion induced by soluble type IV collagen takes place in distinct, separable phases: an initial convex, symmetrical outpouching, caused by localized Ca(2+)-activated actin depolymerization and osmotic flux, followed by an extension with an irregular shape, which requires G protein-mediated actin polymerization.

The role of the extracellular matrix in tumor cell metastasis.

The extracellular matrix is a macromolecular network that surrounds cells and makes up a significant portion of their microenvironment. During the processes of invasion and metastasis, tumor cells encounter interstitial stroma and basement membranes many times. In fact, tumor cell response to extracellular matrix components appears to be a major determinant of metastatic potential.

Tumor cell interactions with the extracellular matrix during invasion and metastasis.

Recent findings have produced great strides in developing an understanding of the molecular events involved in processes necessary for tumor cell invasion and subsequent metastasis formation. This information has been useful in developing new targets for therapeutic intervention such as disruption of tumor cell attachment by peptide analogues of cell adhesion molecules and the use of protease inhibitors to limit extracellular matrix proteolysis required for tumor cell invasion. Future efforts must focus on how the events of cell attachment, matrix proteolysis, and cell migration are controlled and integrated. This requires a better understanding of the transcriptional controls and cell signaling mechanisms that are involved in these events. Preliminary findings suggest that cell-matrix interactions influence gene expression and that the protease inhibitor balance can greatly influence cell-matrix interactions. Therefore it appears that all three steps in the invasive process are linked and interdependent. While this complicates the study of these processes, it is our belief that understanding this interdependence is critical for further development of metastasis research.

Tumor cell motility.

Tumor cell motility is required for invasion and metastasis. The locomotory machinery of the cell includes cell projections called pseudopodia which are regulated by a complicated linkage between cell surface receptors or sensors and the internal cytoskeleton. Recently a new class of motility stimulating cytokines have been identified. These cytokines can function as autocrine motility factors and require a pertussis toxin sensitive G protein pathway to transduce a random motile response.

Cell motility, a principal requirement for metastasis.

In studying the role of motility in the metastasis of tumor cells, we have described an autocrine motility factor. This agent, which stimulates random motility, probably contributes to the initial dissociation of the cells from the primary tumor mass. Extracellular matrix components, via several different mechanisms, may facilitate the crossing of biological barriers by the cells prior to the entry into the circulation. In locating at new sites, the tumor cells may be induced to exit from the circulation in response to attractants such as IGFs that could emanate from the target organ. These same growth factors could then stimulate cellular proliferation for another metastatic cycle. It is quite probable that detection of AMF may provide a new tool in cancer diagnosis. The complete characterization of AMF may also yield valuable therapeutic approaches: design of low molecular size antagonists of the attractants and antibodies that might be effective therapeutically as well as diagnostically. It seems clear, in any event, that immobilizing the tumor cell may be a crucial step in inhibiting metastasis.

Characteristics of invasive and noninvasive human colorectal adenocarcinoma cells.

A human colon adenocarcinoma cell line (LoVo) established from a lymph node metastasis was used to study properties associated with invasive tumor cells. Human amniotic membranes were used as invasion barriers to select highly invasive and noninvasive subpopulations of cells from the parent LoVo line. Enriched subpopulations were compared with respect to parameters associated with invasion. The invasive cells were more invasive in vitro and more highly sialylated than either the parental or noninvasive line. Invasive cells migrated more strongly in vitro toward gradients of soluble and insoluble laminin, and their augmented migration correlated with increased adhesion and spreading on laminin-coated substrata. Invasive cells also had the highest level of specific laminin-binding activity. Thus, the invasive cells were shown to possess specific properties that correlated with their successful traversal of the human amnion membrane.

Signal transduction for chemotaxis and haptotaxis by matrix molecules in tumor cells.

Transduction of signals initiating motility by extracellular matrix (ECM) molecules differed depending on the type of matrix molecule and whether the ligand was in solution or bound to a substratum. Laminin, fibronectin, and type IV collagen stimulated both chemotaxis and haptotaxis of the A2058 human melanoma cell line. Peak chemotactic responses were reached at 50-200 nM for laminin, 50-100 nM for fibronectin, and 200-370 nM for type IV collagen. Checkerboard analysis of each attractant in solution demonstrated a predominantly directional (chemotactic) response, with a minor chemokinetic component. The cells also migrated in a concentration-dependent manner to insoluble step gradients of substratum-bound attractant (haptotaxis). The haptotactic responses reached maximal levels at coating concentrations of 20 nM for laminin and type IV collagen, and from 30 to 45 nM for fibronectin. Pretreatment of cells with the protein synthesis inhibitor, cycloheximide (5 micrograms/ml), resulted in a 5-30% inhibition of both chemotactic and haptotactic responses to each matrix protein, indicating that de novo protein synthesis was not required for a significant motility response. Pretreatment of cells with 50-500 micrograms/ml of synthetic peptides containing the fibronectin cell-recognition sequence GRGDS resulted in a concentration-dependent inhibition of fibronectin-mediated chemotaxis and haptotaxis (70-80% inhibition compared to control motility); negative control peptide GRGES had only a minimal effect. Neither GRGDS nor GRGES significantly inhibited motility to laminin or type IV collagen. Therefore, these results support a role for the RGD-directed integrin receptor in both types of motility response to fibronectin. After pretreatment with pertussis toxin (PT), chemotactic responses to laminin, fibronectin, and type IV collagen were distinctly different. Chemotaxis to laminin was intermediate in sensitivity; chemotaxis to fibronectin was completely insensitive; and chemotaxis to type IV collagen was profoundly inhibited by PT. In marked contrast to the inhibition of chemotaxis, the hepatotactic responses to all three ligands were unaffected by any of the tested concentrations of PT. High concentrations of cholera toxin (CT; 10 micrograms/ml) or the cAMP analogue, 8-Br-cAMP (0.5 mM), did not significantly affect chemotactic or haptotactic motility to any of the attractant proteins, ruling out the involvement of cAMP in the biochemical pathway initiating motility in these cells. The sensitivity of chemotaxis induced by laminin and type IV collagen, but not fibronectin, to PT indicates the involvement of a PT-sensitive G protein in transduction of the signals initiating motility to soluble laminin and type IV collagen.(ABSTRACT TRUNCATED AT 400 WORDS)

Insulin-like growth factors stimulate chemotaxis in human melanoma cells.

Insulin and insulin-like growth factors stimulate motility in the highly metastatic human melanoma cell line, A2058. Insulin-like growth factor-I (IGF-I) is the most potent with a maximal response at a concentration of 10 nM compared to the activities of insulin and insulin-like growth factor-II (IGF-II) which peak at 300-400 nM. Using checkerboard analysis, the responses to IGF-I and insulin are predominantly chemotactic, although insulin had a significant chemokinetic component. Pertussis toxin does not inhibit the response to any of these polypeptides. However, in previous studies, it was shown that the motile response to autocrine motility factor from these same A2058 cells was markedly inhibited by pertussis toxin. 125I-labelled IGF-I binds saturably and specifically to the A2058 cells. Scatchard analysis indicates a high binding affinity (Kd approximately 3 x 10(-10) M) and an estimated 5000 receptors/cell. These studies indicate that in addition to their mitogenic properties, certain growth factors may profoundly enhance metastasis of tumor cells by their ability to induce motility.

Prostacyclin inhibits 5-hydroxytryptamine release but stimulates thromboxane synthesis during cardiopulmonary bypass.

The antiaggregating agent prostacyclin (PGI2) was infused into ten dogs during cardiopulmonary bypass (CPB) to minimize thrombocytopenia and platelet dysfunction. The animals were anesthetized, placed on mechanical ventilation and underwent thoracotomy. After heparinization with 300 u/kg, animals were assigned to control (n=5) or PGI2 treated groups (n=5). Thoracotomy and then CPB decreased platelet numbers to below 30,000/mm3 (p less than 0.05) and fibrinogen to less than 150 mg/dl (p less than 0.05). PGI2 at 100 ng/kg.min was infused for the 2 h period of CPB. PGI2 infusion did not prevent these changes, but did prevent platelet serotonin release. In the control group after CPB, platelet serotonin fell from the baseline value of 1.11 microgram/10(9) to 0.35 microgram/10(9) platelets (p less than 0.05). In contrast, PGI2 treatment resulted in a serotonin increase to 2.27 micrograms/10(9) platelets (p less than 0.05). Thromboxane B2 concentrations of platelets and plasma rose during CPB (p less than 0.05). Surprisingly, PGI2 infusion accentuated this rise in platelet and plasma thromboxane B2 (p less than 0.05). These data indicate that during CPB, an infusion of PGI2: 1) does not prevent thrombocytopenia; 2) increases platelet serotonin uptake despite, 3) an associated rise in platelet and plasma thromboxane B2.