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Doctoral Dissertation Announcement
Candidate: Denise L. Smith
Doctor of Philosophy
Department: Biological Sciences
Title: Characterization of the AP-1 and NF-kappaB Transcription Factors in the U-87 MG Astrocytoma Cell Line
Dr. Bruce Bejcek, Chair
Dr. John Geiser
Dr. Alex Enyedi
Dr. Cindy Miranti
Date: Thursday, October 23, 2008 1:00 p.m. - 3:00 p.m.
3723 Wood Hall
Growth factors, signaling molecules, and transcription factors are frequently mutated in cancer, and PDGF has been shown to be over-expressed in all glioma grades. In the anaplastic astrocytoma cell line U-87 MG, the over-expression of PDGF has been shown to lead to over-expression of transcription factors AP-1 and NF-κB, and characterization of the individual proteins in each transcription factor was assessed.
Proteins were knocked out using transient RNAi on all proteins, as well as induced stable cell line RNAi for four of the proteins. Western blots showed that each protein was decreased, which demonstrates that each protein was present in these cells and that the RNAi worked. Photomicrographs of the cells were taken revealing that, in all cases, neurosphere formation was absent when the RNAi was present suggesting a role in neurosphere formation and/or maintenance. Cell growth was decreased in all knockouts except JunB and p52 suggesting a role in cell growth and/or anti-apoptosis. Morphological changes, such as greater adherence to the plate, were shown in cells with c-Jun, c-Fos, c-Rel, p52, and p65 knocked out, suggesting a role in anchorage independent growth. Reporter gene assay showed that AP-1 was reduced when all proteins except JunB were knocked out and NF-κB was reduced in varying amounts when all proteins were knocked out, suggesting that all proteins are involved in dimer formation of the transcription factors except JunB.
Further assays were performed on the stable cell lines. Growth assays corroborated the photomicrographs showing that cell growth was decreased when c-Jun, c-Fos, c-Rel and p65 was knocked out. Caspase 3 and 7 levels were elevated as was cleaved PARP showing cell death was via apoptosis. Nestin levels were decreased suggesting that differentiation was occurring in the neurospheres, and neurosphere numbers were decreased in size and number as well as delayed in onset when any of the four proteins were knocked out. These proteins share many of the characteristics of cancer and could provide future therapeutic targets.