QUANTITATION OF DNA TOPOISOMERASE-II-ALPHA MESSENGER-RIBONUCLEIC-ACIDLEVELS IN A SMALL-CELL LUNG-CANCER CELL-LINE AND 2 DRUG-RESISTANT SUBLINES USING A POLYMERASE CHAIN REACTION-AIDED TRANSCRIPT TITRATION ASSAY

BACKGROUND: We have modified a polymerase chain reaction (PCR)-aided t ranscript titration assay (1) in order to allow quantitation of low am ounts of DNA topoisomerase II alpha mRNA in small RNA samples. EXPERIM ENTAL DESIGN: The titration assay was used to quantitate the amount of DNA topoisomerase II alpha mRNA in a human small cell lung carcinoma cell line, GLC(4) and its drug-resistant sublines, GLC(4)/ADR and GLC( 4)/CDDP. These cell lines show differences in DNA topoisomerase II alp ha protein level and DNA topoisomerase II enzyme activity. To validate the titration assay, the results were compared with the results of a DNA topoisomerase II enzyme activity assay and DNA topoisomerase II al pha northern and western blotting assays. RESULTS: Using the titration assay, we were able to quantitate DNA topoisomerase II alpha mRNA on a picogram level starting with less than 1 mu g of total RNA/cell line . GLC(4)/ADR showed a markedly decreased DNA topoisomerase II alpha mR NA level that seemed to be unchanged in GLC(4)/CDDP when compared with the parental cell line. The results obtained with this assay are conf irmed by the western blot data and are not in contradiction with the n orthern blot results obtained for the three cell lines. CONCLUSIONS: T he DNA topoisomerase II alpha titration assay is a highly sensitive ne w technique to study the role of DNA topoisomerase II alpha in drug re sistance and may help to identify cancer types and patients most likel y to respond to DNA topoisomerase II targeted drugs. The decrease in D NA topoisomerase II alpha protein level and DNA topoisomerase II activ ity in GLC(4)/ADR may result from transcriptional down regulation of D NA topoisomerase II alpha.