Document Details

Document Type:   Dissertation
Title:   The Expression and Function of PPAR and HIF-1 in Human Melanoma
Author:   Caroline Nicole Mills
College:   Joan C. Edwards School of Medicine
Degree Program:   Biomedical Sciences, Ph.D.
Degree:   Doctor of Philosophy
Committee Director:   Dr. Richard Niles
Document Availability:   Document available for World-Wide access.
Date of Defense:   03/13/2007

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. PPARα 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 PPAR 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. 

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