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Potentiation of etoposide-induced cytotoxicity and DNA damage in CCRF-CEM cells by pretreatment with non-cytotoxic concentrations of arabinosyl cytosine

  • Original Articles
  • DNA Damage, Cytotoxicity, CCRF-CEM Cells, VP16, ARA-C
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Summary

Pretreatment of the human lymphoblastoid cell line CCRF-CEM with 0.02 μm arabinosyl cytosine (ara C) enhances both the cytotoxic and the DNA-damaging effects of etoposide. This concentration of ara C is itself non-cytotoxic and results in no detectable DNA damage as measured by alkaline elution. Ara C pretreatment results in the synchronisation of cells, a 24-h pretreatment resulting in the accumulation of cells in the early S phase. The sensitivity of cells to etoposide-induced cytotoxicity was increased 2.5 times and DNA damage was enhanced 1.66 times by this pretreatment. Maximal potentiation of etoposide-induced DNA damage (2.06-fold increase) was observed after 48 h continuous treatment with ara C, but no further enhancement of cytotoxicity occurred. Cell-cycle analysis demonstrated that 48 h ara C treatment resulted in the accumulation of cells in the late S/G2M phase. Cells returned to a normal cell-cycle distribution within 24 h of the removal of ara C, and the potentiation of etoposide activity was then reduced to a 1.3- to 1.4-fold level. DNA damage induced by etoposide following ara C pretreatment was qualitatively identical to that produced by etoposide alone, suggesting a mechanism involving topoisomerase II. To investigate this possibility, we measured topoisomerase II protein levels by immunoblotting. Measurement of topoisomerase II levels in whole-cell lysates of ara C-pretreated cells showed a 3- to 5-fold increase in topoisomerase levels relative to total protein content. This suggests that elevated enzyme levels may be responsible for the increased sensitivity of ara C-pretreated cells to etoposide.

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Abbreviations

ara-C:

cytosine β-d-arabinofuranoside

m-AMSA:

4′-(9-acridinylamino)-methanesulfon-m-anisidide

DMSO:

dimethylsulphoxide

MTT:

3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide

SSBs:

single-strand breaks

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Author notes

  1. Christine M. Chresta

    Present address: Molecular Cell Pathology, Royal Free Hospital School of Medicine, University of London, Rowland Hill Street, NW3 2PF, London, UK

Authors and Affiliations

  1. Department of Oncology, University College & Middlesex School of Medicine, 91 Riding House Street, W1P 8BT, London, UK

    Christine M. Chresta, Raymond Hicks, John A. Hartley & Robert L. Souhami

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  1. Christine M. Chresta
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  2. Raymond Hicks
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This work was supported by a grant from the North East Thames Regional Health Authority

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Chresta, C.M., Hicks, R., Hartley, J.A. et al. Potentiation of etoposide-induced cytotoxicity and DNA damage in CCRF-CEM cells by pretreatment with non-cytotoxic concentrations of arabinosyl cytosine. Cancer Chemother. Pharmacol. 31, 139–145 (1992). https://doi.org/10.1007/BF00685101

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