REDUCED ACTIVITY OF TOPOISOMERASE-II IN AN ADRIAMYCIN-RESISTANT HUMANSTOMACH-ADENOCARCINOMA CELL-LINE

A human stomach-adenocarcinoma cell line (MKN-45) was selected for res istance to Adriamycin by stepwise exposure to increasing concentration s of this agent. The resulting cell line (MKN/ADR) exhibited a high le vel of cross-resistance to topoisomerase II (topo II)-targeted drugs s uch as Adriamycin, mitoxantrone, and etoposide but showed no cross-res istance to other chemotherapeutic agents such as cisplatin, carboplati n, 5-fluorouracil, or mitomycin-C. P-glycoprotein encoded by the mdr-1 gene was not overexpressed in the MKN/ADR cell line. The doubling tim e of the MKN/ADR cell line (2.1 days) increased only slightly as compa red with that of the MKN cell line (1.7 days). The patterns of cross-r esistance to various chemotherapeutic agents led us to examine the cel lular contents of topo II in both the drug-sensitive and the drug-resi stant cells. Extractable topo II enzyme activity was 3-fold lower in M KN/ADR cells as compared with the parental MKN cells. Levels of topois omerase I (topo I) catalytic activity were similar in both wild-type M KN and drug-resistant MKN/ADR cells. Southern-blot analysis of genomic DNA probed with topo II alpha or II beta showed no sign of either gen e rearrangement or hypermethylation. Northern-blot analysis revealed t hat both topo II alpha and topo II beta mRNA transcripts were essentia lly identical in the MKN and MKN/ADR cells. In contrast, Western-blot analysis revealed an approximately 20-fold lower level of topo II alph a in drug-resistant cells as compared with drug-sensitive cells, where as topo II beta levels were similar in both lines. Moreover, the amoun t of in vivo topo II alpha-DNA covalent complexes formed in the presen ce of etoposide was also approximately 20-fold lower in drug-resistant cells. No mutation was detected in the promoter region of the topo II alpha gene in resistant cells as compared with sensitive cells. Thus, low levels of topo II alpha polypeptide cannot be ascribed to changes in the mRNA levels. Collectively, the data suggest that a quantitativ e reduction in topo II alpha may contribute to the resistance of MKN c ells to Adriamycin and other topo II-targeted drugs.