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DNA topoisomerase II inhibition and gene amplification in V79/B7 cells
A Di Leonardo1, P Cavolina, A Maddalena
1Dipartimento di Biologia Cellulare e dello Sviluppo, A. Monroy, University of Palermo, Italy.
Abstract:
Topoisomerase II inhibitors such as etoposide (VP16) are able to stabilize the enzyme-DNA complex by trapping the topoisomerase on DNA without affecting its strand-break activity. To test if this inhibition resulting in chromosomal breakage via double-strand breaks could underlie gene amplification, we performed VP16 treatments followed by selection for PALA resistance in V79/B7 Chinese hamster cells. We found that VP16 induced PALA-resistant cells very efficiently, and in a dose-dependent manner. On the other hand VP16 in combination with 3-aminobenzamide (3AB), an inhibitor of poly(ADP-ribose) polymerase involved in DNA repair, reduced the frequency of PALA-resistant cells. Cytogenetic analysis revealed a higher number of chromosomal aberrations in VP16-treated cells than in cells treated with VP16 plus 3AB. These results suggest a correlation between frequency of chromosomal aberrations and frequency of PALA-resistant cells, and are consistent with models which consider chromosomal breakage as an important step in initiating gene amplification.
Insights
Etoposide (VP16) treatment effectively induces gene amplification by causing DNA double-strand breaks. Inhibiting DNA repair with 3-aminobenzamide (3AB) reduces this amplification, suggesting breakage initiates gene amplification.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- Topoisomerase II inhibitors, like etoposide (VP16), stabilize enzyme-DNA complexes.
- This stabilization traps topoisomerase on DNA without altering its strand-break activity.
Purpose of the Study:
- To investigate if VP16-induced chromosomal breakage underlies gene amplification.
- To determine the role of DNA repair mechanisms in VP16-induced gene amplification.
Main Methods:
- V79/B7 Chinese hamster cells were treated with VP16.
- Cells were subsequently selected for PALA resistance.
- Combined treatments included VP16 and 3-aminobenzamide (3AB), a poly(ADP-ribose) polymerase inhibitor.
- Cytogenetic analysis was performed to assess chromosomal aberrations.
Main Results:
- VP16 treatment efficiently induced PALA-resistant cells in a dose-dependent manner.
- Combining VP16 with 3AB significantly reduced the frequency of PALA-resistant cells.
- VP16 treatment led to a higher number of chromosomal aberrations compared to VP16 plus 3AB treatment.
Conclusions:
- A correlation exists between chromosomal aberration frequency and PALA-resistant cell frequency.
- Chromosomal breakage is a critical initiating step in gene amplification.
- DNA repair pathways may modulate the frequency of gene amplification events.