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Differential changes in genome structure and expression of the mdr gene family in multidrug-resistant murine
P M Fracasso1, C A Slapak, S Nair
1Department of Medicine, New England Medical Center, Boston, MA, USA.
Abstract:
The genome structure and expression of mdr genes were examined in multidrug-resistant sublines of two different murine (DBA/2J) Friend erythroleukemia cell lines, PC4 and C7D, derived by stepwise exposure to increasing concentrations of adriamycin beginning with 5 ng/ml. The PC4 cell lines selected in higher drug concentrations (80-1280 ng/ml) demonstrated amplification of all three mdr genes with preferential amplification of mdr3. Overexpression of the mdr2 and mdr3 genes accompanied their genomic amplification; however, expression of mdr1 was not seen despite amplification. In the C7D cell lines selected with higher drug concentrations (40-160 ng/ ml), amplification and overexpression of mdr1 and mdr2 without mdr3 was observed. Increased expression of mdr1 occurred prior to gene amplification. The distribution of mdr-specific genes in micrococcal nuclease-generated chromatin fractions differing in transcriptionally active sequences and proteins was different between the parent and drug-resistant sublines. An enrichment (two- to threefold) of mdr3 genes in the H1-depleted mononucleosome fraction enriched for actively transcribed genes (e.g., globin) was detected by Southern analysis of chromatin fractions in PC4-80 cells (selected in 80 ng/ml of adriamycin and overexpressing mdr3), compared to the parental cells. mdr3 enrichment was also detected using a new PCR-based method, which examined mdr3 genes and repetitive sequences. Of note, the H1-depleted chromatin fraction from PC4-20 showed enrichment of the mdr3 gene, although mdr3 expression was not detected in the cell line. These studies showed a different pattern of gene amplification and overexpression in genetically related erythroleukemia cell lines selected for resistance to the same chemotherapeutic agent. A change in chromatin organization of mdr genes preceded overexpression and amplification of the mdr3 gene.
Insights
Multidrug resistance (mdr) gene expression and amplification patterns differ between related cell lines. Chromatin changes in mdr genes precede their overexpression and amplification, impacting drug resistance.
Area of Science:
- Molecular Biology
- Cancer Research
- Genetics
Background:
- Multidrug resistance (mdr) is a major challenge in cancer chemotherapy.
- Understanding the genetic and epigenetic mechanisms underlying mdr is crucial for developing effective treatments.
Purpose of the Study:
- To investigate the genome structure and expression of mdr genes in multidrug-resistant murine erythroleukemia cell lines.
- To compare the patterns of mdr gene amplification and overexpression in related cell lines selected with the same chemotherapeutic agent.
Main Methods:
- Stepwise exposure of Friend erythroleukemia cell lines (PC4 and C7D) to increasing concentrations of adriamycin.
- Analysis of mdr gene amplification using Southern analysis and PCR-based methods.
- Assessment of mdr gene expression and chromatin organization in different cellular fractions.
Main Results:
- PC4 cell lines showed amplification of all three mdr genes, with preferential amplification of mdr3 and its overexpression.
- C7D cell lines exhibited amplification and overexpression of mdr1 and mdr2, but not mdr3.
- Increased mdr1 expression preceded gene amplification in C7D cells.
- Changes in chromatin organization of mdr genes were observed in drug-resistant sublines, with enrichment of mdr3 in actively transcribed chromatin fractions.
Conclusions:
- Genetically related cell lines display distinct patterns of mdr gene amplification and overexpression.
- Alterations in chromatin organization of mdr genes appear to precede their amplification and overexpression.
- These findings highlight the complex interplay between genomic alterations, epigenetic modifications, and drug resistance in cancer cells.