A PCR-aided transcript titration assay (PATTY) to measure topoisomerase I gene expression in human tumor specimens

Topoisomerase I (topo I) inhibitors are promising anticancer agents with de monstrated activity against a wide range of solid tumors. Quantitative info rmation on topol mRNA levels in tumor biopsies may predict response to topo I inhibitors. Materials and methods: A polymerase chain reaction aided tra nscript titration assay (PATTY) was developed to allow quantitation of topo l mRNA in small samples. Concentrations of topol mRNA in total RNA samples were estimated by RT-PCR analysis in a human small cell lung cancer (SCLC) cell line (GLC,) and its topotecan (GL(2)C/SK and F) and camptothecin (GL(2 )C/Campt) resistant sublines, human nonsmall cell lung cancer (NSCLC) and o varian carcinoma samples. Results: Topol PATTY showed a decreased topo I mR NA level in GLC(2)/SK and F (4.5 pg/100 ng total RNA) and GLC,/Campt (2.2 p g/100 ng total RNA), respectively, compared to the parent cell line GLC(2) (5.4 pg/100 ng total RNA). Topol protein levels as measured by Western blot ting were compatible with topol mRNA levels. Median (range) topol mRNA leve ls were 3.23 (2.33-5.10) pg/100 ng total RNA in resected NSCLC specimen (n= 6), and 2.03 (0.54-0.95) pg/100 ng total RNA in resected ovarian cancer spe cimen (n=6). Conclusion: We conclude that topol PATTY is a new assay that q uantitates topol mRNA levels in cell lines and small tumor samples.