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105 MEASURING TOXICITY, INCORPORATION, AND REPAIR OF 5-HYDROXYMETHYL-2′-DEOXYURIDINE IN CELLS.
  1. D. K. Rogstad1,
  2. J. Herring1,
  3. K. N. Rogstad1,
  4. A. Burdzy1,
  5. L. C. Sowers1
  1. 1Loma Linda University School of Medicine, Loma Linda, CA.

Abstract

5-Hydroxymethyl-2′-deoxyuridine (HmdU) is among the most common endogenous oxidative DNA lesions and is repaired by more than one DNA glycosylase in human cells. This DNA-repair pathway may be exploited for use in cancer chemotherapy. Exogenous delivery of HmdU to cells in culture leads to its incorporation into the DNA and subsequent repair, resulting in death by apoptosis if the HmdU load is too heavy for the cell. Both incorporation into the DNA and repair of HmdU out of the DNA are necessary for cell death to occur. To measure the incorporation into and subsequent repair of HmdU out of the DNA of cells in culture, we have developed a highly sensitive and specific method using stable isotope dilution and gas chromatography-negative ion chemical ionization-mass spectrometry (GC-NICI-MS) for measuring HmdU levels found in DNA. Using the osteosarcoma cell line U2OS as a model system, we treat cells with HmdU, harvest cells, and isolate DNA. Hydrolysis of the DNA with formic acid releases the free bases, and we convert the base 5-hydroxymethyluracil (HmU) into its methyl ester, 5-methoxymethyluracil, subsequently derivatizing with 3,5-bis(trifluoromethyl)benzyl bromide to yield a stable compound. When analyzing trace levels of HmdU deoxynucleoside standard, this derivatization process yields a detection limit of approximately 500 attomoles (5 × 10−16 mol) on column. We demonstrate in U2OS cells that toxicity of exogenously delivered HmdU is proportional to its incorporation into the DNA, and we show that repair of HmdU out of the DNA is occurring. Application of our method in cell lines with varying sensitivities to HmdU treatment will allow us to gain further insight into the mechanism of and resistance to this potential chemotherapeutic agent.

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