Oncogene Expression in Transformed Cells

Naudé, Johan Alfred Leonard te Water

Oncogene Expression in Transformed Cells

Thesis / Dissertation

1994

Publisher

University of Cape Town

Department

Division of Medical Biochemistry

Faculty

Faculty of Health Sciences

Abstract

This study investigated the expression of the c-fos gene in two cell lines. The parent cell line is a normal human diploid fibroblast cell line, WI-38. The derived cell line, WI-38 CT-1, is a heteroploid cell line which exhibits many of the characteristics common to transformed cells; it was derived from the parent cell line by y-irradiation. The c-fos gene is an essential component of the mechanisms governing cellular proliferation, and was shown by Northern blotting to be expressed at a very low level in the transformed cell line, when compared to the parent cell line. Defining the nature of this facet of the behaviour of the transformed cell line was the major thrust of this work. It was demonstrated that the low expression of the c-fos mRNA in the WI-38 CT-1 cell line was probably due to there being a decreased transcription of the c-fos gene. This proposition was supported by there being a similar decrease in the expression of the EGRl gene in the transformed cell line. The expression of this gene is known to be regulated in a manner similar to that of the c-fos gene. Further work showed that this occurred in the presence of a vigorous response to serum stimulation in the transformed cell line, as measured by the total rates of RNA synthesis following serum stimulation. The decreased c-fos response to serum stimulation was shown not to be limited by the expression of receptors on the cell surface of the WI-38 CT-1 cell line, by employing agents such as phorbol esters and the calcium ionophore that bypass this route. The use of these agents, which act via discrete regions of the c-fos promoter, also demonstrated that the inability of the transformed cell line to respond to serum stimulation with a vigorous c-f os excursion was a consequence of a global depression of c-fos transcription, as all elements involved in the expression of the c-fos gene were equally influenced. This study has also demonstrated that bradykinin, a vasoactive peptide, which is involved in mediating the response of cells to inflammation, and which has been shown to be mitogenic for fibroblasts, was able to cause induction of c-fos mRNA expression in both cell lines. This is a novel finding. Furthermore, there were no sequence differences in the c-fos promoter in either cell line which might account for the observed difference in c-fos gene expression. The sequence of the c-fos promoter in both cell lines was shown to differ from that originally reported for this region: however, these differences were shown not to involve any of the protein-binding sequences described for this promoter. In vitro analysis of the DNA-protein interactions showed that there was a qualitative similarity in the patterns produced by gel shift analysis in both cell lines using the c-fos promoter as a probe. Higher resolution mapping of these interactions, using the methylation interference assay, showed that the DNA-binding activities involved were similar to those described for the serum response factor (SRF) and the cAMP response element (CRE): these sequences, and their cognate DNA-binding activities, have been previously identified as being major determinants of c-fos gene expression. Transfection of a c-fos promoter-dependent reporter gene into both cell lines showed an equivalent induction. This similarity was sustained for different reporter gene constructs and inducing agents. This dissertation reports a phenomenological study of the expression of the c-fos gene in two related human cell lines. It has defined the comparative behaviour of the c-fos gene in these cell lines, and has suggested that the nuclear atypia present in the transformed cell line might be responsible for the differential expression of the c-fos gene in these two cell lines. It has used the expression of the c-fos gene to illustrate the inherent complexities of cellular transcriptional regulation and the differences implicit in related but behaviourally distinct cell lines. It has suggested that the main thesis of this study might be relevant to some aspects of the behaviour of metastatic cells.

Keywords

c-fos gene

oncogene

Reference:

Collections

PhD / Doctoral

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