Date of Award

1983

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Pharmaceutical Sciences

First Advisor

Clinton O. Chichester

Abstract

The transformation from carcinoma in situ to invasive carcinoma occurs when tumor cells traverse extracellular matracies allowing them to move into parenchymal tissues. Tumor invasion may be aided by the secretion of collagen and elastin degrading proteases from tumor and tumor-associated cells. In this study ~ the production of Type I and Type V collagen degrading activities and elastolytic activities by DLD-1 human colon carcinoma cells, B16-F10 murine melanoma cells, and normal human dermal fibroblasts was examined. DLD-1 cells and normal fibroblasts produced similarly high levels of collagenolytic activity. DLD-1 cells also produced high levels of elastinolytic activity; this activity was found exclusively in the extracellular medium. DLD-1 cells and normal fibroblasts produced more collagen and elastin degrading activity than did B16-F10 melanoma cells, a cell line characterized as highly metastatic. The Type I and Type V collagenolytic activities from DLD-1 cells were separated and characterized using DEAE cellulose chromatography and gel filtration chromatography. The Type I collagenolytic activity appeared to be an anionic protein at pH 8.3, and two forms of the enzyme were detected, one with a molecular weight of 60,000 and the other with a molecular weight of 35,000 daltons. The Type V collageno-ytic activity was a cationic protein at pH 8.3 and two forms of this activity were detected, one with a molecular weight of 80,000 and the second with a molecular weight of 54,000 daltons. The effect of the polar solvent, dimethylformamide (DMF) on the production of collagen and elastin degrading activities was studied using cultured DLD-1 cells. DMF treated cells produced significantly higher levels of Type I collagenolytic, Type V collagenolytic and cellassociated elastase activities than did control cells. DMF treatment had no significant effect on media elastase levels. Treatment of DLD-1 cells with DMF elevated the cellular levels of protein and RNA while these same cells had less DNA per cell. DMF treated cells were also able to synthesize protein at a significantly faster rate than control cells. This increased rate of protein synthesis may account for part of the increased production of connective tissue degrading enzymes seen in DMF treated cells.However, the effect of DMF on enzyme production was still present when the cells were treated with cycloheximide. The failure of cycloheximide to prevent the DMF induced increase in enzyme activity suggests that the effect of DMF is not entirely dependent on protein synthesis. These data suggest that DLD-1 human colon carcinoma cells produce at least two different collagen degrading enzymes and that DMF treatment may facilitate the invasive movement of cancer cells during the matastatic process by increasing the secretion of connective tissue degrading enzymes.

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