Resistance-associated factors in human small-cell lung-carcinoma GLC4 sub-lines with increasing adriamycin resistance

Previous studies have shown that the in vitro-selected adriamycin-resistant human small-cell lung-carcinoma cell line GLC4-ADR150 displays multidrug resistance as the result of 3-fold decreased DNA-topoisomerase II (topo II) activity and a 6-fold reduction in adriamycin accumulation. Not the MDR1 gene, but the MRP gene, was over-expressed in this cell line. The aim of our study was to establish which of these drug-resistance-associated factors are already involved in drug resistance occurring at early steps of selection with adriamycin. To address this question, changes in expression of topo II alpha/topo II beta, MRP and drug accumulation were measured along with adriamycin resistance (from 2- to 10- to 150-fold) and in a partial revertant cell line (10-fold resistant). Topo II alpha and II beta mRNA and protein levels were decreased in the resistant sub-lines, except in the 10-fold-resistant cell line. Cellular daunorubicin accumulation was decreased 1.2- to 5-fold with increasing resistance. MRP mRNA was over-expressed in all resistant sub-lines, with a marked increase in the 10-fold-resistant cells (level of expression as high as in the GLC4-ADR150 cells). Expression of an ATP-binding 190-kDa membrane protein and Western-blot analysis with anti-MRP anti-serum ASPKE, was in accordance with the expression of MRP mRNA in all cell lines. Expression of MRP mRNA and protein, however, was not proportional with the decrease in drug accumulation in all resistant sub-lines. This study also shows that drug accumulation, topo II and MRP expression were all changed at the earliest stage of resistance development of GLC4 cells upon adriamycin selection.