Altered Drug Interaction and Regulation of Topoisomerase IIb: Potential Mechanisms Governing Sensitivity of HL-60 Cells to Amsacrine and Etoposide

Topoisomerase II (topo II), an enzyme essential for cell viability, is present in mammalian cells as the aand b-isoforms. In human leukemia HL-60/S or HL-60/doxorubicin (DOX)0.05 cells, the levels of topo IIaor b-protein were similar in either asynchronous exponential or synchronized cultures. Although topo IIa was hypophosphorylated in HL-60/DOX0.05 compared with HL-60/S cells, both overall and site-specific hyperphosphorylation of topo IIb was apparent in HL-60/DOX0.05 compared with HL-60/S cells. The phosphorylation of topo IIa and not b was enhanced in the S and G2 1 M phases of HL-60/S cells. In contrast, an increase in the phosphorylation of topo IIb compared with a was apparent in the G1 and S phases of HL-60/DOX0.05 cells. The cytotoxicity and depletion of topo IIa or b in cells treated with drug for 1 h revealed that mole-formole, amsacrine was 2-fold more effective than etoposide in killing HL-60/S or HL-60/DOX0.05 cells and in depleting the b versus a topo II protein. Present results demonstrate that: 1) hyperphosphorylation of topo IIb in HL-60/DOX0.05 cells may be a compensatory consequence of the hypophosphorylation of topo IIa to maintain normal topo II function during proliferation, and 2) enhanced sensitivity of HL-60/S or HL-60/DOX0.05 cells to amsacrine may be due to the preferential interaction and depletion of topo IIb. The DNA topoisomerases (topo) alter DNA topology for the processing of genetic material and are key targets for the clinically important antineoplastic agents amsacrine (mAMSA) and etoposide (VP-l6) (Chen and Liu, 1994; Watt and Hickson, 1994; Wang, 1996). Although topo I exists as a single 97-kDa protein, topo II has an aand b-isoform with molecular mass of 170 and 180 kDa, respectively (Watt and Hickson, 1994; Wang, 1996). Although a substantial literature exists on the regulation, function, and drug interactions of the a-isoform, the role of the b-isoform has received substantially less attention (Chen and Liu, 1994; Watt and Hickson, 1994; Wang, 1996). A role for topo IIb in cell differentiation was originally proposed by Woessner et al. (1990, 1991). More recently, the altered regulation of topo II and the pronounced up-regulation of topo IIb during all trans-retinoic-induced differentiation of human leukemia HL-60 cells has been reported (Aoyama et al., 1998b). Previous studies (Cornarotti et al., 1996; Dereuddre et al., 1997) have suggested that the aand b-isoforms of topo II may represent distinct targets that govern differential sensitivity to drugs that poison the enzyme. A more recent study (Herzog et al., 1998) has provided evidence that high levels of resistance to m-AMSA in a subline of HL-60 is correlative with the absence of detectable levels of the topo IIb protein. A notable observation of this study was the finding that the absence of topo IIb does not interfere with cell proliferation (Herzog et al., 1998). HL-60 cells that exhibit increased resistance to doxorubicin (DOX) have been isolated (Ganapathi et al., 1996a). Although these resistant cells exhibit decreased drug accumulation due to overexpression of P-glycoprotein, the expression of resistance to topo II poisons is correlative not with intracellular drug levels but with decreased drug-stabilized topo II-DNA cleavable complex formation (Ganapathi et al., 1996a; Aoyama et al., 1998) and functional alterations in topo IIa. The reduced DNA damage also has been found to be related to site-specific hypophosphorylation of topo IIa (Aoyama et al., 1998). Although these changes in drug accumulation and hypophosphorylation of topo II are possibly linked, the differential sensitivity to m-AMSA compared with This work was supported by U.S. Public Health Service Grant CA35531 and Grant CA74939 from the Department of Health and Human Services (to K.A.H., D.R.G., M.A., Y.Y., M.K.G., and R.G.), and by the Imperial Cancer Research Fund (I.D.H.). ABBREVIATIONS: topo, topoisomerase(s); m-AMSA, amsacrine; VP-16, etoposide; HL, human leukemia; DOX, doxorubicin; PAGE, polyacrylamide gel electrophoresis. 0026-895X/99/061340-06$3.00/0 Copyright © The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 56:1340–1345 (1999). 1340 at A PE T Jornals on July 6, 2017 m oharm .aspeurnals.org D ow nladed from VP-16 remains unexplained in the HL-60 cells. In the present study, we have investigated the regulation of topo IIb as well as the differential sensitivity of the aand b-isoforms to m-AMSA and VP-16 with isoform-specific antisera in sensitive (HL-60/S) and DOX-resistant (HL-60/DOX0.05) HL-60 cells. Results suggest that unlike topo IIa, both overall and site-specific hyperphosphorylation of topo IIb is observed in the HL-60/DOX0.05 cells. Furthermore, in both the HL-60/S and HL-60/DOX0.05 cells, m-AMSA was .2-fold more effective than VP-16 in topo IIb-stabilized DNA cleavable complex formation (based on band depletion experiments) and cytotoxicity in a soft-agar colony assay. Materials and Methods The wild-type HL-60 (HL-60/S) cells were obtained from Dr. Andrew Yen, College of Veterinary Medicine, Cornell University, Ithaca, NY. Cultures of HL-60/S cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum and 2 mM L-glutamine (BioWhittaker, Walkersville, MD) at 37°C in a humidified 5% CO2 plus 95% air atmosphere. The resistant subline of HL-60 developed by culturing the wild-type cells in increasing concentrations of 0.025 to 0.05 mg/ml DOX has been described previously (Ganapathi et al., 1996b). The DOX-resistant subline (HL-60/DOX0.05) was maintained in the absence of DOX during experimentation. Doubling time in vitro of the HL-60/S and HL-60/DOX0.05 cells was 18 to 20 h. The enrichment of cells in G1, S, and G2 1 M phases of the cell cycle was carried out by centrifugal elutriation (Hengstschlager et al., 1997) in a J2–21 centrifuge equipped with a JE-6 rotor (Beckman Coulter, Fullerton, CA). Briefly, cells (2 3 10) were loaded at a rotor speed of 2000 and 1875 rpm for the HL-60/S and HL-60/DOX0.05 cells, respectively. Fractions were collected with incremental increases in flow rate. The cells in each fraction were analyzed for cell cycle phase distribution by flow cytometry (Kawamura et al., 1996). Fractions containing cells in the cell cycle phase of interest were pooled and used for experiments on topo II protein levels and phos-