Related Experiment Video
Updated: Oct 7, 2026
Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells
Published on: February 24, 2014
Molecular characterisation of camptothecin-induced mutations at the hprt locus in Chinese hamster cells
E Balestrieri1, R Zanier, F Degrassi
1Centre of Evolutionary Genetics, National Research Council, c/o Department of Genetics and Molecular Biology, La Sapienza University, Via degli Apuli 4, 00185 Rome, Italy.
Abstract:
The capacity of the topoisomerase I inhibitor camptothecin (CPT) to induce single locus mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene and the DNA changes underlying induced mutations were analysed in Chinese hamster ovary cells. Camptothecin treatments increased hprt mutations up to 50-fold over the spontaneous levels at highly cytotoxic doses. Genomic DNA was isolated from 6-thioguanine resistant clones and subjected to multiplex PCR to screen for gross alterations in the gene structure. The molecular analysis revealed that deletion mutants represented 80% of the analysed clones, including total hprt deletion, multiple and single exon deletions. Furthermore, a fraction of the analysed clones showed deletions of more than one exon that were characterised by the absence of non-contiguous exons. These data show that single locus mutations induced by camptothecin are characterised by large deletions or complex rearrangements rather than single base substitutions and suggest that the recombinational repair of camptothecin-induced strand breaks at replication fork may be involved in the generations of these alterations at the chromatin structure level.
Insights
Camptothecin (CPT), a topoisomerase I inhibitor, significantly increases mutations in the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene. These CPT-induced mutations primarily involve large deletions and complex rearrangements, not single base changes.
Area of Science:
- Genetics
- Molecular Biology
- Toxicology
Background:
- Topoisomerase I inhibitors like camptothecin (CPT) are used in cancer therapy.
- Understanding their genotoxic effects is crucial for assessing safety and efficacy.
- The hypoxanthine-guanine phosphoribosyltransferase (hprt) gene is a common target for mutation analysis.
Purpose of the Study:
- To investigate the capacity of camptothecin (CPT) to induce mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene.
- To characterize the underlying DNA changes responsible for CPT-induced mutations.
Main Methods:
- Chinese hamster ovary cells were treated with camptothecin (CPT).
- Genomic DNA from 6-thioguanine resistant clones was isolated.
- Multiplex PCR was used to screen for gross alterations in the hprt gene structure.
Main Results:
- Camptothecin (CPT) treatment increased hprt mutations up to 50-fold over spontaneous levels at highly cytotoxic doses.
- Molecular analysis revealed that 80% of analyzed clones were deletion mutants, including total, multiple, and single exon deletions.
- A fraction of clones showed deletions of non-contiguous exons, indicating complex rearrangements.
Conclusions:
- Camptothecin (CPT)-induced single locus mutations are characterized by large deletions or complex rearrangements, rather than single base substitutions.
- Recombinational repair of CPT-induced DNA strand breaks at replication forks may generate these chromatin alterations.
