Jianming Han

Date of Award

2004

Degree Name

Doctor of Philosophy

College

Graduate School of Education and Professional Development

Type of Degree

Ph.D.

Document Type

Dissertation

First Advisor

Richard M. Niles

Second Advisor

Beverly Delidow

Third Advisor

Vernon Reichenbecher

Fourth Advisor

Donald Primerano

Fifth Advisor

Todd Green

Abstract

Protein Kinase C (PKC) has been implicated in many cellular responses. Of the 11 isotypes in this family, seven are expressed in B16 mouse melanoma cells − PKCα, PKCδ, PKCε, PKCθ, PKCλ, PKCζ, and PKCµ.

Treatment of B16 cells with retinoic acid (RA) results in growth inhibition and differentiation. Simultaneously, RA induces a 6-8 fold increase of PKC α protein level, but only about 2-fold increase of PKC enzyme activity. The over- expression of wild type PKCα in B16 cells mimicked the action of RA. The over- expression of enzymatically inactive PKCα, either open or closed conformation, also inhibited B16 cell anchorage-dependent and –independent growth. The percent reduction in growth rate correlated with the expression level of enzymatically inactive PKCα. The over-expression of enzymatically inactive PKCα did not affect the phosphorylation status of other PKC isozymes expressed in B16, suggesting that it was not interfering with their functional activity. Bisindolylmaleimide, a selective PKC enzyme inhibitor, did not reverse the effect of RA on B16 cell growth and differentiation. Cumulatively, these data suggest that PKCα has a non-enzymatic function in an RA-mediated cell signaling pathway which results in growth arrest and differentiation.

PKCλ and PKCµ protein levels were decreased about 50% after 48h of RA treatment. PKCλ protein level was found directly correlating with the growth rate of B16 cells. In order to study the function of PKCλ, the full length mouse PKCλ cDNA was cloned and expressed in B16 cells. The anchorage-dependent growth rate of B16 cell clones that over-expressed PKCλ was greater than wild type cells. These data indicate that PKCλ may be part of a pathway leading to enhanced B16 cell proliferation. There are no consistent changes in the levels of cell cycle regulatory proteins in B16 cell clones which overexpress PKCλ. However, the protein level of cyclin-dependent kinase inhibitors p21 was downregulated and p27 upregulated by RA treatment. The p27 protein level was increased at 2-h after RA treatment. Future studies need to investigate how p21 and p27 are regulated by RA, and to elucidate the role of PKCλ in growth control using inducible dominant-negative PKCλ constructs.

Subject(s)

Vitamin A.

Cancer cells - Regulation.

Cancer cells - Growth.

Retinoids.

Protein Kinase C.

Download

Share

COinS