Purification of DNA topoisomerase I from the spleen of a patient with non-Hodgkin's lymphoma.
DNA topoisomerase I (topo I) was purified 124-fold from the spleen of a patient which was diffusely infiltrated by malignant lymphoma. Extracts prepared from the lymphoma tissue demonstrated elevated topo I activity. The purification uses a new two step chromatographic method involving hydroxylapatite and Fast Protein Liquid Chromatography mono S. The purified enzyme has a molecular weight of 67 kilodaltons as determined by sodium dodecyl sulfate denaturing gel electrophoresis. Western blotting confirms the identity of the protein as topo I. The purified lymphoma topo I has a similar specific activity as topo I isolated by the same procedure from normal placenta. By using a new quantitative radiolabeled DNA relaxation assay, it was estimated that each molecule of topo I is able to relax 9.6 molecules of supercoiled DNA per min at 30C. In addition it was found that both the placental topo I and the lymphoma topo I were each 50% inhibited by 8 microns camptothecin and that the drug stimulates the lymphoma topo I to cleave supercoiled plasmid DNA. These findings indicate that the elevation of topo I measured in crude extracts of this human malignant lymphoma can be entirely accounted for by the increase in topo I protein in the tumor. Furthermore, the sensitivity of the lymphoma topo I towards camptothecin suggests that therapy with topo I directed agents might be beneficial in this tumor.
Florell
S R
SR
Department of Pathology, University of Utah Health Sciences Center, Salt Lake City 84132, USA.
Martinchick
J F
JF
Holden
J A
JA
D000368:Aged; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D015153:Blotting, Western; D002166:Camptothecin / Q000494:pharmacology; D004264:DNA Topoisomerases, Type I / Q000302:isolation & purification*; D004278:DNA, Superhelical / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D006801:Humans; D008228:Lymphoma, Non-Hodgkin / Q000201:enzymology*; D008297:Male; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D013160:Splenic Neoplasms / Q000201:enzymology*; D059004:Topoisomerase I Inhibitors
D000972:Antineoplastic Agents, Phytogenic; D004278:DNA, Superhelical; D009363:Neoplasm Proteins; D059004:Topoisomerase I Inhibitors; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin