Studies on interaction of Plasminogen with Balb/c 3T3 and SV3T3 Cells in Culture

Author: Tobler, Jerry

Year: 1978

Degree: Dissertation (Ph.D.)

Advisor: Stroud, Robert M.

Committee Member: Unknown, Unknown

Option: Chemistry

DOI: 10.7907/mqz2-kv25

Abstract

The existence of a specific transformed cell surface-plasminogen interaction which has been implicated in many phenotype changes that occur in cells upon transformation was studied. In addition, the fate of the plasmin in cell culture medium activated by a cell-derived plasminogen activator was investigated.

As an initial step, the plasminogen binding to Balb/c 3T3 and SV3T3 cells was studied using 125I-labeled dog plasminogen. The binding appears to be independent of the protease-dependent morphological change which is exhibited by many transformed cells. The 125I- plasminogen which bound to the SV3T3 cells was completely degraded during three days of incubation to macromolecules which were the same size as the large and small chains of active plasmin, and to smaller fragments including 3-iodo-L-tyrosine. The plasminogen which bound to the 3T3 cells was only partially degraded to 3-iodo-L-tyrosine with intermediate conversion to plasmin-size peptides.

The results of a sublethal cell-surface trypsinization assay, developed to determine whether the processing of plasminogen by cells was a cell-surface phenomenon or an internal process, suggest that the cell-associated plasminogen was primarily bolll1d to the surfaces of the 3T3 and SV3T3 cells while the macromolecular degradation products were inside the cells.

Therefore, the 3T3 and SV3T3 cells bound, endocytosed and degraded serum plasminogen. The overall rate of this processing was approximately twice as fast for the SV3T3 cells as the 3T3 cells. This binding and processing of plasminogen does not appear to be a specific plasma membrane-plasminogen interaction and is known to be independent of the protease-dependent morphological change.

Essentially none of the plasminogen in the 3T3 growth medium was activated to plasmin, while a significant amount of the plasminogen in the SV3T3 growth medium was activated to plasmin, and subsequently complexed with a serum inhibitor of 47,000 molecular weight. This inhibitor is covalent, forming a hydroxylamine-dissociable bond.

To investigate whether any active plasmin remained in SV3T3 growth medium, a model system of urokinase in situ activation of plasminogen designed to mimic the levels of plasminogen activation observed in a 48-hour incubation of plasminogen with SV3T3 cells was developed. Some of the plasmin in this model system incorporated 32P-labeled di-isopropylfluorophosphate, implying that active plasmin exists in SV3T3 cell culture medium and thus may be responsible for the protease-dependent morphological change expressed by these cells and other phenotypic changes which occur upon virus transformation.

Files