Effects of wild-type p53 expression on the quantity and activity of topoisomerase IIalpha and beta in various human cancer cell lines.
The p53 null HL-60 cell line was transfected with plasmids coding for either the wild-type p53 or mutant p53 gene. The stable expression of wild-type p53 resulted in a significant increase in sensitivity to the topoisomerase II poisons etoposide and doxorubicin, but not to the topoisomerase II inhibitors razoxane and ADR-529. HL-60 cells expressing wild-type p53 demonstrated 8- to 10-fold more VP-16 induced DNA breaks by the alkaline elution assay. The effect of inducible expression of wild-type p53 was also studied in the p53 null erythroblastoid cell line K562 and in the human squamous carcinoma cell line SqCC. The inducible expression of wild-type p53 in the K562 cell line resulted in a 3-fold increase in sensitivity to VP-16. The quantity of topoisomerase IIalpha was not altered by the transfection as determined by immunoblotting, while the amount of the beta isoform was increased 2.5-fold in HL-60 cells. The topo II catalytic activity present in nuclear extracts was measured as the decatenation of kinetoplast DNA, and found to be unaltered by p53 expression. Immunostaining for topoisomerase IIalpha was substantially diminished in both stable and inducible wild-type p53 expressing cells when three different antibodies were used (two polyclonal and one monoclonal). However, the addition of VP-16 resulted in a rapid appearance of nuclear fluorescence for topoisomerase IIalpha. No changes in topoisomerase IIbeta immunostaining were observed. These results suggest that an epitope for topoisomerase IIalpha is concealed in cells expressing wild-type p53 and that a complex between topoisomerase IIalpha and p53 may be disrupted by the addition of antitumor drugs.
Hochhauser
D
D
Molecular Pharmacology and Therapeutics Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Valkov
N I
NI
Gump
J L
JL
Wei
I
I
O'Hare
C
C
Hartley
J
J
Fan
J
J
Bertino
J R
JR
Banerjee
D
D
Sullivan
D M
DM
D000951:Antigens, Neoplasm; D015153:Blotting, Western; D002294:Carcinoma, Squamous Cell / Q000378:metabolism; D002384:Catalysis; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004268:DNA-Binding Proteins; D018922:HL-60 Cells; D006454:Hemoglobins / Q000032:analysis; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020014:K562 Cells; D008856:Microscopy, Fluorescence; D011233:Precipitin Tests; D013997:Time Factors; D059005:Topoisomerase II Inhibitors; D014162:Transfection; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
D000951:Antigens, Neoplasm; D004268:DNA-Binding Proteins; D006454:Hemoglobins; D007527:Isoenzymes; D059005:Topoisomerase II Inhibitors; D016159:Tumor Suppressor Protein p53; D004250:DNA Topoisomerases, Type II