Date of Award

2007

Degree Name

Doctor of Philosophy

College

Graduate School of Education and Professional Development

Type of Degree

Ph.D.

Document Type

Dissertation

First Advisor

Richard Niles

Second Advisor

Beverly Delidow

Abstract

The first part of my dissertation focuses on the expression and function of PPARs in human melanoma. I found that the A375 cells were significantly growth inhibited in response to PGJ2 and troglitazone treatment. HEMn-LP showed significant growth inhibition in response to troglitazone. I found that PPARγ and PPARδ mRNA is present in both the SK-Mel 28 and A375 cells. The relative level of PPARα mRNA expression is highest in SK-Mel 28 cells, ~3 fold higher relative to both the normal human melanocytes and A375 cells. PPARγ protein was ~50% higher in both SK-Mel 28 and A375 cells relative to the HEMn-LP. PPAR α protein levels were highest in the A375 cells. Consistent 80% knockdown of PPARα was achieved through siRNA treatment; however, there was no change in cellular morphology. There was also no decrease in expression of a direct PPARα target, MCAD. Therefore, a reasonable conclusion is that the increased expression of PPARα in SK-Mel28 cells is not contributing to its in vitro transformed phenotype.

Hypoxia inducible factor 1α, HIF-1α, is a transcription factor that has been shown to be a master regulator of oxygen homeostasis. A splice variant of HIF-1α, HIF-1α785, is missing exon 11 from its oxygen dependent degradation domain. This region encodes the lysine that is critical for enhancing HIF-1α degradation. The role of HIF-1α in the progression of human melanoma has not been fully elucidated. Here, I show for the first time that in human melanoma, HIF-1α is expressed endogenously with no external stimuli under normoxic conditions. In cell lines derived from RGP, VGP, and metastatic phases of human melanoma progression, the relative amounts of HIF-1α and HIF-1α785 mRNA increase as a function of malignant progression. The expression levels have been verified by qPCR and western blot. Overexpression of HIF-1α or HIF-1α785 in SbCl2 cells leads to increased anchorage independent growth, with HIF-1α785 having the greater impact. In WM9 cells, inhibition of HIF-1α by siRNA significantly inhibits matrigel invasion and anchorage independent growth in soft agar. These results show that in human melanoma, HIF-1α and HIF-1α785 seem to function to increase tumorgenicity.

Subject(s)

Melanoma.

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