Topoisomerase II and drug resistance in leukemic cells

Abstract: Mechanisms for resistance to chemotherapy include among other, elevated p-glycoprotein (p-gp) level and/or altered topoisomerase II level (topo II). The present study focused on topo 11 expression, and the enzyme related in vitro drug sensitivities in human myeloid leukemic K562 cell line and isolated peripheral mononuclear cells from patients with acute myelogenous leukemia (AML), and with chronic lymphocytic leukemia (CLL). P-gp protein level was also investigated. K562 cells and isolated mononuclear cells were treated in vitro with topo II interactive agents at different concentrations. In order to determine intracellular etoposide accumulation, drug induced DNA damage and topo Ilalpha/beta mRNA level, we established a high-performance liquid chromatographic (HPLC) assay with electrochemical detection, a rapidly performed DNA unwinding technique and a real time RT-PCR method, respectively. The SDS/KCI assay for measurement of drug stabilized topo 11-DNA complex, Western blot for topo 11 and p-gp protein levels, MTT and FMCA assays for etoposide cytotoxicity and immunostaining for subcellular topo Il isoenzymes distribution were also applied. The K562 cell line study showed that both topo IIalpha and Beta were the targets of etoposide, teniposide, mitoxantrone and amsacrine. The topo 11-DNA complex formation was related to DNA damage (r 2=0.9, p=0.0001), but did not necessarily cause cytotoxicity, suggesting that subsequent events following DNA fragmentation were crucial for cell death. In clinical studies, we found that leukemic cells from patients with AML had more complex formation (p=0.07) and DNA damage (p=0.001) than cells from patients with CLL. Topo Ila protein level was undetectable in patient samples. The topo IIbeta levels in p-gp positive AML cells were significantly higher than that in p-gp negative AML cells (p=0.001). The topo 110 expression correlated with p-gp expression in AML (p=0.7, p=0.0004) but not in CLL cells. P-gp positive leukemic cells had lower etoposide accumulation than the pop negative cells (p=0.01). About 26% (12/46) of all samples had detectable topo IIalpha mRNA expression and 56% (26/46) had detectable topo IIbeta mRNA. Experimental results were compared with treatment response in patients with AML. We found elevated p-gp protein expression in the resistant group and undetectable p-gp expression in the remission group (p=0.02). However, there was no difference between these groups neither concerning topo 11 protein nor mRNA expressions. Cells from healthy blood donors had low DNA damage and undetectable topo 11 and p-gp protein levels. Our results have shown a previously unknown co-expression of topo HP and p-gP protein in leukemic cells from patients with AML. Drug induced DNA damage did not translate to in vitro cytotoxicity, suggesting that topo II-DNA complex formation is essential, but not the only critical step to achieve drug induced cell death. Our limited data did not support a relationship between topo II mRNA and protein levels and chemotherapy response in AML.