Abstract

Cytokines have a profound effect on immune modulation, thus playing a significant role in cancer gene therapy. A variety of human carcinomas secrete transforming growth factor-beta (TGF-β), a cytokine with potent immunosuppressive properties. Suppressing TGF-β could be a key to successfully combating malignancies, such as gliomas, mammary, and colon cancers, that over-express TGF-β. Conversely, interleukin-12 (IL-12) is a potent immunostimulatory cytokine capable of regulating T and NK cell-mediated cytotoxic responses (Trinchieri, 1994) during an anti-tumor response. Activation of the immune system with such immunostimulatory cytokines renders it inert to the suppressive effects of TGF-β. To better understand these processes our lab has made use of a unique syngeneic tumor model. H238 is a Herpes simplex type-2 transformed murine fibrosarcoma, which produces TGF-β. We have demonstrated strong expression of TGF-β1, 2, and 3 from H238 cells, providing a mechanism for tumorigenesis (Goldman et ah, 1996). To inhibit H238 tumor development, our research strategy is focusing on a novel delivery approach using murine IL-12 and antisense TGF-βl.

It was hypothesized that co-expression of antisense TGF-βl and IL-12 by H238 cells would synergistically enhance the BALB/c mouse’s immune system and, thereby, potentially abolish tumor development. To test this hypothesis, two specific aims were achieved. Firstly, we constructed and transfected plasmid vectors which express IL-12 and/or antisense TGF-β1 into H238 cells, thereby establishing three new modified H238 cell lines. The growth kinetics of these modified cells were evaluated in culture and found to have no difference in growth compared to control H238 cells. Expression assays using ELISA revealed a slight reduction in TGF-β1 produced by cells transfected with the antisense TGF-β1 containing plasmid vectors and strong expression of IL-12 p70 from cells transfected with the IL-12 containing plasmid vectors. In fact, supernatant collected from cells and diluted 1:250 revealed IL-12 p70 expression levels approaching 800 μg/ml after 72 hours.

We began our second specific aim to evaluate the tumorigenicity and immunogenicity of this strategy in the BALB/c mouse system. An analysis of mouse weights pre-injection and post-sacrifice along with careful qualitative observations revealed no significant cytotoxic effects of IL-12 in any of the mice injected with IL-12 secreting cells. The mice injected with cells transfected with an antisense TGFβ1 probe (pBmas) showed a significantly enhanced tumor progression compared to wild-type and plasmid control groups, in vivo. However, absolutely no tumors formed in the mice injected with cells transfected with plasmids containing murine IL-12 followed up to 18 days after the first palpable tumors appeared in the control groups. In addition, complete tumor regression was observed in a single IL-12 mouse after formation of a very small tumor. Injection of H238 wild-type cells into mice, previously immunized with H238 cells genetically modified to secrete IL-12 (pBIL-12H238), revealed no tumor formation in any of the mice compared to the control group. This may have implications into IL- 12’s role on establishing protective immunity (immunological memory).

Thus, we report IL-12 secreting cells to be highly immunogenic causing complete tumor rejection in an immunocompetent BALB/c host. The murine tumor model (H238) being a syngeneic system, has allowed further investigation into: 1) the effects of TGF-β and IL-12 on the host’s immune system, and 2) the possibility of using this novel approach as a DNA vaccine in human tumors expressing TGF-β.

LLU Discipline

Microbiology and Molecular Genetics

School

Graduate School

First Advisor

James D. Kettering

Second Advisor

Lora M. Green

Third Advisor

Daila S. Gridley

Degree Name

Master of Science (MS)

Degree Level

M.S.

Year Degree Awarded

2001

Date (Title Page)

8-2001

Language

English

Library of Congress/MESH Subject Headings

Neoplasms -- immunology; Neoplasms -- therapy; Immunogenetics; Interleukin-12 -- physiology; Transforming Growth Factor beta; Gene Therapy; Immunotherapy, Adoptive; Killer Cells, Natural

Type

Thesis

Page Count

xx; 182

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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