Abstract
We have analysed the contribution of several parameters, e.g. drug accumulation, MDR1 P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP) and topoisomerase (topo) II, to drug resistance in a large set of drug-resistant variants of the human non-small-cell lung cancer cell line SW-1573 derived by selection with low concentrations of doxorubicin or vincristine. Selection with either drug nearly always resulted in MDR clones. The resistance of these clones could be explained by reduced drug accumulation and was associated with a decrease rather than an increase in the low MDR1 mRNA level. To test whether a decrease in MDR1 mRNA indirectly affected resistance in these cells, we introduced a MDR1-specific hammerhead ribozyme into wild-type SW-1573 cells. Although this led to a substantial reduction in MDR1 mRNA, it did not result in resistance. In all resistant clones we found an altered form of the multidrug resistance-associated protein (MRP), migrating slightly slower during SDS-polyacrylamide gel electrophoresis than MRP in parental cells. This altered MRP was also present in non-P-gp MDR somatic cell hybrids of the SW-1573 cells, demonstrating a clear linkage with the MDR phenotype. Treatment of crude cellular membrane fractions with N-glycanase, endoglycosidase H or neuraminidase showed that the altered migration of MRP on SDS-PAGE is due to a post-translational modification. There was no detectable difference in sialic acid content. In most but not all doxorubicin-selected clones, this MDR phenotype was accompanied by a reduction in topo II alpha mRNA level. No reduction was found in the clones selected with vincristine. We conclude from these results that selection of the SW-1573 cell line for low levels of doxorubicin or vincristine resistance, predominantly results in MDR with reduced drug accumulation associated with the presence of an altered MRP protein. This mechanism can be accompanied by other resistance mechanisms, such as reduced topo II alpha mRNA in case of doxorubicin selection.
Highlights
In somatic cell fusions we have shown that the multidrug resistance (MDR) phenotype, the reduced drug accumulation and the reduction in MDR1 P-gp mRNA are transferred together to drug-sensitive SW-1573 cells, but that the alteration in topo IIa is not genetically linked to the non-P-gp MDR phenotype (Eijdems et al, 1992)
All clones were analysed for their level of mRNA for MDRI P-gp and topo IIa and a representative subset of ten clones was chosen and analysed in detail
We conclude that most clones have a MDR phenotype of the non-P-gp variety, as MDRJ mRNA is reduced rather than elevated in most of them
Summary
Transfected with the xl subunit of the murine sodium/ Digitonin (20 JAM) was added 5 min before the end of the potassium exchanger (Eijdems et al, 1992). This clone has incubation time with daunorubicin. The sensitivity for MDR drugs plemented with 10% growth medium, and after two cold was unaffected by the presence of ouabain in the culture washes the cells were transferred to liquid scintillation fluid medium. The non-P-gp MDR cell line 1R50b was isolated Opti-Phase III (LKB, Bromma, Sweden) and radioactivity from the drug-sensitive parental cell line SI by a multistep was measured. Values were corrected for the amount of doxorubicin selection up to 50 nM
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