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We have employed cesium sulfate density gradient centrifugation to separate RNA and DNA of L1210 cells labeled with [3H]fluorodeoxyuridine. We have analyzed nucleotide and nucleoside digests of purified DNA from the [3H]fluorodeoxyuridine-labeled cells and demonstrate by reverse phase and anion exchange high pressure liquid chromatography the presence of tritium radioactivity co-migrating with fluorodeoxyuridine 5'-monophosphate or fluorodeoxyuridine. These observations demonstrate the internucleotide incorporation of fluorodeoxyuridine in DNA and suggest a new mechanism of action for this cytotoxic and mutagenic agent.

We have employed cesium sulfate density gradient analysis to monitor the incorporation of 1-beta-D-arabinofuranosylcytosine (ara-C) into L1210 cellular nucleic acid. This methodology permits a distinct separation of RNA and DNA under nondegrading conditions. The results demonstrate specific incorporation of ara-C into DNA with less than 1% of total incorporation into nucleic acid being detectable in RNA. Moreover, we have found a highly significant relationship (p < 0.0001) between the incorporation of ara-C into DNA with the loss of clonogenic survival. This relationship had not been established due to the previous use of alkaline conditions that degrade (ara-C) DNA. Our results suggest that the incorporation of ara-C into DNA is one mechanism responsible for producing lethal cellular events.

We recently demonstrated a highly significant relationship between the incorporation of 1-beta-D-arabinofuranosylcytosine (ara-C) into L1210 DNA and the loss of clonogenic survival. These studies have now been extended to the human promyeloblast (HL-60) cell line and myeloblasts from a patient with acute myelogenous leukemia. Our results demonstrate: (i) the specific internucleotide incorporation of ara-C into human myeloblast DNA; (ii) the lability of [3H]ara-C-labeled DNA to alkali which necessitates the use of nondegrading assay conditions; and (iii) a highly significant relationship (P less than 0.0001) between the loss of clonogenicity of these cells and the extent of ara-C incorporation. These findings suggest that the incorporation of ara-C into DNA is one of the initial events leading to cell lethality. This method is applicable to clinical samples of bone marrow and peripheral blood as an in vitro assay for studying the sensitivity of cell populations to this drug.

We have employed cesium sulfate density gradient analysis to monitor the incorporation of 1-beta-D-arabinofuranosylcytosine (ara-C) into L1210 cellular nucleic acid. This methodology permits a distinct separation of RNA and DNA under nondegrading conditions. The results demonstrate specific incorporation of ara-C into DNA with less than 1% of total incorporation into nucleic acid being detectable in RNA. Moreover, we have found a highly significant relationship (p0.0001) between the incorporation of ara-C into DNA with the loss of clonogenic survival. This relationship had not been established due to the previous use of alkaline conditions that degrade (ara-C) DNA. Our results suggest that the incorporation of ara-C into DNA is one mechanism responsible for producing lethal cellular events.

Cytosine arabinoside (Ara-C) is the most effective agent in the treatment of acute myelogenous leukemia. This agent incorporates in leukemic cell DNA, and the extent of this incorporation correlates with loss of clonogenic survival. The incorporated Ara-C residue behaves as a relative DNA chain terminator, and the extent of (Ara-C)DNA formation correlates with inhibition of DNA synthesis. The incorporation of Ara-C into DNA requires the formation of Ara-CTP, and previous measurements of this metabolite have also been correlated with cytotoxicity. Because it is clinically relevant to define biochemical parameters predictive of Ara-C cytotoxicity, the present studies were undertaken to determine the relationship among Ara-CTP pools, formation of (Ara-C)DNA, and loss of clonogenic survival. The results demonstrate that the incorporation of Ara-C into DNA is the single most powerful predictor of cell lethality. Furthermore, although there is a correlation between Ara-CTP pools or continuous cellular exposure to Ara-CTP and cell kill, these relationships are less significant than that obtained with formation of (Ara-C)DNA. The extent of Ara-C incorporation into DNA can be predicted by the product of the Ara-CTP level and time (T), thus supporting the concept that Ara-C incorporation is dependent on continuous exposure to the triphosphate metabolite. These findings support the formation of (Ara-C)DNA as a highly predictive parameter of lethal cellular events.

We have employed cesium sulfate density gradient centrifugation to separate RNA and DNA of L1210 cells labeled with [3H]fluorodeoxyuridine. We have analyzed nucleotide and nucleoside digests of purified DNA from the [3H]fluorodeoxyuridine-labeled cells and demonstrate by reverse phase and anion exchange high pressure liquid chromatography the presence of tritium radioactivity co-migrating with fluorodeoxyuridine 5'-monophosphate or fluorodeoxyuridine. These observations demonstrate the internucleotide incorporation of fluorodeoxyuridine in DNA and suggest a new mechanism of action for this cytotoxic and mutagenic agent.