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Date of Award
Restricted Thesis: Campus only access
Bachelor of Science
Dr. Valerie M. Kish
Glioblastoma multiforme (GBM) is the deadliest form of brain cancer, with survival after diagnosis typically under 1 year. One of the reasons GBM is so lethal is the fact that it is often resistant to radiation treatment and chemotherapy. In approximately 30% of glioblastomas, the tumor suppressor gene p53 is mutated. The p53 protein is essential as a transcription factor that controls cell cycle arrest and apoptosis by activating other proteins such as p21 and bax. If the p53 gene is mutated, it may be unable to activate these proteins that give the cell time to repair DNA damage or cause cell death. In order to find a method to treat this disease, it is necessary to characterize the functional status of glioblastomas in addition to the genotypic status. It is for this purpose that a functional assay for p53 has been developed. The functional assay involves cloning the p53 gene from a cell line into a eukaryotic expression vector, co-transfection cells that are null for the p53 gene with the vector containing the p53 gene and a reporter vector, and assaying for luciferase activity to assess p53 functionality. Two cell lines, T98G and U251, both ofwhich contain mutantp53, have been cloned and the clones have been sequenced. T98G clones 7 and 9 contained the full length p53 gene in the sense orientation and were determined to contain the expected mutation at codon 237 of ATG(Met)-->ATA(Ile) and two other mutations at codons 88 (Ala-->Ala) and 89 (Thr-->Ala). U251 clones 5, 9, 12, and 16 contained the full-length p53 gene in the sense orientation and clone 14 contained the full-length p53 gene in the antisense direction. All five of these clones contained the expected mutation at codon 273 of CGT(Arg)-->CAT(His). The p53 gene from U251 clone 12 was re-cloned into a new eukaryotic expression vector, pTRE2, which was more efficient for luciferase assay. In addition, wild-type p53 from the U87 glioblastoma cell line has been cloned into pTRE2, in the sense and antisense direction to obtain clones for luciferase assay controls.
Hull, Melissa A., "Development of a p53 functional assay for characterization of T98G, U251, and U87glioblastoma cell lines" (2001). Honors Theses. 507.