Quantitation of DNA topoisomerase IIalpha and beta in human leukaemia cells by immunoblotting.
DNA topoisomerase II (topo II) is an essential nuclear enzyme and is the target for etoposide, which is used in the therapy of childhood acute lymphoblastic leukaemia (ALL). Topo II exists as two isoforms referred to as topo IIalpha and topo IIbeta. To determine whether cellular levels of topo IIalpha and beta are an important factor in determining drug sensitivity/resistance requires accurate, precise measurements of the two isoforms. We have developed a quantitative Western blotting method to accurately measure the absolute amounts of human topo IIalpha and beta, using recombinant human topo IIalpha and beta as standards. This quantitative method has been used to assess the efficiency of several commonly used topo II extraction protocols. The extractable amount of topo IIalpha and beta was found to be salt-dependent. However extraction using the optimal salt concentration was found to be as efficient as extraction with DNase I/Rnase A digestion and SDS solubilisation. Using the optimum extraction procedure and the quantitative immunoblotting method, topo IIalpha and beta was quantified in cell lines, peripheral blood lymphocytes and in lymphoblasts from children with newly diagnosed ALL.
Padget
K
K
School of Biochemistry and Genetics, The Medical School, University of Newcastle upon Tyne, UK.
Pearson
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AD
Austin
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CA
D000293:Adolescent; D000328:Adult; D000818:Animals; D000951:Antigens, Neoplasm; D000970:Antineoplastic Agents / Q000494:pharmacology; D015153:Blotting, Western; D002051:Burkitt Lymphoma / Q000201:enzymology; D002648:Child; D002675:Child, Preschool; D004250:DNA Topoisomerases, Type II / Q000032:analysis* / Q000172:deficiency / Q000276:immunology / Q000302:isolation & purification; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006801:Humans; D007527:Isoenzymes / Q000032:analysis* / Q000172:deficiency / Q000276:immunology / Q000302:isolation & purification; D019169:Jurkat Cells / Q000201:enzymology; D020014:K562 Cells / Q000201:enzymology; D007938:Leukemia / Q000201:enzymology* / Q000473:pathology; D015459:Leukemia-Lymphoma, Adult T-Cell / Q000201:enzymology; D008297:Male; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000276:immunology / Q000302:isolation & purification; D014411:Neoplastic Stem Cells / Q000187:drug effects / Q000201:enzymology*; D009994:Osmolar Concentration; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000188:drug therapy / Q000201:enzymology; D011817:Rabbits; D011994:Recombinant Proteins / Q000276:immunology; D012965:Sodium Chloride / Q000032:analysis; D059004:Topoisomerase I Inhibitors; D014407:Tumor Cells, Cultured / Q000201:enzymology
D000951:Antigens, Neoplasm; D000970:Antineoplastic Agents; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D009363:Neoplasm Proteins; D011994:Recombinant Proteins; D059004:Topoisomerase I Inhibitors; D012965:Sodium Chloride; D004250:DNA Topoisomerases, Type II