Regulation of murine melanoma metastasis by protein kinase C (PKC) delta.

摘要

Protein kinase C (PKC) delta has been shown to regulate many cellular activities involved in metastastatic process. Studies have shown that over-expression of PKC delta increases the metastatic potential of a highly metastatic murine melanoma cell line, and a mammary carcinoma cell line (La Porta et al 2000 and Kiley et al. 1999).; In this study, we examined the effects of transfection with wild-type or mutant PKC delta genes on metastasis of B16F1 melanoma cells. Cells are transfected with either wild-type delta or delta with a substitution of phenylalanine for tyrosine 155 (Y155F). We examined the effects on (1) metastasis, (2) tumor cell (TC) retention in the lung, (3) cell size, (4) invasion and adhesion to biological matrices, (5) proliferation and/or viability under adverse conditions including (a) limiting serum concentrations with or without cell crowding, and (b) exposure to cytotoxic agents and (6) patterns of subcellular localization. Results of this study show that over-expression of wild-type delta in B16F1 cells increases: the number and size of viable tumor cells/clumps 48 hrs after intravenous inoculation, number and size of metastatic pulmonary nodules several weeks later, in vitro adhesion of TC to matrigel, and consistently increases proliferation when compared to the control or Y155F mutant, but has only a slight or negligible effect on cell viability. However, over-expression of Y155F mutant delta had little or no effect on TC retention, metastasis, and adhesion but markedly increased TC survival in the presence of all cytotoxic agents and exhibited striking alterations in subcellular localization. Interestingly, the effect of mutant Y155F on proliferation was highly dependent upon serum concentration and cell crowding, but often resulted in less proliferation than wildtype delta and showed diminished viability.; In summary, the collective results in this thesis demonstrates that PKC delta regulates B161F1 metastasis and suggests that phosphorylation of intact tyrosine 155 can markedly and differentially effect many of the key regulatory events essential to the metastatic cascade.