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Resistance-associated factors in human small-cell lung-carcinoma GLC4 sub-lines with increasing adriamycin resistance
C H Versantvoort1, S Withoff, H J Broxterman
1Department of Medical Oncology, Free University Hospital, Amsterdam, The Netherlands.
Abstract:
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.
Insights
Early adriamycin resistance in lung cancer cells involves changes in drug accumulation, DNA-topoisomerase II (topo II) activity, and multidrug resistance-associated protein (MRP) gene expression. These factors are altered even at the initial stages of resistance development.
Area of Science:
- Molecular Biology
- Cell Biology
- Pharmacology
Background:
- Multidrug resistance (MDR) in cancer is a significant clinical challenge.
- Adriamycin resistance in human small-cell lung carcinoma (GLC4-ADR150) is linked to decreased DNA-topoisomerase II (topo II) activity and reduced adriamycin accumulation.
- Overexpression of the multidrug resistance-associated protein (MRP) gene, not MDR1, is observed in resistant cell lines.
Purpose of the Study:
- To investigate which drug-resistance factors are involved in the early stages of adriamycin selection.
- To determine the correlation between adriamycin resistance levels, topo II activity, MRP expression, and drug accumulation.
- To analyze changes in resistant sub-lines and a partially revertant cell line.
Main Methods:
- Measurement of adriamycin resistance levels (2- to 150-fold).
- Quantification of DNA-topoisomerase II alpha/beta mRNA and protein levels.
- Assessment of cellular daunorubicin accumulation.
- Analysis of MRP mRNA and protein expression using Western blotting.
- Evaluation of an ATP-binding 190-kDa membrane protein.
Main Results:
- Topo II alpha and beta mRNA/protein levels decreased in resistant sub-lines (except 10-fold resistant).
- Cellular daunorubicin accumulation decreased with increasing resistance (1.2- to 5-fold).
- MRP mRNA was over-expressed in all resistant sub-lines, with a significant increase in the 10-fold resistant cells.
- MRP expression correlated with mRNA levels but not always with decreased drug accumulation.
- Drug accumulation, topo II, and MRP expression were altered at the earliest stage of adriamycin resistance.
Conclusions:
- Drug accumulation, DNA-topoisomerase II, and MRP expression are all implicated in the initial development of adriamycin resistance in GLC4 cells.
- MRP gene overexpression is an early event in adriamycin resistance development.
- The interplay between these factors contributes to multidrug resistance in lung cancer.
