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DIFFERENTIAL EXPRESSION OF GENES BETWEEN
NICKEL COMPOUNDS/3-METHYLCHOLANTHRENE-TRANSFORMED,
AND NON-TRANSFORMED, C3H/10T1/2 Cl 8 MOUSE EMBRYO CELL LINES,
AND THIOL REGULATION OF VEGF-A AND VEGF-A RECEPTORS
IN HUMAN RPE CELLS
by
Aruni Therese De Silva
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(PATHOBIOLOGY)
December 2009
Copyright 2009 Aruni Therese De Silva
Object Description
| Title | Differential expression of genes between nickel compounds/3-methylcholanthrene-transformed, and non-transformed, C3H/10T1/2 cl 8 mouse embryo cell lines, and thiol regulation of VEGF-A and VEGF-A receptors in human RPE cells |
| Author | Silva, Aruni Therese De |
| Author email | adesilva@usc.edu; arunidesilva@gmail.com |
| Degree | Doctor of Philosophy |
| Document type | Dissertation |
| Degree program | Pathobiology |
| School | Keck School of Medicine |
| Date defended/completed | 2009-07-21 |
| Date submitted | 2009 |
| Restricted until | Unrestricted |
| Date published | 2009-08-17 |
| Advisor (committee chair) | Landolph, Joseph R., Jr. |
| Advisor (committee member) |
Hinton, David Widelitz, Randy Mosteller, Raymond |
| Abstract | Molecular mechanisms of neoplastic cell transformation by insoluble Nickel (Ni) compounds have not yet been elucidated, and are the subject of this work.; Epithelial cell transformant factor-2 (Ect-2) gene is amplified and differentially expressed between non-transformed and nickel or 3-methylcholanthrene (MCA)-transformed 10T1/2 mouse fibroblastic cell lines leading to higher steady-state levels of Ect-2 mRNA and protein in all transformed cells. Ect-2 proto-oncogene catalyzes GDP exchange on Rho proteins, and regulates cytokinesis. We hypothesized there would be higher steady-state levels of microtubules in transformed cell lines. The hypothesis was tested by staining cell lines with fluorescent alpha or beta tubulin antibody and analyzed using confocal microscopy and the volocity image analysis program. Compared to the 10T1/2 cells, the average intensity of all cells stained silmutaneuosly for alpha and beta tubulin was 1.5 - 2.1 and 1.1 - 2.5 folds higher.; We identified the unknown down-regulated R2-2 and R2-3 gene fragments in transformed cell lines as fragments of beta centaurin-2 and FAD synthetase genes. Beta centaurin-2 protein regulates disassembly of microfilaments. We hypothesized there would be higher steady-state levels of microfilaments in all transformed cells. Hypothesis was tested by staining non-transformed and Ni-/MCA-transformed cell lines with phalloidin-conjugated- Tetramethylrhodamine B isothiocyanate (TRITC) against actin microfilaments, and quantitating the intensity of microfilament staining and cross-sectional area of cells. Compared to the 10T1/2 cells, we found an average intensity of 1.7 - 2.3 folds higher for microfilaments in all transformed cell lines, and a 1.3 - 1.7 fold for all transformed cells stained simultaneously, compared to non-transformed cells. Aggregation of microfilaments and a rounded or star-shaped morphology was observed for all transformed cells. All transformed cells analyzed had a cross-sectional area of 1.9 - 2.5 folds larger than that of non-transformed 10T1/2 cells. Neoplastic cell transformation is partly due to the amplification of the Ect-2 gene and the simultaneous inactivation of the beta centaurin-2 gene, leading to cytoskeletal derangements. Together, higher steady-state levels of microtubules and microfilaments leads to significant changes in cell shape, effects on gene expression and on induction and maintenance of morphological, Anchorage Independence (AI), and neoplastic cell transformations in transformed cells. |
| Keyword | neoplastic cell transformation; alpha tubules; beta tubules; microfilaments; cytoskeletal derangements; nickel |
| 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-m2568 |
| Contributing entity | University of Southern California |
| Rights | Silva, Aruni Therese De |
| 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-Silva-3108 |
| Archival file | uscthesesreloadpub_Volume26/etd-Silva-3108.pdf |
Description
| Title | Page 1 |
| Contributing entity | University of Southern California |
| Full text | DIFFERENTIAL EXPRESSION OF GENES BETWEEN NICKEL COMPOUNDS/3-METHYLCHOLANTHRENE-TRANSFORMED, AND NON-TRANSFORMED, C3H/10T1/2 Cl 8 MOUSE EMBRYO CELL LINES, AND THIOL REGULATION OF VEGF-A AND VEGF-A RECEPTORS IN HUMAN RPE CELLS by Aruni Therese De Silva A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (PATHOBIOLOGY) December 2009 Copyright 2009 Aruni Therese De Silva |
