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Effects of antileukemia agents on nuclear matrix-bound DNA replication in CCRF-CEM leukemia cells
D J Fernandes1, C Smith-Nanni, M T Paff
1Department of Biochemistry, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, North Carolina 27103.
Abstract:
The effects of various antileukemic agents on DNA replication associated with the nuclear matrix were investigated in CCRF-CEM leukemia cells. Residual nuclear matrices were prepared by sequential treatment of nuclei with 1.5 M NaCl, DNase I, and Triton X-100 and contained 1-5, 10, and 37% of the total nuclear DNA, protein, and phospholipid, respectively. In control cells pulse-labeled for 45 s with [3H]thymidine, the specific activity of nascent DNA was four-fold greater in the nuclear matrix fraction relative to the specific activity of the high salt-soluble (nonmatrix) DNA fraction. Pulse-labeling and reconstitution experiments indicated that this enrichment of newly replicated DNA on the nuclear matrix did not result from aggregation of nascent DNA with the matrix. A 2-h incubation of tumor cells with either 0.1 microM teniposide (VM-26), 0.2 microM VM-26, or 0.5 microM amsacrine (m-AMSA) reduced the relative specific activity of nascent DNA on the nuclear matrix by 59, 61, and 54%, respectively, compared to control cells. In contrast hydroxyurea and cytosine arabinoside, at concentrations that markedly inhibited total nuclear DNA synthesis, did not decrease the relative specific activity of newly replicated DNA on the matrix. The results provide evidence that the antiproliferative effects of the DNA topoisomerase II inhibitors, VM-26 and m-AMSA, are localized on the nuclear matrix of CCRF-CEM leukemia cells.
Insights
Antileukemic drugs teniposide (VM-26) and amsacrine (m-AMSA) target DNA replication at the nuclear matrix in leukemia cells. Other agents inhibiting DNA synthesis did not show this specific localization effect.
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- The nuclear matrix plays a crucial role in DNA replication.
- Understanding how antileukemic agents interact with the nuclear matrix is vital for cancer therapy.
Purpose of the Study:
- To investigate the effects of antileukemic agents on DNA replication associated with the nuclear matrix in leukemia cells.
- To determine if specific antileukemic drugs localize to the nuclear matrix during DNA replication.
Main Methods:
- CCRF-CEM leukemia cells were used.
- Nuclear matrices were isolated using sequential treatments (NaCl, DNase I, Triton X-100).
- Nascent DNA synthesis was measured using [3H]thymidine pulse-labeling.
Main Results:
- Newly replicated DNA showed a four-fold enrichment in the nuclear matrix fraction.
- Teniposide (VM-26) and amsacrine (m-AMSA) significantly reduced nascent DNA activity on the nuclear matrix.
- Hydroxyurea and cytosine arabinoside did not affect the relative specific activity of nascent DNA on the nuclear matrix.
Conclusions:
- The antiproliferative effects of DNA topoisomerase II inhibitors VM-26 and m-AMSA are localized on the nuclear matrix.
- This localization suggests a specific mechanism of action for these drugs in leukemia cells.