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Topoisomerase II cleavable complex formation within DNA loop domains
1Department of Experimental Oncology, Hollings Cancer Center, Medical University of South Carolina, Charleston, SC 29425, USA.
Abstract:
The distribution of VM-26 (Teniposide)-stabilized cleavable complexes within DNA loops bound to the nuclear matrix was determined to provide further insights into the mode of DNA synthesis inhibition by VM-26. Covalent binding of [(3)H]VM-26 was 9-fold greater per milligram of nuclear matrix protein compared with high salt-soluble nonmatrix protein of CEM cells. The ratio declined from 9-fold in CEM cells to 4-fold in drug-resistant VM-1/C2 cells, which have decreased nuclear matrix DNA topoisomerase IIalpha. VM-26 induced a concentration-dependent increase in the frequency of cleavable complex formation with actively replicating matrix DNA. At 25 microM VM-26, the frequency was 32 +/- 2 (SEM) complexes per 10(6) bp of replicating matrix DNA compared with 13 +/- 2 (SEM) complexes per 10(6) bp of nonreplicating DNA in the matrix fraction. VM-26 at concentrations as high as 25 microM stabilized less than 3 complexes per 10(6) bp in the various nonmatrix DNA domains, since the nonmatrix DNA comprises the DNA loop domains that are distal to the matrix-bound replication sites. A negligible frequency of cleavable complex formation was detected in both the matrix and nonmatrix DNA domains of drug-resistant VM-1/C2 cells. Compared with untreated control cells, VM-26 induced an accumulation of nascent DNA in the nuclear matrix fraction of CEM cells but decreased the amount of nascent DNA in the nonmatrix fraction. The extensive cleavable complex formation on matrix replicating DNA stalled most of the replication forks within 1 kb of the replication sites on the nuclear matrix. The results provide evidence that nascent DNA bound to the nuclear matrix is an important site of VM-26 cleavable complex formation, and that these complexes inhibit DNA synthesis by blocking the movement of nascent DNA away from replication sites on the nuclear matrix.
Insights
VM-26 (Teniposide) stabilizes DNA cleavable complexes, primarily at nuclear matrix replication sites, inhibiting DNA synthesis by stalling replication forks. This mechanism is less effective in drug-resistant cells with reduced topoisomerase IIalpha.
Area of Science:
- Molecular Biology
- Cancer Research
- Biochemistry
Background:
- VM-26 (Teniposide) is a topoisomerase II inhibitor used in cancer therapy.
- Understanding its precise mechanism of DNA synthesis inhibition is crucial for optimizing its use.
- The nuclear matrix plays a role in DNA replication and organization.
Purpose of the Study:
- To investigate the distribution of VM-26-stabilized cleavable complexes within DNA loops bound to the nuclear matrix.
- To elucidate the role of the nuclear matrix in VM-26-induced DNA synthesis inhibition.
- To compare VM-26 activity in drug-sensitive and drug-resistant cancer cells.
Main Methods:
- Quantification of covalent [(3)H]VM-26 binding to nuclear matrix and nonmatrix proteins.
- Analysis of cleavable complex formation frequency in replicating and non-replicating DNA fractions.
- Assessment of nascent DNA distribution in nuclear matrix and nonmatrix fractions following VM-26 treatment.
Main Results:
- VM-26 showed significantly higher covalent binding to nuclear matrix proteins compared to nonmatrix proteins.
- VM-26 induced a concentration-dependent increase in cleavable complexes on actively replicating matrix DNA.
- Drug-resistant cells exhibited reduced cleavable complex formation and lower topoisomerase IIalpha levels in the nuclear matrix.
Conclusions:
- Nascent DNA associated with the nuclear matrix is a primary target for VM-26 cleavable complex formation.
- VM-26 inhibits DNA synthesis by impeding nascent DNA movement away from nuclear matrix replication sites.
- Differences in nuclear matrix-bound topoisomerase IIalpha contribute to VM-26 resistance.