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Altered gene expression in human leukemia K562 cells selected for resistance to etoposide
1Department of Pharmacology, University of Pittsburgh School of Medicine, PA 15261.
Abstract:
Sublines of K562 human leukemia cells were selected for resistance (30- to 80-fold) to etoposide by continuous exposure to 0.5 microM VP-16. Two etoposide-resistant cell lines, K/VP.5 and K/VP.5-1, showed a 5-fold reduction in levels of topoisomerase II alpha protein compared with K562 cells. Northern analysis indicated a 2.5-fold reduction in topoisomerase II alpha mRNA in etoposide-resistant cell lines, due in part to a 1.7-fold decrease in topoisomerase II mRNA stability with no change in transcription rate. Immunoblotting assays of electrophoresed cell lysates from VP-16-treated cells revealed less drug-induced covalent topoisomerase II/DNA adducts in resistant than in sensitive cells, suggesting a functional alteration in resistant cell topoisomerase II. Recent reports of specific topoisomerase II DNA binding sites near the promoter sites of growth response genes and alterations of gene expression in cells treated with topoisomerase II inhibitory drugs led to experiments to determine if the apparent functional alterations of topoisomerase II were accompanied by changes in the regulation of these genes. Therefore, the expression of several growth response genes was compared by northern analysis in parental K562 and both VP-16-resistant cell lines. Basal levels of c-myc were comparable for all three cell lines, but levels of c-jun and c-fos were elevated 2- to 4-fold in VP-16-resistant cell lines. Increased levels of c-fos and c-jun were not a result of altered rates of transcription, as determined by nuclear run-off assays. Exposure of both sensitive and resistant cells to 200 microM VP-16 for 5 hr resulted in no further changes in topoisomerase II mRNA levels but caused an additional 2- to 3-fold elevation in the level of c-jun mRNA, indicating that altered basal levels of this gene were not due to deregulation of this gene. Acquired VP-16 resistance in K/VP.5 and K/VP.5-1 cells was accompanied by reduced levels and altered activities of DNA topoisomerase II as well as changes affecting the expression of genes important for growth and differentiation.
Insights
Etoposide resistance in leukemia cells involves decreased topoisomerase II (DNA topoisomerase II) levels and altered DNA topoisomerase II activity. This resistance is linked to changes in growth-related gene expression, impacting cell growth and differentiation.
Area of Science:
- Molecular Biology
- Cancer Research
- Genetics
Background:
- Etoposide (VP-16) is a chemotherapy drug that targets DNA topoisomerase II.
- Drug resistance in cancer cells is a significant clinical challenge.
- DNA topoisomerase II plays a critical role in DNA replication and gene expression.
Purpose of the Study:
- To investigate the molecular mechanisms underlying etoposide resistance in K562 human leukemia cells.
- To determine the impact of etoposide resistance on DNA topoisomerase II levels, activity, and gene expression.
Main Methods:
- Selection of etoposide-resistant K562 cell lines (K/VP.5 and K/VP.5-1).
- Analysis of DNA topoisomerase II alpha protein and mRNA levels using immunoblotting and Northern analysis.
- Assessment of DNA topoisomerase II/DNA adducts.
- Comparison of growth response gene expression (c-myc, c-jun, c-fos) via Northern analysis and nuclear run-off assays.
Main Results:
- Etoposide-resistant cell lines exhibited reduced DNA topoisomerase II alpha protein and mRNA levels.
- mRNA stability, not transcription rate, contributed to decreased DNA topoisomerase II alpha mRNA.
- Resistant cells showed reduced drug-induced covalent DNA topoisomerase II/DNA adducts, indicating altered enzyme activity.
- Elevated basal levels of c-jun and c-fos were observed in resistant cells, independent of transcription rate changes.
Conclusions:
- Acquired etoposide resistance in K562 cells is associated with decreased DNA topoisomerase II levels and altered enzyme activity.
- Changes in DNA topoisomerase II are accompanied by altered expression of key growth-related genes (c-jun, c-fos).
- These molecular alterations likely contribute to the observed resistance and affect cellular growth and differentiation pathways.