The Netherlands Cancer Institute is running a DNA microarray facility where this technology is used in fundamental research projects and studies on patient material. We are using the Unigene 40k human gene library (Research Genetics) to monitor tumour-specific expression profiles. We aim to find prognostic markers that predict treatment outcome. For these studies, we have available an extensive, well-documented tumour bank that has been collected over the past 20 years. Currently, large numbers of tumour samples and healthy control tissues are being prepared for analysis. To gain more insight into the significance of tumour-specific gene expression profiles, we are in the process of generating microarray data on important tumour-related aspects of the eukaryotic cell, for example its cell division cycle. In the last decade many of the key regulators of the cell cycle have been identified. The role of the retinoblastoma protein family in regulating the activity of the E2F transcription factors (potent inducers of the G1-S transition of the cell cycle) is now well established. Activation or repression of a series of genes is essential for a smooth passage through each cell cycle phase. Among the target genes of E2F transcription factors are cell-cycle control genes (for example, genes encoding cyclin E and the pocket protein p107, a relative of the retinoblastoma tumour-suppressor gene product pRb) and proteins involved in the actual duplication of the genome (for example DNA pola, TK and CDC6). So far, five E2Fs have been cloned that can bind members of the pocket protein family (pRb, p107 and p130). We set out to identify new target genes of E2Fs by generating monoclonal NIH3T3 cell lines that overexpress a particular E2F species. We used DNA microarrays to screen for fluctuations in mRNA levels of known and unknown genes and found that one major target of E2F1 was insulin-like growth factor-2 (IGF-2). NIH3T3 cells overexpressing E2F1 and growing under low serum conditions show very low levels of IGF-2 expression. A plausible explanation for the frequently observed programmed cell death in E2F1-overexpressing cells after serum withdrawal now becomes apparent. It is known that IGF growth factors can inhibit the apoptotic signals in cells. The fact that IGF-2 levels drop as a consequence of E2F1 overexpression might hamper the resistance of the cells to apoptosis. It might therefore be that overexpression of E2F1 triggers apoptosis and levels of the rescuing IGF-2 are too low to prevent the cells from undergoing apoptosis. During the conference we will present our progress with the new glass-based arrays we currently apply to find more E2F target genes, to study cell cycle-related phenomena and to identify tumour-specific expression profiles.
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