Characterization of the AP-1 and NF-Kappa B Transcription Factors in the U-87 MG Astrocytoma Cell Line
Date of Award
Doctor of Philosophy
Dr. Bruce Bejcek
Dr. John Geiser
Dr. Alex Enyedi
Dr. Cindy Miranti
Growth factors, signaling molecules and transcription factors are frequently mutated in cancer and platelet-derived growth factor (PDGF) has been shown to be over-expressed in most gliomas. In the astrocytoma cell line U-87 MG, the over-expression of PDGF has been shown to lead to over-expression of transcription factors activator-protein-1 (AP-1) and nuclear factor-kappa B (NF-κB), and it is believed that the individual subunits of AP-1 (c-Jun, JunB, JunD, c-Fos, FosB, Fra-1, and Fra-2) and NF-κB (c-Rel, p50, p52, p65, and RelB) play different roles in proliferation, survival and differentiation in the U-87 MG cell line.
To assess these different roles, transient short hairpin RNA (shRNA) for all transcription factor subunits, as well as induced stable shRNA for c-Jun, c-Fos, p65, and c-Rel were generated to suppress gene expression. Protein levels were decreased for each subunit of the two transcription factors when the shRNA was present or induced and that each subunit was present in this cell line.
Photomicrographs of the cells showed c-Rel, RelB, p65, c-Jun, c-Fos, Fra-1, and Fra-2 had fewer cells when transient shRNA or induced stable shRNA was present. Also when the transient shRNA or induced stable shRNA was present, no neurospheres were present. Photos of stained neurospheres revealed onset delay, smaller and fewer neurospheres when the stable shRNA was induced. AP-1 and NF-κB transcription factor activity was reduced when any transient shRNAs or induced stable shRNA was present except for JunB.
In the presence of c-Jun, c-Fos, c-Rel, or p65 induced stable shRNA growth was reduced, caspase 3 and 7, and cleaved PARP levels were increased, and nestin levels were decreased. These results suggest that c-Jun, c-Fos, Fra-1, Fra-2, p65, c-Rel, and RelB have a role in proliferation and/or evasion of apoptosis. The changes in neurospheres and nestin levels suggest a role for c-Jun, c-Fos, c-Rel, and p65 in neurosphere formation and/or maintenance and differentiation respectively. These results further illustrate that the different subunits do have different roles in the U-87 MG cells.
Smith, Denise L., "Characterization of the AP-1 and NF-Kappa B Transcription Factors in the U-87 MG Astrocytoma Cell Line" (2008). Dissertations. 817.