Laura Masiero

Control of type IV collagenase activity by components of the urokinase–plasmin system: a regulatory mechanism with cell‐bound reactants

The urokinase‐type plasminogen activator (uPA) and the matrix‐degrading metalloproteinases MMP‐2 and MMP‐9 (type IV collagenases/gelatinases) have been implicated in a variety of invasive processes, including tumor invasion, metastasis and angiogenesis. MMP‐2 and MMP‐9 are secreted in the form of inactive zymogens that are activated extracellularly, a fundamental process for the control of their activity. The physiological mechanism(s) of gelatinase activation are still poorly understood; their comprehension may provide tools to control cell invasion. The data reported in this paper show multiple roles of the uPA–plasmin system in the control of gelatinase activity: (i) both gelatinases are associated with the cell surface; binding of uPA and plasmin(ogen) to the cell surface results in gelatinase activation without the action of other metallo‐ or acid proteinases; (ii) inhibition of uPA or plasminogen binding to the cell surface blocks gelatinase activation; (iii) in soluble phase plasmin degrades both gelatinases; and (iv) gelatinase activation and degradation occur in a dose‐ and time‐dependent manner in the presence of physiological plasminogen and uPA concentrations. Thus, the uPA–plasmin system may represent a physiological mechanism for the control of gelatinase activity.

Down-regulation of tumour gelatinase/inhibitor balance and preservation of tumour endothelium by an anti-metastatic ruthenium complex

The anti‐metastatic ruthenium complex Na[trans‐RuCl4(DMSO)1m] was given i.p. at 22 and 44 mg/kg/day, on days 8–13 after tumour implantation, to mice carrying s.c. implants of MCa mammary carcinoma. The aim of the study was to compare the effects on lung metastasis formation with those on primary tumour cells. This investigation was based on flow cytometry analysis after propidium iodide and acridine orange staining, histology of tumour parenchyma and RT‐PCR analysis for the type‐IV collagenases MMP‐9 and MMP‐2 and their respective inhibitors TIMP‐1 and TIMP‐2 mRNAs. Na[trans‐RuCl4(DMSO)1m] is not cytotoxic for tumour cells but has the capacity of interacting with nucleic acids, giving a general reduction of nucleic acid content as shown by a marked reduction of acridine orange staining and a tendency to a reduction of DNA polyploidy with marked reduction of 8n and 4n cell populations. Na[trans‐RuCl4(DMSO)1m] also influences a proteolytic system which has the potential of degrading the basement membrane and has been related to metastatic aggressiveness: it markedly reduces, in a dose‐dependent manner, MMP‐2/TIMP‐2 balance, but not that of MMP‐9/TIMP‐1. The different enzyme/inhibitor mRNA levels between untreated and treated tumours seem to be unaffected by tumour‐infiltrating lymphocytes and are paralleled by the maintenance of connective tissue around blood vessels in the tumour mass. Correspondingly, lung metastasis formation is markedly reduced, to less than 10% of that seen in controls.

72-kilodalton type IV collagenase, type IV collagen, and Ki 67 antigen in serous tumors of the ovary : A clinicopathologic, immunohistochemical, and serological study

The immunohistochemical expression of 72-kDa type IV collagenase [matrix-metalloproteinase (MMP)-21], basement membrane component type IV collagen and proliferation-related antigen Ki 67 were investigated in 43 benign, borderline, and malignant serous tumors of the ovary. The results were compared with the histotypes of ovarian serous tumors and with their clinical behavior. Serum evaluation of MMP-2 was performed in 14 patients with cystadenocarcinoma and the data compared with that of a control group. The basement membrane (BM) was continuous in benign cystadenomas and in some borderline tumors, whereas it was discontinuous or absent in other borderline tumors, in borderline tumors with microinvasion, and cystadenocarcinomas. The percentage of MMP-2- and Ki 67-expressing cells increased from cystadenomas to borderline tumors, being the highest in malignant tumors; a frequent basal disposition of the MMP-2 cytoplasmic granules also was observed in cystadenocarcinomas. Statistical analysis demonstrated that MMP-2 expression was inversely related to BM integrity. Serum MMP-2 values did not differ from that of the control group. Cox regression analysis showed that tumor stage and grade were significant prognostic factors, whereas MMP-2 and Ki 67 immunohistochemical expression added no further significant information to the prognosis. The investigators conclude that the correlation between increasing MMP-2 expression and BM alteration gives support to the hypothesis of a direct role of the metalloproteinase in the process of destructive stromal invasion. MMP-2, type IV collagen, and Ki 67 immunodetection varied according to the histologic classification of ovarian serous tumors. However, neither these factors nor the serum evaluation of MMP-2 appear useful as prognostic predictors in this series.

Mg++-induced endothelial cell migration: Substratum selectivity and receptor-involvement

The activation of endothelial cells during angiogenesis requires cell spreading and migration. These processes are influenced by extracellular signals such as chemoattractants from the local microenvironment. We have shown previously that transmembrane Ca++ influx is necessary for motility and cell spreading, thus we hypothesized that the extracellular divalent cations Mg++ and Ca++ may regulate human umbilical vein endothelial cell (HUVEC) spreading and act as chemoattractants. Studies demonstrated that extracellular Mg++ induced a statistically better spread phenotype when cells were plated on multiple extracellular matrix substrata; Ca++ promoted cell spreading only on vitronectin. Mg++ but not Ca++ acted as a potent chemoattractant when HUVEC migrated on gelatin- and type IV collagen- but not on vitronectin-coated filters. A checkerboard analysis of migration showed that Mg++ induces both chemokinetic and chemotactic migration peaking at 0.1 and 10 mM, respectively. An equivalent effect of oligomycin was seen on motility to Mg++ or to vascular endothelial growth factor (VEGF) in extracellular Mg++-free conditions, ruling out an exclusive role for Mg++ as a migration energy producer. The Mg++-stimulated chemotaxis was inhibited >60% by pertussis toxin, d-erythrosphingosine, and tyrphostin B48, but unaffected by cholera toxin exposure. These data suggest that Mg++-induced chemotaxis may be promoted through a Gi protein-coupled receptor pathway with a requirement for protein kinase C activity and protein tyrosine phosphorylation. Thus, Mg++ may be a newly recognized receptor-mediated chemoattractant for endothelial cells.

Synthesis and refolding of human TIMP‐2 from E. coli, with specific activity for MMP‐2

Tissue inhibitors of metalloproteinase (TIMPs) are inhibitory counterparts of collagenases, containing 12 cysteine residues paired to six internal disulphide bridges. TIMP‐2, an inhibitory protein of 72 kDa gelatinase/type IV collagenase (MMP‐2), was expressed in Escherichia coli as a fusion protein with a 34 amino acid NH2‐linked tail containing six consecutive histidine residues. The protein was purified in a single‐step using an ion metal affinity column (IMAC) in denaturing conditions. The immobilized fusion TIMP‐2 protein was refolded at a high concentration in the column, producing about 5 mg of protein per litre of bacterial cells. It shows specific binding and inhibitory activity against MMP‐2, but has no effect against 92 and 45 kDa gelatinases.

Interaction of hydro- or lipophilic phthalocyanines with cells of different metastatic potential.

A highly metastatic (4R) and a nonmetastatic (RE4) transformed rat embryo fibroblast cell line were incubated with lipid-soluble Zn(II)-phthalocyanine (ZnPc) and its water-soluble tetrasulphonated derivative (ZnPcTS) and compared for phthalocyanine uptake. The hydrophobic liposome-delivered ZnPc showed a significantly greater uptake by both cell lines than did ZnPcTS. Moreover, the two phthalocyanines appear to interact with cells according to different pathways, as suggested by the different temperature-dependence of the binding process and the different inhibitory action exerted by selected serum proteins, such as lipoproteins and heavy proteins. Under all experimental conditions, the two cell lines exhibited similar interactions with ZnPc and ZnPcTS, suggesting that heterogeneity of the tumor cell population has a minor influence on the accumulation of photosensitizers.

Regulation of cell spreading during differentiation in the muscarinic M5 receptor tumor-suppressor model

Activation of the muscarinic receptor in Chinese hamster ovary (CHO) cells results in a reversal of the malignant phenotype for which spreading into a bipolar, fibroblastic morphology is a marker. The process of morphologic change requires multiple events, including alterations in adhesions to substrates and cytoskeletal re‐arrangement. In this report, we demonstrate the calcium‐dependent involvement of p125FAK in this cellular shape change using an inhibitor of ligand‐induced calcium influx, carboxyamido‐triazole (CAI). p125FAK becomes tyrosine‐phosphorylated after exposure to the agonist carbachol (CC), reaching maximal phosphorylation prior to initiation of cellular shape change at 1 hr into CC exposure (386 ± 103%). Phosphorylation remained elevated through the shape change (4–12 hr). CHOm5 cell exposure to the Ca2+‐mobilizing agents maitotoxin and ionomycin also resulted in p125FAK phosphorylation. Inhibition of Ca2+ influx with CAI, an inhibitor of ligand‐induced Ca2+ influx, had little effect on CC‐induced phosphorylation but partially inhibited ionomycin‐mediated p125FAK phosphorylation. While the intracellular Ca2+ chelator BAPTA failed to prevent CC‐induced p125FAK tyrosine phosphorylation, it inhibited phosphorylation due to ionomycin. CC induced Ca2+‐independent binding of phosphorylated p125FAK selectively to the C‐terminal SH2 domain of phosphatidylinositol‐3′‐kinase (PI3K). Further, CC, maitotoxin and ionomycin induced in vitro kinase activity of p125FAK for the exogenous substrate poly(Glu4Tyr1). Kinase activity stimulated by all 3 agonists was inhibited by pre‐incubation with either CAI or BAPTA. Our results indicate that increasing intracellular Ca2+ can stimulate both p125FAK autophosphorylation and kinase activity; however, p125FAK phosphorylation in response to CC also may be induced through a Ca2+‐independent pathway. Int. J. Cancer 72:362–368, 1997.