Quantitation of DNA topoisomerase II alpha messenger ribonucleic acid levels in a small cell lung cancer cell line and two drug resistant sublines using a polymerase chain reaction-aided transcript titration assay.
BACKGROUND
We have modified a polymerase chain reaction (PCR)-aided transcript titration assay (1) in order to allow quantitation of low amounts of DNA topoisomerase II alpha mRNA in small RNA samples.


METHODS
The titration assay was used to quantitate the amount of DNA topoisomerase II alpha mRNA in a human small cell lung carcinoma cell line, GLC4 and its drug-resistant sublines, GLC4/ADR and GLC4/CDDP. These cell lines show differences in DNA topoisomerase II alpha 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 alpha 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 micrograms of total RNA/cell line. GLC4/ADR showed a markedly decreased DNA topoisomerase II alpha mRNA level that seemed to be unchanged in GLC4/CDDP when compared with the parental cell line. The results obtained with this assay are confirmed by the western blot data and are not in contradiction with the northern blot results obtained for the three cell lines.


CONCLUSIONS
The DNA topoisomerase II alpha titration assay is a highly sensitive new technique to study the role of DNA topoisomerase II alpha in drug resistance and may help to identify cancer types and patients most likely to respond to DNA topoisomerase II targeted drugs. The decrease in DNA topoisomerase II alpha protein level and DNA topoisomerase II activity in GLC4/ADR may result from transcriptional down regulation of DNA topoisomerase II alpha.
Withoff
S
S
Department of Internal Medicine, University Hospital Groningen, The Netherlands.
Smit
E F
EF
Meersma
G J
GJ
van den Berg
A
A
Timmer-Bosscha
H
H
Kok
K
K
Postmus
P E
PE
Mulder
N H
NH
de Vries
E G
EG
Buys
C H
CH
D000951:Antigens, Neoplasm; D001483:Base Sequence; D015152:Blotting, Northern; D018288:Carcinoma, Small Cell / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins; D004351:Drug Resistance; D015870:Gene Expression; D006801:Humans; D007527:Isoenzymes / Q000235:genetics*; D008175:Lung Neoplasms / Q000378:metabolism*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism*; D011994:Recombinant Proteins; D017186:Titrimetry; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D000951:Antigens, Neoplasm; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D007527:Isoenzymes; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D011994:Recombinant Proteins; D004250:DNA Topoisomerases, Type II