title: Simultaneous quantitation of topoisomerase II alpha and beta isoform mRNAs in lung tumor cells and normal and malignant lung tissue.
abstract: Certain drugs used in the treatment of lung cancer and other human malignancies are cytotoxic because of their ability to interact with the two isoforms of topoisomerase II (topo II), topo IIalpha and topo IIbeta. As part of an effort to evaluate the contribution of topo II alterations to drug sensitivity and resistance in lung cancer, we have developed a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay to measure levels of topo II alpha and beta mRNAs simultaneously using a single pair of primers with sequences common to both isoforms. The PCR products derived from the topo II alpha and beta mRNAs are both 446 bp but have different electrophoretic mobilities in a nondenaturing polyacrylamide gel, allowing sensitive, rapid quantitation when the products are radiolabeled with [35S]-dATP. Using this RT-PCR method, poly(A+) RNA from 13 non-small cell lung cancer (NSCLC) cell lines was analyzed. The results obtained indicated that the cell lines express a wide range of topo II alpha mRNA levels (12-fold) and topo IIbeta mRNA levels (5.5-fold). Tumor and normal lung tissues from 25 patients with NSCLC were also examined. In the tumor samples, the levels of the topo II alpha and beta mRNAs were similar. However, mean topo IIalpha mRNA levels in the tumors were approximately 7-fold higher than those of the paired normal lung tissues. In contrast, topo IIbeta mRNA levels were similar in both tumor and normal lung. Topo II alpha and beta mRNA levels were both significantly lower in the squamous cell tumors than in the adenocarcinoma samples. Topo IIbeta mRNA levels in the squamous cell tumors were also significantly lower than those in paired normal lung tissue. The RT-PCR method described is reliable and convenient, and for the first time, makes the rapid simultaneous direct comparison of topo IIalpha and topo IIbeta mRNA levels feasible in large numbers of clinical samples.
authors.affiliation: Cancer Research Laboratories, Queen's University, Kingston, Ontario, Canada.
authors.forename: S E
authors.initials: SE
authors.lastname: Mirski
authors.forename: T
authors.initials: T
authors.lastname: Voskoglou-Nomikos
authors.forename: L C
authors.initials: LC
authors.lastname: Young
authors.forename: R G
authors.initials: RG
authors.lastname: Deeley
authors.forename: B G
authors.initials: BG
authors.lastname: Campling
authors.forename: J H
authors.initials: JH
authors.lastname: Gerlach
authors.forename: S P
authors.initials: SP
authors.lastname: Cole
mesh_terms: D000951:Antigens, Neoplasm; D001483:Base Sequence; D002289:Carcinoma, Non-Small-Cell Lung / Q000201:enzymology* / Q000235:genetics; D004250:DNA Topoisomerases, Type II / Q000235:genetics*; D004268:DNA-Binding Proteins; D006801:Humans; D007527:Isoenzymes / Q000235:genetics*; D008168:Lung / Q000201:enzymology*; D008175:Lung Neoplasms / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D014407:Tumor Cells, Cultured
chemical_list: D000951:Antigens, Neoplasm; D004268:DNA-Binding Proteins; D007527:Isoenzymes; D012333:RNA, Messenger; D004250:DNA Topoisomerases, Type II