GENE EXPRESSION STUDIES OF LYMPHOMA CELLS TREATED WITH ANTIESTROGEN

Abstract

The studies described in this thesis set out to examine certain antiestrogen effects that are independent of the estrogen receptor (ER) for their expression. Experiments were performed therefore on an ER-negative murine lymphoma cell line, EL4, in order to eliminate any ER-mediated biological activity of the antiestrogen, clomiphene. The concentration of clomiphene used in these studies was largely submicromolar to reflect in vivo concentrations of clomiphene were shown previously to be cytostatic for EL4 cells. Additionally, since little is known of the long term biological effects of antiestrogens on cells in culture, EL4 cells were grown in the continuous presence of a subtoxic concentration (1 X 10-8 M) clomiphene for more than one year (EL4T cells), and compared with untreated controls (EL4 cells). Cell cycle analysis showed an accumulation of EL4T cells in the G1 phase which was coupled with a reduction of cells in the S phase and G2 phase when compared to EL4 cells. This suggested a discrete block at the G1-S boundary or in the late G1 phase induced by clomiphene. The ability of clomiphene to perturb the distribution of EL4T cells in discrete phases of the cell cycle cycle led to studies of differential gene expressions. The technique adopted was first developed by Liang and Pardee (1992) for differential display of mRNA. In the original method, mRNA was first reverse transcribed into subsets of cDNA were using oligo(dT) primers anchored by two bases at the 3’ end. Subsets of cDNA were next amplified in polymerase chain reactions containing the anchored oligo(dT) primer, and arbitrary decamer (i.e. the primer pair) and 35S-dATP. PCR products were visualized by autoradiography. During the course of these studies however, contamination of the thermal cycler by 35S was discovered. The problem of volatile contaminants arising from 35S-labelled radio-chemicals was also documented by other laboratories. In view of this potential hazard, a non-radioactive method was developed to display PCR products which were visualized by electrophoresis on 5 mm slab gels of low-melting agarose. Of 32 primer pairs tested, only four pairs displayed a total of eleven mRNAs that appeared to be expressed more intensely in one cell population than in the other. Northern analysis was next performed to confirm differential gene expression. Probes isolated from the apparently differentially displayed fragments were hybridized against total RNA and EL4 and EL4T cells. Only one of the eleven probes (probe 11) hybridized as a single band on the Northern blot. The other probes either hybridizes to ribosomal RNA or did not hybridise at all. Such false-positive results and artefacts were now known to be common among practitioners of this method. As a trial, probe 11 (0.5 kb) was sequenced by direct cycle sequencing. This resulted in 163 bases of readable sequence. The base sequence was translated into protein sequences in two open reading frames. Database homology search using the first open reading frame gave several possible homologies, while the second open reading frame was partially but consistently homologous to immunoglobulins.