Zhaodong Xu

Abstract

Insulin-like growth factors (IGFs) are potent mitogens for a variety of cancer cells in vitro. In breast, prostate and neuroblastoma cancer cells, it has been suggested that IGF-II plays a paracrine/autocrine role. However, information on cell-type -specific IGF-II expression in vivo is limited. In situ hybridization and immunohistochemistry were carried out to determine the cell type expressing IGF-II in different tumor tissues. Both IGF-II mRNA and protein were localized to malignant cells, and expression in the stroma was minimal in all the tumors. The data are consistent with the hypothesis that cancer cell growth is regulated by IGF-II, and therefore IGF-II is a potential target for cancer therapy.

RNA enzymes (ribozymes) which selectively cleave RNA targets via base-pairing interactions can serve as therapeutic agents. We constructed IGF-II ribozymes and stably transfected them in prostate cancer PC-3 cells. Single- and double-hammerhead ribozymes were synthesized and cloned into the pTZU6+27 or pcDNA vectors. In vitro studies showed that both single- and double-ribozymes cleaved the ~ 140 bases and /or ~1 kilo bases IGF-II RNA substrates examined, while the mutant ribozymes did not. Kcat/Km for cleaving the shorter IGF-II substrate by double ribozyme and single ribozyme was 4772 and 1546 M-1S-1, respectively, suggesting that double ribozyme was ~3 fold more efficient in cleaving the IGF-II substrate than the single ribozyme in vitro. PC-3 stable transfectants expressing single ribozyme or double ribozyme, under control of U6 promoter, reduced the endogenous IGF-II mRNA and cell growth compared to mutant ribozyme transfectants. Similarly, PC-3 stable transfectants expressing single ribozyme, under control of CMV promoter, reduced the endogenous IGF-II mRNA and IGF-II protein secretion compared to vector-control cells. Furthermore, PC-3 cells expressing single ribozyme grew poorly under serum-free or 2% FCS conditions as judged by growth curves, supporting our hypothesis that IGF-II plays a critical role in prostate cancer cell growth, and thus provides a basis for developing a potential gene therapy for cancer.

LLU Discipline

Microbiology and Molecular Genetics

Department

Microbiology, Molecular Biology and Biochemistry

School

Graduate School

First Advisor

Yoko Fujita-Yamaguchi

Second Advisor

Daisy De Leon

Third Advisor

Keiichi Itakura

Fourth Advisor

James Kettering

Fifth Advisor

John J. Rossi

Sixth Advisor

John Termini

Degree Name

Doctor of Philosophy (PhD)

Degree Level

Ph.D.

Year Degree Awarded

1998

Date (Title Page)

6-1998

Language

English

Library of Congress/MESH Subject Headings

Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Receptors, Insulin-Like Growth Factor I -- genetics; Receptors, Insulin-Like Growth Factor II -- genetics; RNA, Catalytic -- metabolism; Gene Expression; Catalysis.

Type

Dissertation

Page Count

xii; 203

Digital Format

PDF

Digital Publisher

Loma Linda University Libraries

Usage Rights

This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.

Collection

Loma Linda University Electronic Theses and Dissertations

Collection Website

http://scholarsrepository.llu.edu/etd/

Repository

Loma Linda University. Del E. Webb Memorial Library. University Archives

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