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Resistance to paraquat in a mammalian cell line
Abstract:
Paraquat-resistant variants were isolated in Chinese hamster ovary (CHO) cells by stepwise increases in paraquat concentrations. Three series of selective experiments gave variants which appeared to be using one or several different mechanisms of resistance. In all variants tested (PQ-1, PQ-2, PQ-3, PQ-2X and PQ-3X of series 1), radioactively labeled paraquat was taken up by the cells. These variants exhibited no unusual resistance to either oxygen or radiation, nor were increases found in the activities of free-radical scavenging enzymes. They had extra DNA (3-12%) and an unusual acrocentric marker chromosome which was common to all of the variants but never observed in the parental cells. Double minutes were observed in 29% of metaphases of the PQ-3 variant. One of the resistant lines exhibited evidence of an intrinsic chromosomal instability, a phenotype that could conceivably facilitate gene amplification. Selection series 2 and 3 were designed to further evaluate gene amplification as a mechanism of resistance. These variants exhibited high frequencies (40-100%) of tetraploidy or hypotetraploidy with loss of chromosomes and varying frequencies of double minutes (10-75% of metaphases). In two of the variants the same marker chromosome which was observed in the series 1 variants was seen. Two other lines exhibited a variant of this marker, incorporating it into a metacentric chromosome. It may be that gene amplification facilitates resistance to paraquat and that both stable and unstable methods of amplifying genes are used.
Insights
Researchers developed paraquat-resistant Chinese hamster ovary (CHO) cells using stepwise selection. These variants showed increased DNA and chromosomal abnormalities, suggesting gene amplification as a resistance mechanism.
Area of Science:
- Cell Biology
- Genetics
- Toxicology
Background:
- Paraquat is a widely used herbicide with significant toxicity.
- Understanding cellular resistance mechanisms is crucial for safety and therapeutic development.
- Chinese hamster ovary (CHO) cells are a standard model for genetic and toxicological studies.
Purpose of the Study:
- To investigate the mechanisms of paraquat resistance in CHO cells.
- To identify genetic and chromosomal alterations associated with paraquat resistance.
- To evaluate the role of gene amplification in conferring resistance.
Main Methods:
- Stepwise selection of paraquat-resistant variants in CHO cells.
- Analysis of paraquat uptake using radiolabeled compounds.
- Assessment of resistance to oxygen, radiation, and free-radical scavenging enzyme activity.
- Cytogenetic analysis including DNA content, marker chromosomes, and double minutes.
- Evaluation of chromosomal instability and ploidy changes.
Main Results:
- Isolated paraquat-resistant variants (PQ-1, PQ-2, PQ-3, PQ-2X, PQ-3X) showed paraquat uptake but no increased resistance to oxygen, radiation, or scavenging enzymes.
- Variants exhibited increased DNA content (3-12%) and a common acrocentric marker chromosome not present in parental cells.
- Double minutes were observed in some variants, indicating potential gene amplification.
- Further selection experiments revealed high frequencies of tetraploidy/hypotetraploidy and varying double minutes, supporting gene amplification.
- Chromosomal instability was observed, potentially facilitating gene amplification.
Conclusions:
- Gene amplification appears to be a key mechanism conferring paraquat resistance in CHO cells.
- Both stable and unstable methods of gene amplification may be employed.
- The identified marker chromosome and chromosomal instability are significant findings in paraquat resistance research.