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Published on: June 14, 2018
Enhanced gene amplification in human cells knocked down for DNA-PKcs
Alberto Salzano1, Nino Kochiashvili, Solomon G Nergadze
1Dipartimento di Genetica e Microbiologia "Adriano Buzzati-Traverso", Università di Pavia, Via Ferrata 1, 27100 Pavia, Italy.
Inhibiting DNA-PKcs in human cells enhances gene amplification, particularly through double minutes, a key mechanism for drug resistance. This highlights DNA-PKcs
Area of Science:
- Molecular Biology
- Genetics
- Cancer Research
Background:
- Gene amplification is crucial for oncogene activation and drug resistance in tumors.
- Amplified DNA exists as intra-chromosomal arrays or extra-chromosomal elements called double minutes.
- Previous studies in rodent cells showed enhanced intra-chromosomal amplification when DNA-PKcs is deficient.
Purpose of the Study:
- To investigate the role of DNA-PKcs in gene amplification in human cells.
- To determine the effect of DNA-PKcs inhibition on drug resistance mechanisms.
- To establish human cell models for studying gene amplification.
Main Methods:
- Utilized human HeLa cell lines with constitutively inhibited DNA-PKcs expression via shRNAs.
- Assessed sensitivity to ionizing radiation.
- Quantified chromosomal aberrations and methotrexate resistance rates.
- Analyzed the mechanism of methotrexate resistance, focusing on dihydrofolate reductase gene amplification.
Main Results:
- DNA-PKcs inhibited cell lines exhibited increased sensitivity to ionizing radiation.
- A significant increase (6-18 times) in chromosomal aberrations and methotrexate-resistant colonies was observed.
- The primary mechanism for methotrexate resistance was extra-chromosomal amplification of the dihydrofolate reductase gene.
- Inhibition of DNA-PKcs favored gene amplification via double minute formation in human cells.
Conclusions:
- Constitutive inhibition of DNA-PKcs in human cells promotes gene amplification, particularly through double minutes.
- This suggests DNA-PKcs plays a critical role in suppressing extra-chromosomal amplification.
- Human cell lines with inhibited DNA-PKcs expression serve as valuable models for studying gene amplification mechanisms and their role in drug resistance.
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