Texas Medical Center
gomez, fabiola c.
MECHANISM-BASED STRATEGIES TO ENHANCE THE ACTIONS OF A.
Degree: PhD, 2010, Texas Medical Center
Heat shock protein 90 (HSP90) is an abundant molecular chaperone that regulates the functional stability of client oncoproteins, such as STAT3, Raf-1 and Akt, which play a role in the survival of malignant cells. The chaperone function of HSP90 is driven by the binding and hydrolysis of ATP. The geldanamycin analog, 17-AAG, binds to the ATP pocket of HSP90 leading to the degradation of client proteins. However, treatment with 17-AAG results in the elevation of the levels of antiapoptotic proteins HSP70 and HSP27, which may lead to cell death resistance. The increase in HSP70 and HSP27 protein levels is due to the activation of the transcription factor HSF-1 binding to the promoter region of HSP70 and HSP27 genes. HSF-1 binding subsequently promotes HSP70 and HSP27 gene expression. Based on this, I hypothesized that inhibition of transcription/translation of HSP or client proteins would enhance 17-AAG-mediated cytotoxicity. Multiple myeloma (MM) cell lines MM.1S, RPMI-8226, and U266 were used as a model. To test this hypothesis, two different strategies were used.
For the first approach, a transcription inhibitor was combined with 17-AAG. The established transcription inhibitor Actinomycin D (Act D), used in the clinic, intercalates into DNA and blocks RNA elongation. Stress inducible (HSP90á, HSP70 and HSP27) and constitutive (HSP90â and HSC70) mRNA and protein levels were measured using real time RT-PCR and immunoblot assays. Treatment with 0.5 µM 17-AAG for 8 hours resulted in the induction of all HSP transcript and protein levels in the MM cell lines. This induction of HSP mRNA levels was diminished by 0.05 µg/mL Act D for 12 hours in the combination treatment, except for HSP70. At the protein level, Act D abrogated the 17-AAG-mediated induction of all HSP expression levels, including HSP70. Cytotoxic evaluation (Annexin V/7-AAD assay) of Act D in combination with 17-AAG suggested additive or more than additive interactions.
For the second strategy, an agent that affected bioenergy production in addition to targeting transcription and translation was used. Since ATP is necessary for the proper folding and maturation of client proteins by HSP90, ATP depletion should lead to a decrease in client protein levels. The transcription and translation inhibitor 8-Chloro-Adenosine (8-Cl-Ado), currently in clinical trials, is metabolized into its cytotoxic form 8-Cl-ATP causing a parallel decrease of the cellular ATP pool. Treatment with 0.5 µM 17-AAG for 8 hours resulted in the induction of all HSP transcript and protein levels in the three MM cell lines evaluated. In the combination treatment, 10 µM 8-Cl-Ado for 20 hours did not abrogate the induction of HSP mRNA or protein levels. Since cellular bioenergy is necessary for the stabilization of oncoproteins by HSP90, immunoblot assays analyzing for expression levels of client proteins such as STAT3, Raf-1, and Akt were performed. Immunoblot assays detecting for the phosphorylation status of the translation repressor 4E-BP1, whose activity is modulated by upstream…
Advisors/Committee Members: Varsha Gandhi, Ph.D., Juan Fueyo-Margareto, M.D., Peng Huang, M.D. Ph.D..
Subjects/Keywords: 17-allylamino-17demethoxygeldanamycin; 8-chloro-adenosine; actinomycin D; heat shock proteins; heat shock factor; multiple myeloma; transcription inhibitor; Cancer Biology; Therapeutics
to Zotero / EndNote / Reference
APA (6th Edition):
gomez, f. c. (2010). MECHANISM-BASED STRATEGIES TO ENHANCE THE ACTIONS OF A. (Doctoral Dissertation). Texas Medical Center. Retrieved from http://digitalcommons.library.tmc.edu/utgsbs_dissertations/31
Chicago Manual of Style (16th Edition):
gomez, fabiola c. “MECHANISM-BASED STRATEGIES TO ENHANCE THE ACTIONS OF A.” 2010. Doctoral Dissertation, Texas Medical Center. Accessed September 21, 2018.
MLA Handbook (7th Edition):
gomez, fabiola c. “MECHANISM-BASED STRATEGIES TO ENHANCE THE ACTIONS OF A.” 2010. Web. 21 Sep 2018.
gomez fc. MECHANISM-BASED STRATEGIES TO ENHANCE THE ACTIONS OF A. [Internet] [Doctoral dissertation]. Texas Medical Center; 2010. [cited 2018 Sep 21].
Available from: http://digitalcommons.library.tmc.edu/utgsbs_dissertations/31.
Council of Science Editors:
gomez fc. MECHANISM-BASED STRATEGIES TO ENHANCE THE ACTIONS OF A. [Doctoral Dissertation]. Texas Medical Center; 2010. Available from: http://digitalcommons.library.tmc.edu/utgsbs_dissertations/31