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 demonstrated activity against a wide range of solid tumors. Quantitative information on topol mRNA levels in tumor biopsies may predict response to topo I inhibitors.


METHODS
A polymerase chain reaction aided transcript titration assay (PATTY) was developed to allow quantitation of topol 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 (GL2C/SK and F) and camptothecin (GL2C/Campt) resistant sublines, human non-small cell lung cancer (NSCLC) and ovarian carcinoma samples.


RESULTS
Topol PATTY showed a decreased topo I mRNA level in GLC2/SK and F (4.5 pg/100 ng total RNA) and GLC,/Campt (2.2 pg/100 ng total RNA), respectively, compared to the parent cell line GLC2 (5.4 pg/100 ng total RNA). Topol protein levels as measured by Western blotting were compatible with topol mRNA levels. Median (range) topol mRNA levels 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 specimen (n = 6).


CONCLUSIONS
We conclude that topol PATTY is a new assay that quantitates topol mRNA levels in cell lines and small tumor samples.
Meersma
G J
GJ
Department of Medical Oncology, University Hospital Croningen, The Netherlands.
Bakker
M
M
Groen
H J
HJ
Van der Zee
A G
AG
Jensen
P B
PB
Giaccone
G
G
De Vries
E G
EG
Smit
E F
EF
D015152:Blotting, Northern; D015153:Blotting, Western; D004264:DNA Topoisomerases, Type I / Q000235:genetics*; D005260:Female; D006801:Humans; D008175:Lung Neoplasms / Q000201:enzymology; D009369:Neoplasms / Q000201:enzymology*; D010051:Ovarian Neoplasms / Q000201:enzymology; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis*; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D004264:DNA Topoisomerases, Type I