Study of the Influence on Growth and Apoptosis Anduced by Transduction of Bioactive TGF-b1 cDNAin TGF-b1 Sensitive Ovarian Cancercell-lines.

Project Details

Description

1. Aim study the effect of TGF-131 on the growth and apoptosis of ovarian cancer cells after introduction of the corresponding cDNA in these cells.To compare this with the effect of exogenous TGF-~31 Introduction TGF-B1 belongs to a superfamily of growth factors (TGF-13's, Activins, Bone Morphogenic Proteins's, BMP and Decapentaplegic Product, DPP) with a wide range of biological functions(1). In normal ovarian epithelium TGF-O n and its cellular receptors function as an autocrine growth-suppressive circuit(2,3). TGF-131 binds to a complex of two transmembrane serine/ threonine kinases, induces their phosphorylation, which results in the activation of signal transduction cascades, including phosphorylation of the Smad proteins(4,5) and eventually interaction with the AP-1 system. In cancer cells this growth suppressive circuit is often inactivated due to mutations of components of the signal transduction cascade(6). These mutations not only favor cell growth, but also probably play a role in the pathogenesis of certain cancers(7).In prior work we have demonstrated that some ovarian cancer cell lines have conserved their sensitivity to exogeneous TGF-31 although they produce insufficient active TGF-131 to suppress their own growth(8). Even if the TGF-31 secreted under a latent form (associated with "latency associated protein" or "LAP") by these cells would be completely activated in vivo, this would still yield insufficient concentrations to suppress cellular growth(8). These sensitive cell lines could be used as a model for the development of a molecular treatment strategy which subsequently could be applicable to a still larger proportion of in vivo ovarian cancers, as primary ovarian cancer cultures have kept their sensitivitity to TGF-61(9). Administration of TGF-131 peptide in vivo at an effective antitumoral dose is impossible, because lethal in animals. In the roposed project, we want to examine whether expression of TGF-81 by means of gene transfer can inhibit the growth of ;ell lines with a functional signal transduction in vitro and subsequently in vivo. The first steps in this project have been realted in preparatory work:1. Using site-directed mutagenesis we have created a mutant TGF-B1 cDNA which encodes an active form of TGF-31 which does not need further extracellular activation(10). 2. We have verified the export and functionality of this construct: comparative in vitro transfection plasmid transfection experiments have been performed with mutant and wildtype TGF- 31 expression constructs on 293 cells. The conditioned media of the transfected cells was added to the sensitive Mink CCL64 cell line. Based on cell counts an important growth inhibition (> 90 %) has been observed in cells exposed to conditioned media of TGF-131A transfected cells in comparison with conditioned media of the controls, including 293 cells transfected with wildtype TGF-131 (summarizing figure appendix 1, unpublished data). This work was done by a doctoral student (VUB, Masters and PhD program in Medical and Pharmaceutical Research), Mr. Ziad Zeinoun, under the supervision of a molecular biologist, dr. Erik Teugels, PhD.
AcronymOZR475
StatusFinished
Effective start/end date1/01/0031/12/03

Flemish discipline codes

  • Basic sciences

Keywords

  • Cancer Research