Nonradioactive quantitative detection of gene expression : methodological studies in normal and malignant tissuesutilizing digital image analysis

Abstract

This thesis describes the development and adaptation of gene expression analysis to archival biopsies allowing retrospective investigation of thymidylate synthase (TS) gene expression without the need for preemptive sample recovery. The ability to effectively isolate messenger ribonucleic acid (mRNA) from formalin-fixed paraffin embedded material for RNA analysis, and the nearly universal accessibility of this material opens the archives of hospital pathology departments to all sorts of gene expression studies. The methods developed in this thesis are based on the reverse-transcriptase polymerase chain reaction (RT-PCR) amplification of complimentary deoxyribonucleic acid (cDNA) obtained from different tissues. The quantification of the PCR amplicons is by computer assisted digital image analysis (DIA). As model systems five different processes are investigated including the development of a practical DIA workstation for nonradioactive detection of gene expression. The workstation is first applied to the analysis of the transcriptional regulation of interferon-gamma (IFN-g) gene expression in interleuken-2 (IL-2) stimulated natural killer cells (NK) by histamine and catalase in the presence and absence of autologous monocytes. The same system is then applied to the analysis of TS gene expression in flash-frozen biopsies. Next, a solid-phase system of isolating and concentrating mRNA from formalin-fixed paraffin embedded biopsies with reusable oligo(dT)25 paramagnetic beads was developed. The method uses the repeated re-elution and concentration of mRNA isolated from the same portion of an archival biopsy.Intracellular concentrations of TS protein are studied in surgical biopsies from normal and cancerous human tissue originating from the GI-tract prior to the administration of 5FU and compared to TS gene expression. The concentration of TS is measured with tritium release assays, and the TS gene expression levels for both flash-frozen and formalin-fixed archival tissues are determined with the PCR based methodology previously described. The concentrations of TS mRNA varied considerably between the different tumor types examined, however the TS concentrations were consistently higher in tumor than in normal tissue. The use of oligo(dT)25 paramagnetic beads allowed for the isolation of intact mRNA even from archival tissues high in ribonucleic acidase (RNase) activity such as liver. The TS gene expression levels strongly correlated to enzyme activity for both flash-frozen and formalin-fixed material.In conclusion, these new methodologies should allow for the possibility to accurately determine TS gene expression using archival material in large retrospective studies. The use of competitive semi-quantitative RT-PCR, the 96-well microtiter format and multi-channel pipette with a ìstructuredî pipetting protocol throughout each experiment were permissible in a clinical setting and proved not only amenable to the potential large-scale screening of biopsy material but permitted greater precision, accuracy and reproducibility by reducing the number of steps required to perform an experiment.