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Isolation and characterization of DNA sequences amplified in multidrug-resistant hamster cells
Abstract:
The mechanism by which mammalian cells acquire resistance to chemotherapeutic agents has been investigated by using molecular genetic techniques. LZ and C5, two independently derived multidrug-resistant Chinese hamster cell lines, share specific amplified DNA sequences. We demonstrate that commonly amplified DNA sequences reside in a contiguous domain of approximately equal to 120 kilobases (kb). We report the isolation of this DNA domain in cosmid clones and show that the level of amplification of the domain is correlated with the level of resistance in multidrug-resistant cell lines. The organization of the amplified domain was deduced by a unique approach utilizing in-gel hybridization of cloned DNA with amplified genomic DNA. We show that the entire cloned region is amplified in adriamycin-resistant LZ cells and independently derived, colchicine-resistant C5 cells. A mRNA species of approximately equal to 5 kb is encoded by a gene located within the boundaries of this region. Genomic sequences homologous to the 5-kb mRNA span over 75 kb of the amplified DNA segment. The level of expression of this mRNA in multidrug-resistant cells is correlated with the degree of gene amplification and the degree of drug resistance. Our results strongly suggest that the 5-kb mRNA species plays a role in the mechanism of multidrug resistance common to the LZ and C5 cell lines.
Insights
Multidrug resistance in mammalian cells involves amplified DNA sequences. A specific 120-kilobase DNA domain and a 5-kilobase mRNA are amplified, correlating with increased drug resistance.
Area of Science:
- Molecular biology
- Genetics
- Cell biology
Background:
- Mammalian cells can develop resistance to multiple chemotherapeutic agents.
- Understanding the genetic mechanisms of multidrug resistance is crucial for cancer treatment.
- Previous studies suggested amplified DNA sequences might be involved in drug resistance.
Purpose of the Study:
- To investigate the molecular genetic basis of multidrug resistance in mammalian cells.
- To identify and characterize amplified DNA sequences in multidrug-resistant cell lines.
- To determine the role of specific genes and their expression in conferring drug resistance.
Main Methods:
- Utilized molecular genetic techniques to study drug-resistant Chinese hamster cell lines (LZ and C5).
- Isolated a 120-kilobase amplified DNA domain using cosmid cloning.
- Employed in-gel hybridization to analyze the organization of the amplified DNA domain.
- Quantified mRNA expression levels using molecular hybridization techniques.
Main Results:
- Two multidrug-resistant cell lines (LZ and C5) share a common amplified DNA domain of approximately 120 kilobases (kb).
- The level of amplification of this DNA domain directly correlates with the degree of drug resistance observed in the cell lines.
- A 5-kb mRNA species, encoded by a gene within this amplified region, shows increased expression correlating with gene amplification and drug resistance.
Conclusions:
- The 120 kb amplified DNA domain and its encoded 5 kb mRNA are strongly implicated in the mechanism of multidrug resistance.
- Gene amplification and subsequent overexpression of the 5 kb mRNA play a significant role in conferring resistance to chemotherapeutic agents.
- These findings provide insights into the genetic underpinnings of multidrug resistance, potentially guiding future therapeutic strategies.