Ying Huang

Date of Award

2003

Degree Name

Biomedical Sciences

College

Joan C. Edwards School of Medicine

Type of Degree

Ph.D.

Document Type

Dissertation

First Advisor

Richard M. Niles

Second Advisor

Beverly Delidow

Third Advisor

Vernon E. Reichenbecher

Fourth Advisor

Elsa Mangiarua

Fifth Advisor

Don Primerano

Abstract

Our laboratory has been studying the effects of all-trans retinoic acid (atRA) on the growth and differentiation of the B16 mouse melanoma cells. RA inhibits both anchorage-dependent and –independent growth and stimulates melanin production in B16 cells, which are accompanied by an increase in AP-1 activity. To further investigate the RA-induced AP-1 activity, I established clones of B16 stably expressing an AP-1-luciferase reporter gene. Treatment of these clones with phorbol dibutyrate (PDB), a known activator of AP-1, rapidly increased AP-1 activity which reached its maximum level at 2-4 h and returned to baseline level by 24 h. In contrast, RA treatment resulted in a slow increase in AP-1 activity that reached a maximum level at 48 h and was maintained for the duration of the treatment. I tested the importance of this increase in AP-1 activity for the biological effects of RA by establishing clones which stably express a dominant-negative version of the Fos gene (A-Fos), a component of the AP-1 complex. Clones expressing A-Fos have greatly diminished AP-1 activity and a markedly decreased sensitivity to RA-induced growth inhibition and differentiation. Our observations suggest that RA-induced AP-1 activity plays an important role in the biological changes mediated by RA in B16 cells.

The AP-1 transcription complex consists of homodimers and heterodimers of Jun and Fos family members, or ATF2 bZIP proteins. Malignant B16 melanoma cells have a much higher amount of ATF2 compared to non-malignant mouse melanocytes. Over 70% of the ATF2 protein in B16 cells is in the phosphorylated form, while only 16% of the total ATF2 is phosphorylated in the mouse melanocytes. RA treatment of B16 cells for 48 h decreased ATF2 phosphorylation by 60%. Investigation of the signaling pathway involved in RA- dependent decrease in ATF2 phosphorylation demonstrated that RA inhibits activation of the MAP kinase p38. These data suggest that RA inhibits ATF2 phosphorylation most likely via its ability to block the activation of p38. In addition, RA increases the sensitivity of B16 cells to the chemotherapeutic drug, taxol, likely by the way of decreasing ATF2 phosphorylation.

Subject(s)

Melanoma - Cytopathology.

Cancer - Research.

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