We have examined features of DNA topoisomerase II (topo II) isoforms in human leukemic CCRF-CEM cells and two teniposide-resistant sublines, CEM/VM-1 and CEM/VM-1-5. They are about 40- to 50-fold and 150- to > 400-fold resistant to teniposide, respectively, and have increased levels of cross-resistance to other complex-stabilizing topo II inhibitors. Topo II activity in these lines is reduced in proportion to their resistance. However, both sublines carry two identical point mutations in topo II alpha cDNA that have recently been found to confer resistance to etoposide and m-AMSA in transfected yeast cells. Although these data provide a strong molecular basis for this type of multidrug resistance, these findings alone cannot explain the increased level of resistance and cross-resistance, and the further decreased cellular topo II activities in the most resistant CEM/VM-1-5 cells compared to CEM/VM-1 cells. In this study we found that (1) topo II beta is not expressed in CEM/VM-1-5 cells; (2) a 160-kDa protein was consistently detected and coimmunoprecipatated only in nuclear extracts of CEM cells; (3) when nuclear extracts from all three cell lines were incubated at 37 degrees C, an immunoreactive band of 140-kDa appeared by 60-90 min only in samples of CEM cells, not in those of CEM/VM-1 and CEM/VM-1-5 cells; and (4) the in vivo phosphorylation level of topo II alpha protein was decreased > or = 2-fold in both resistant cell lines, compared to that of CEM cells. Thus, cell lines selected for the altered topo II-associated multidrug resistance phenotype may contain multiple alterations in both topo II isoforms.(ABSTRACT TRUNCATED AT 250 WORDS)