Retinoic acid inhibition of human melanoma cell invasion through a reconstituted basement membrane and its relation to decreases in the expression of proteolytic enzymes and motility factor receptor.

Treatment of four A375 human melanoma sublines (A375, A375P, A375P-5, A375M), exhibiting distinct metastatic potentials in vivo, with beta-all-trans-retinoic acid in vitro caused a dose- and time-dependent inhibition of the ability of these cells to penetrate Matrigel-coated filters using a reconstituted basement membrane invasion assay. The possible mechanisms of action responsible for the antiinvasive effect were further investigated, and the data showed that compared with untreated cells the retinoic acid-treated cells: (a) secreted lower levels of collagenolytic enzymes, as demonstrated by a decreased ability of the cells to degrade [3H]proline-labeled type IV collagen substrate and by a reduction in the activity of a secreted Mr 64,000 collagenolytic enzyme detected in type IV collagen-containing polyacrylamide gels; (b) expressed lower levels of the human type IV collagenase mRNA (except in the A375P cells), as detected by Northern blot analysis; (c) exhibited decreased levels of tissue plasminogen activator activity, as demonstrated by a chromogenic assay; (d) were 10-40% less adhesive to a reconstituted basement membrane matrix, as determined by a 60-min Na2(51)CrO4-labeled cell attachment assay; (e) exhibited an increase in the high affinity metastasis-associated cell surface laminin receptor, as determined by flow cytometry after binding of fluorescently labeled laminin receptor antibody; and (f) expressed decreased amounts of gp78, a cell surface receptor for motility factor, demonstrated by immunoblotting and immunofluorescence. Collectively, these data suggest that retinoic acid inhibits tumor cell invasion through a basement membrane-like matrix by suppressing matrix degradation and by altering cell surface receptors.

Quantitation of human melanoma, carcinoma and sarcoma tumor cell adhesion to lymphatic endothelium.

We have used an in vitro adhesion assay to study the interaction of tumor cells with lymphatic endothelium, a dynamic event that leads to tumor metastasis in vivo. 3H-thymidine-labeled human tumor cells from: one primary Ewing sarcoma, two established melanoma cell lines, two colon and two breast carcinomas (one established line and one primary culture of each) were added to 24-well culture dishes containing confluent monolayers of bovine lymphatic endothelium. Radioactivity associated with either the cells in suspension or the attached cells was assessed and compared at frequent intervals up to 360 minutes. Generally, tumor cell attachment increased as a function of time reaching a plateau between 180 and 360 minutes. the modular media system described here facilitates the primary and secondary culture (or co-culture) of a variety of normal and transformed cells. Primary cultures with a rounded morphology (one breast and one colon carcinoma) showed the lowest preferential attachment for lymphatic endothelium. All established cell lines and the primary Ewing sarcoma cell line displayed a more fibroblastic morphology and achieved the highest adhesion profiles. There was a correlation between the malignancy and attachment potential for the melanoma and breast carcinoma cell lines. Collectively, these data show that established tumor cell lines with fibroblastic-like morphology exhibit more rapid adhesion than primary tumor cell cultures with more rounded morphologies. While this property may reflect in vitro selection and/or adaptation, it does correlate with the metastatic propensity for some human tumor cells.

Cytogenetic evidence of gene amplification as a mechanism for tumor cell invasion.

In order to study the process by which human melanoma cells achieve invasion of basement membranes, a modification of the Membrane Invasion Culture System was developed to allow the in vitro collection of human melanoma cell populations that had invaded acellular human amniotic membranes. A significant increase in the number of double-minute chromosomes (DMs) was observed in metaphase nuclei of A375P human melanoma cells which had passed through two amniotic membranes (A375P-2) over that of control cells. Eighteen percent of the first monolayer of A375P-2 cells contained 1-89 DMs/cell, whereas 3-8.3% of the control A375P cells contained 1-10 DMs/cell. There was a rapid loss of DMs in A375P-2 cells as a function of passage number. After 25 days in tissue culture, the incidence of DMs had essentially dropped below the control range. These data indicate that an unstable gene amplification event may be part of the process by which melanoma cells execute invasion through basement membranes.

Factor XI antigen and activity in human platelets.

Washed platelets, contaminated with less than 0.20% plasma factor XI, were examined for the presence of factor XI antigen and activity. These platelets contained a factor-XI-like coagulant activity (0.67 +/- 0.11 U/10(11) platelets) that remained constant after successive washes. By means of indirect immunofluorescence, a monospecific antibody to factor XI showed specific staining of both normal platelets and platelets from patients deficient in plasma factor XI. Radiolabeled Triton extracts of washed platelets and labeled purified factor XI solutions were analyzed for factor XI antigen by Staph A immunoprecipitation analysis using antibody to purified plasma factor XI followed by SDS gel electrophoresis. On unreduced gels, the platelet material ran as a single band having an apparent molecular weight of 220,000 daltons, whereas purified plasma factor XI gave a single band at 160,000 daltons. On reduced gels, the platelet material analyzed as a single band at 52,000 daltons, whereas purified factor XI gave a single band of 80,000 daltons. Analysis of a partially purified factor XI preparation from platelets by immunoelectrophoresis revealed that the platelet preparation displayed a slightly lower cathodal electrophoretic mobility at pH 8.6 than did plasma factor XI and yet appeared to possess complete antigenic identity with plasma factor XI. These results indicate that platelets possess a form of factor XI that exists as a disulfide-linked 52,000-dalton tetramer in contrast to the plasma form that circulates as a 80,000-dalton disulfide-linked dimer.