Related Experiment Videos
Cloning and characterization of DNA sequences amplified in multidrug-resistant Djungarian hamster and mouse cells
A V Gudkov1, O B Chernova, A R Kazarov
1Laboratory of Oncovirology, Cancer Research Center, Moscow, U.S.S.R.
Abstract:
Five recombinant phages containing different parts of genomic regions amplified in multidrug-resistant (MDR) cells have been isolated from genomic libraries of colchicine- and actinomycin D-resistant Djungarian hamster cells. Fragments of these clones together with a part of Chinese hamster mdr gene (plasmid pDR4.7 a gift of Dr. I. Roninson) were used as hybridization probes to study composition and variations of amplified DNA in a large number of MDR cell lines. Two of the six probes used (pC52 and pDR4.7) showed DNA amplification in a large number of MDR cell lines tested (commonly amplified clones) regardless of their origin (Djungarian hamster or mouse), type of selective agent used (colchicine, actinomycin D, or anthracyclines), and mode of selection (in vitro or in vivo). These clones hybridized with two different RNA transcripts (pDR4.7, 5kb; pC52, 3.5 kb) that were overproduced in MDR cells. Degrees of amplification of both commonly amplified sequences correlated with levels of resistance in all but one of the cell lines. Other cloned sequences (sporadically amplified clones) were amplified to different extents (but never greater than the commonly amplified sequences) in some of the Djungarian hamster MDR cell lines. Such differential amplification is not the result of heterogeneity of cell population since 20 cell clones tested showed identical ratios of amplification of different amplified sequences. Sporadically amplified sequences usually coamplified with the commonly amplified ones at first steps of selection, but then they would cease to amplify and, at the later stages of selection, they could even be completely deamplified. It seems that disappearance of unnecessary parts of amplicons is a regular process accompanying stepwise gene amplification.
Insights
Researchers identified commonly amplified DNA sequences in multidrug-resistant (MDR) cells, which correlate with drug resistance levels. These findings shed light on the dynamic process of gene amplification during drug selection.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- Multidrug resistance (MDR) in cancer cells is a significant clinical challenge.
- Gene amplification is a common mechanism underlying MDR.
- Understanding the specific amplified genomic regions is crucial for developing targeted therapies.
Purpose of the Study:
- To isolate and characterize amplified DNA sequences in multidrug-resistant (MDR) cells.
- To investigate the correlation between DNA amplification and drug resistance levels.
- To explore the dynamics of gene amplification during stepwise selection.
Main Methods:
- Isolation of recombinant phages from genomic libraries of resistant cell lines.
- Use of cloned fragments as hybridization probes to analyze amplified DNA.
- Hybridization studies with various MDR cell lines.
- Analysis of RNA transcripts overproduced in MDR cells.
Main Results:
- Two DNA sequences (pC52 and pDR4.7) were commonly amplified across diverse MDR cell lines, irrespective of the selective agent or cell origin.
- Amplification of these common sequences correlated with drug resistance levels.
- Sporadically amplified sequences were observed, showing differential amplification patterns and potential deamplification during later selection stages.
- Coamplification and subsequent deamplification suggest a process of optimizing amplicons during stepwise selection.
Conclusions:
- Commonly amplified DNA sequences play a significant role in conferring multidrug resistance.
- The observed amplification patterns suggest a dynamic and selective process of amplicon evolution.
- Understanding these amplification dynamics may offer new strategies for overcoming drug resistance in cancer treatment.