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IDENTIFYING NOVEL COMBINATION FOR MULTIDRUG-RESISTANT NEUROBLASTOMA WITH TP53 MUTATIONS
by
Jen-Ming Huang
________________________________________________________________
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(CRANIOFACIAL BIOLOGY)
May 2010
Copyright 2010 Jen-Ming Huang
Object Description
| Title | Identifying novel combination for multidrug-resistant neuroblastoma with TP53 mutations |
| Author | Huang, Jen-Ming |
| Author email | jenmingh@usc.edu; jenmingh@gmail.com |
| Degree | Doctor of Philosophy |
| Document type | Dissertation |
| Degree program | Cranio-Facial Biology |
| School | School of Dentistry |
| Date defended/completed | 2010-03-16 |
| Date submitted | 2010 |
| Restricted until | Unrestricted |
| Date published | 2010-05-06 |
| Advisor (committee chair) | Keshelava, Nino |
| Advisor (committee member) |
Paine, Michael L. Shi, Wei Seeger, Robert C. |
| Abstract | Neuroblastoma is a tumor of the sympathetic nervous system in children. Loss of p53 function confers high-level drug resistance in neuroblastoma, and we therefore hypothesize that p53-independent therapies might be a potential therapy against recurrent neuroblastoma. We tested the activity of histone deacetylase (HDAC) inhibitors (vorinostat and depsipeptide), and flavopiridol, a pan-Cdk inhibitor, in a panel of multidrug-resistant (MDR) neuroblastoma cell lines that included lines with wild-type (wt) and transcriptionally active TP53 (n =3), mutated (mt) and loss of function (LOF) TP53 (n = 4) or p14 deletion (n = 1). Cytotoxicity was measured using a fluorescence-based assay of cell viability employing digital imaging microscopy (DIMSCAN), where cytotoxic synergy, expressed as combination index (CI) (CI < 0.8 indicates synergy) was calculated by CalcuSyn software. The combinations of HDAC inhibitors with flavopiridol were synergistic and significantly more cytotoxic (p<0.001) in cell lines with p53 LOF and in wt TP53 cells stably transfected with dominant negative p53 plasmids. In this study, we determined the mechanism and the molecular targets of the vorinostat + flavopiridol drug combination. The combination induced caspase-independent cell death, in the form of mitotic cell death. Our data characterized the mitotic cell death caused by the combination in CHLA-90 (mt TP53) cells. Cell cycle analysis by flow cytometry demonstrated prominent cell cycle arrest in the G2/M phase for a cell line with wt TP53 (SK-N-RA) at 16-20 hours (37%), while cells with mt TP53 (CHLA-90) lost DNA content and entered into the subG1 region at 6-24 hours (25-40% specific cell death). The morphological hallmarks of mitotic cell death, such as defective spindle formation and abnormal cytokinesis, were observed by confocal microscopy after treatment with the vorinostat + flavopiridol combination in CHLA-90 cells.; The combination caused reduction in the expression of G2/M (Cyclin B1) and mitotic proteins (Mad2, MPM2) in cell lines with mt TP53 (CHLA-90, CHLA-172), but not in those with wt TP53 (SK-N-RA, CHLA-136). Plk1 expression was reduced in all treated lines. siRNA knockdown of Plk1 was more lethal in CHLA-90. Moreover, siRNA knockdown of Mad2 and cyclin B1 or Plk1 synergistically reduced the clonogenicity of CHLA-90 cells. We also combined HDAC inhibitors with other Cdk inhibitors (roscovitine and purvalanol A) against MDR neuroblastoma cell lines with mt TP53, and demonstrated significant synergy. Thus, the combination of HDAC inhibition with Cdk inhibitors may be a unique approach to treating cancers with p53-LOF, one that evokes induction of mitotic failure. |
| Keyword | cancer; drug-resistance; neuroblastoma; p53; therapeutics |
| Language | English |
| Part of collection | University of Southern California dissertations and theses |
| Publisher (of the original version) | University of Southern California |
| Place of publication (of the original version) | Los Angeles, California |
| Publisher (of the digital version) | University of Southern California. Libraries |
| Provenance | Electronically uploaded by the author |
| Type | texts |
| Legacy record ID | usctheses-m3024 |
| Rights | Huang, Jen-Ming |
| Repository name | Libraries, University of Southern California |
| Repository address | Los Angeles, California |
| Repository email | http://www.usc.edu/isd/libraries/services/ask_a_librarian/email/ |
| Filename | etd-Huang-3600 |
| Archival file | uscthesesreloadpub_Volume51/etd-Huang-3600.pdf |
Description
| Title | Page 1 |
| Full text | IDENTIFYING NOVEL COMBINATION FOR MULTIDRUG-RESISTANT NEUROBLASTOMA WITH TP53 MUTATIONS by Jen-Ming Huang ________________________________________________________________ A Dissertation Presented to the FACULTY OF THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (CRANIOFACIAL BIOLOGY) May 2010 Copyright 2010 Jen-Ming Huang |
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