Abstract

Transforming growth factor-beta (TGF-p) belongs to a family of immunosuppressive cytokines, capable of regulating macrophages, T, B and NK cells. We have detected the TGF-p transcript being synthesized by H238 cells. This is herpes simplex-Type2 transformed murine fibrosarcoma cell line. The amount of TGF-p formed/106 cells was >2000pg/ml. Previous attempts to control H238 tumorigenicity had sub-optimal efficacies. In addition, studies had shown that the FI238 cell line was not responsive to TGF-p. This project was an attempt to develop an improved modality to reduce H238 tumorigenicity and investigate potential abnormalities in expression of TGF-p receptors in the H238 cell line.

We created expression plasmids encoding transgenes for murine IL-2 and antisense TGF-p and stably transfected H238 cells to create populations/clones of H238 cells expressing these transgenes. In vitro, expression analysis using ELISA and RT-PCR showed that H238 cells are capable of expressing functional IL-2 and, in addition, antisense transfected H238 cells showed a significant decrease in TGF-p production. The ultimate test, was to evaluate the tumorigenicity of these stable transfected cells in an immunocompetent BALB/c host, in comparison to wild type H238 cells. Mice injected with wild type and control H238 cells, showed a 80 - 100% tumor incidence. Mice injected with either antisense TGF-p or murine IL-2 transfected H238 cells showed almost no tumor incidence (0-20%). Mitogen stimulation of splenocytes from tumor bearing and non tumor bearing animals showed significant differences. Splenocytes from non tumor bearing animals displayed a higher stimulation index than from tumor bearing animals, probably alluding to lower amounts of tumor derived systemic TGF-p.

The non-responsiveness of H238 cells to TGF-p was also confirmed. Our results corroborated Uhm, at al (1993), whereby the authors reported similar results. To evaluate this further, we hypothesized that a defect within the TGF-p receptors may be the cause of this inert response. Total H238 RNA was exctracted and mRNA for TGF-p receptors was amplified using primers specific to the Type 1 and Type 2 TGF-p receptors, these were sequenced to confirm their identity. Our sequence analysis concluded that the TGF-p receptors on H238 cells were essentially normal with no critical mutations which would validate either splicing or truncated products. Protein expression was confirmed using Western blot analysis. Whole cell lysate was also resolved into membrane and cytosolic fractions, and these probed with for the type 1 or the type2 receptor. In comparision, to positive cell lysate controls for these receptors, H238 cell lysates showed a comparitively less amount of receptor expression. Receptor expression was further quantitated using a laser scanning cytometer.

Using this procedure, H238 cells showed approximately 50% less expression of the type 2 TGF-p receptor than the type 1 TGF-p receptor. Cytochemical staining for the receptors showed little membrane puncate staining, however, distinct staining was observed in the cytosolic regions of the cells. The lack of pucntate staining may be explained by either the relatively very few numbers of TGF-p receptors expressed/cell or there may be defects in the glycosylation and receptor trafficking. In addtion, the different ratios of the type 1 and type 2 receptor expression may also account for the inertness of H238 cells to TGF-p. This has been shown to be present in other tumor models and may explain the non responsiveness of FI238 cells to TGF-p.

LLU Discipline

Microbiology and Molecular Genetics

Department

Microbiology, Molecular Biology and Biochemistry

School

Graduate School

First Advisor

J. Kettering

Second Advisor

D. Gridley

Third Advisor

I. Fodor

Fourth Advisor

L. Green

Fifth Advisor

D. De Leon

Sixth Advisor

J. Rossi

Degree Name

Doctor of Philosophy (PhD)

Degree Level

Ph.D.

Year Degree Awarded

2001

Date (Title Page)

6-2001

Language

English

Library of Congress/MESH Subject Headings

Transforming Growth Factor beta; Interleukin-2 -- therapeutic Use; Neoplasms, Experimental -- drug therapy; Killer Cells, Lymphokine-Activated -- immunology; Receptors, Transforming Growth Factor beta

Type

Dissertation

Page Count

xii; 236

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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