Our previous studies have shown that a peptide corresponding to the residue sequence 185-203 of the NC1 domain of the ␣3 chain of basement membrane collagen (type IV) inhibits the activation of polymorphonuclear leukocytes. Peptides from the same region of the ␣1, ␣2, ␣4, and ␣5(IV) chains did not exhibit this property. Because of the intimate relationship between metastasizing neoplastic cells and vascular as well as epithelial basement membranes, we measured the cell adhesionpromoting activity of peptides from the NC1 domain of type IV collagen and their effect on proliferation of human melanoma cells. We found that peptide ␣3(IV)185-203 (CNYYSNSYSFWLASLNPER) not only promotes adhesion of human melanoma cells but also inhibits their proliferation. Adhesion increased by 50 -60% over control. Melanoma cell proliferation was inhibited by 40% when cells were grown in a medium containing 5 g/ml peptide for 5 days. Studies showed that replacement of serine in position 189 or 191 by alanine resulted in significantly reduced adhesion. Similarly, serine replacement resulted in reduced ability to inhibit proliferation. Our data suggest that a region of the NC1 domain of the ␣3(IV) chain, contained within the sequence 185-203, not only specifically promotes adhesion but also inhibits proliferation of melanoma cells. These properties appear to be dependent on the presence of the triplet sequence -SNS-(residues 189 -191), which is unique to the ␣3 chain and may represent an important functional epitope.
Sera from patients with poststreptococcal glomerulonephritis (PSGN) known to have antibodies to proteoglycans were studied for the presence of antibodies against other basement membrane (BM) components. BM collagen (type IV) was isolated in the native state by extracting bovine anterior lens capsule (ALC) with 0.5 M acetic acid. The 7-S (collagenous) domain and the NC-1 (noncollagenous) domain of type IV collagen were obtained after bacterial collagenase digestion of ALC followed by gel filtration. Laminin was isolated from the mouse EHS tumor and fibronectin from human plasma. Immunologic studies, using an ELISA and electroimmunoblot, revealed the presence of antibodies that reacted with intact, native type IV collagen and the 7-S collagenous domain of this molecule. Reaction with the NC-1 (noncollagenous) domain was minimal, and not higher than that obtained with control sera. Laminin reaction strongly with the patients' sera, but fibronectin did not. Unlike sera from patients with Goodpasture syndrome, which contain antibodies primarily against the NC-1 (noncollagenous) domain of type IV collagen, sera from patients with acute PSGN contain antibodies against all the major macromolecular components of BM. This difference in immunologic reactivity may account for the observed differences in the pathologic picture at the glomerular level.
Previous studies from our laboratories demonstrated that a peptide from the noncollagenous domain of the alpha3 chain of basement membrane collagen (COL IV), comprising residues 185-203, inhibits polymorphonuclear leukocyte activation and melanoma cell proliferation; this property requires the presence of the triplet -SNS- in residues 189-191 (Monboisse et al., J. Biol. Chem., 269, 25475, 1994; Han et al., J. Biol. Chem., 272, 20395, 1997). In the present study, we demonstrate that whole native COL IV and -SNS- containing synthetic peptides (10 microg/ml) added to culture medium inhibit the proliferation of not only melanoma cells, but also breast-, pancreas- and stomach-tumor cells up to 67%, and prostate tumor cells by 15%. ALC-COL IV at 5 microg/ml was shown to inhibit melanoma cell proliferation maximally at 69% and the alpha3(IV)185-203 peptide inhibited proliferation (62%) maximally at 10 microg/ml. Treatment of the alpha3(IV)185-203 peptide with either a specific mAb or a polyclonal antibody, prepared against the sequence alpha3(IV)179-208, decreased the ability of the peptide to inhibit cell proliferation by 97%, while treatment of ALC-COL IV with the same antibodies inhibited proliferation by 44%. Exposure of the above tumor cells to COL IV or the peptides resulted in an increase of intracellular cAMP that was inhibited by prior treatment of the protein with the above antibodies. To investigate the role of cAMP in the inhibition of cell proliferation, cAMP analogs and inhibitors were used. cAMP analogs mimicked the inhibitory effect of the peptide. Rp-cAMPS, a cAMP competitive inhibitor, suppressed the inhibitory effect of ALC-COL IV and of the cAMP analogs. The protein kinase-A inhibitor H-89 blocked the ability of ALC-COL IV and of the alpha3(IV)185-203 peptide to inhibit tumor cell proliferation. These data suggest that ALC-COL IV, through its alpha3(IV) chain, inhibits tumor cell proliferation utilizing a signal transduction pathway which includes cAMP and cAMP-dependent protein kinase(s).
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