Incorporation in L1210 DNA*

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 centrifugation to separate RNA and DNA of L1210 cells labeled with [3HJfluorodeoxyuridine. We have analyzed nucleotide and nucleoside digests of purified DNA from the [3H]fluorodeoxyuridine-labeled ceIls 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.
Fluorouracil and fluorodeoxyuridine are effective agents in the treatment of certain human epithelial tumors (1)(2)(3). Several mechanisms of action have been proposed for these agents, among which are 1) incorporation of FUra' in RNA with disruption of RNA synthesis and function and 2) conversion to FdUMP with irreversible binding to thymidylate synthetase and inhibition of DNA synthesis by limiting production of dTMP (3-8). Either mechanism could be responsible for the cytotoxic effects of these agents. FUra incorporation in RNA has been correlated with antitumor activity: while the misincorporation of FdUrd in eukaryotic DNA has not been demonstrated previously (9).
The enzymes deoxyuridine-triphosphate nucleotidohydrolase and uracil-DNA-glycosylase are responsible for preventing the incorporation of uracil nucleotides in DNA (10)(11)(12). The nucleotide hydrolase degrades intracellular dUTP, and uracil DNA-glycosylase removes uracil residues incorporated in the DNA strand. These enzymes can also utilize the fluorinated derivatives of uracil as substrates, and this has been suggested as an explanation for the failure to detect FdUrd in cellular DNA (10,11). Although fluorodeoxyuridine-triphosphate and dUTP are hydrolyzed at similar rates, the excision of FUra from DNA by uracil DNA-glycosylase is much less efficient than that of uracil (11). This finding, as well as the * This work was supported in part by Grant CA-19589 from the National Cancer Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.  (14), suggests that FdUrd residues could be identified in mammalian DNA.
We have studied the incorporation of FdUrd in DNA of L1210 murine leukemia cells. The results demonstrate that FdUrd incorporates in internucleotide linkage in DNA.

MATERIALS AND METHODS
Cell Culture-The L1210 cells were grown as a suspension culture in minimum essential medium for suspension supplemented with 10% heat-inactivated dialyzed fetal calf serum, 1% L-glutamine, 100  sulfate. The nucleic acids were purified by phenol extraction and then precipitated by the addition of 1/10 volume of 4 M NaCl and 2 volumes of absolute ethanol. The nucleic acids were analyzed by cesium sulfate gradient centrifugation (15). Digestion of [3HjFdUrd-iabeled Nucleic Acids"Ll210 cells were labeled with [3H]FdUrd for 6 h as described for the incorporation studies. The total nucleic acids were purified as described above and separated by centrifugation on cesium sulfate gradients. The purified DNA was digested to 5'-nucleotides using DNase I and snake venom phosphodiesterase. The digestion of ['HIFdUrd-labeled DNA to 3" nucleotides was performed by the sequential action of micrococcal nuclease and spleen phosphodiesterase. Further digestion of the 5" or 3'-nucleotides to nucleosides was accomplished with bacterial alkaline phosphatase. (All enzymes were purchased from Worthington Biochemicals.) After precipitating the remaining macromolecular species with perchloric acid, nucleotides or nucleosides were analyzed by high pressure liquid chromatography using a Varian Model 5020 gradient liquid chromatograph under reverse phase and anion exchange conditions.
The reverse phase isocratic chromatography was performed on a Varian Micropak MCH-10 column using 0.01 M KH2P04 (pH 5.25) buffer at a flow rate of 1 ml/min. Anion exchange analysis was performed using a Varian Micropak AX-10 column with a gradient elution. Buffer A was 0.01 M KH2PO4 (pH 2.85)/acetonitrile (20/80), and buffer B was 0.01 M KH2P04 (pH 2.85). The elution program utilizing buffer B was as follows: 1) 0% for 10 min, 2) O-lOO% for 10 min, and 3) 100% for 40 min. Each elution was performed after the addition of appropriate markers. The 5-fluoro-2'-deoxycytidine marker was kindly provided by Dr. J. Fox, Sloan-Kettering Institute for Cancer Research, Rye, NY. Fractions were collected during the elution and assayed for tritium counts.

RESULTS
The amount of 13H]FdUrd incorporation into LE10 nucleic acids was monitored by cesium sulfate gradient centrifugation which separates RNA (banding between density 1.62 to 1.68 g/ml) and DNA (banding between density 1.42 and 1.48 g/ ml). Fig. 1 shows the incorporation of r3H]FdUrd and "P in L1210 nucleic acids at various concentrations of FdUrd (10" to M) during an incubation period of 6 h. There is significant incorporation of tritium radioactivity in both the DNA and RNA regions of the gradient. The amount of tritium incorporated in DNA and RNA is dependent on drug concentration. The labeling with 32P serves as a measure of newly 8885 5-Fluoro-2'-deoxyuridine Incorporation in L1210 DNA synthesized RNA and DNA and demonstrates that RNA synthesis is not affected by increasing the concentration of FdUrd, while DNA synthesis is inhibited at each drug level. The incorporation of tritium radioactivity into RNA and DNA also increases with time of exposure, as illustrated in Fig. 2.
It is important to demonstrate that the tritium radioactivity detectable in the DNA region of the Cs2S04 gradient represents [3H]FdUrd. The labeled DNA was digested to nucleosides using DNase I, snake venom phosphodiesterase, and BAP for analysis by high pressure liquid chromatography. The profile seen in Fig. 3A illustrates that the tritium radioactivity co-migrates with FdUrd and not with other metabolites such as fluorouridine or deoxyuridine. Although not shown in this profile, the radioactivity co-migrating with FdUrd does not co-migrate with any of the pyrimidine and purine deoxyribonucleosides or ribonucleosides. Furthermore, the radioactivity does not co-migrate with 5-fluoro-2'-deoxycytidine (Fig. 3B). Similar results are obtained when the DNA fraction is first treated with either NaOH (0.3 N for 6 h at 30 37 "c) or RNase A (0.1 mg/ml, with 10 mM EDTA for 2 h at 37 "C) and is then precipitated with perchloric acid prior to the enzyme digest. These observations confirm that the FdUrd residues are incorporated into DNA and not RNA. Cells were exposed to equimolar M) concentrations of FdUrd and dThd. The amount of each nucleoside incorporated in DNA was determined and the ratio of FdUrd to dThd was 1:900.
The C3H]FdUrd-labeled DNA was also digested to 5'-and 3'-nucleotides to determine whether the FdUrd residues are in internucleotide linkage. As illustrated in Fig. 4, digestion of the labeled DNA with DNase I and snake venom phosphodiesterase results in a peak of tritium radioactivity co-migrating  at 4 h, 0.19 pmol at 8 h, and 0.21 pmol at 12 h. this radioactive peak; however, on further digestion with BAP, there is again a peak shift with the tritium radioactivity comigrating with FdUrd (data not shown).

DISCUSSION
Our previous studies monitoring the incorporation of FUra into the nucleic acids of human tumor cells by cesium sulfate gradient centrifugation demonstrated a significant amount of tritium radioactivity in the RNA and DNA fractions2 (18). This suggested that FUra could incorporate into DNA and preliminary studies with these cell lines indicated that we were detecting FdUrd residues. In order to optimize conditions, we have employed ["HIFdUrd and have monitored the incorporation of this fluorinated nucleoside into the nucleic acids of L1210 cells. Our results demonstrate that the incorporation of tritium radioactivity in both the RNA and DNA zorporation in L1210 DNA 8887 fractions is dependent upon drug concentration and time of exposure.
We have previously demonstrated that the tritium radioactivity detectable in the RNA fraction of the cesium sulfate gradient represents incorporation of [3H]FUra.2 Similar results were obtained with the RNA fraction of L1210 cells labeled with ["HIFdUrd (data not shown).
Analysis of the DNA fraction after digestion to nucleosides demonstrates the co-migration of tritium radioactivity with FdUrd and not with other deoxynucleosides, including dUrd. Furthermore, although the purified DNA fraction might be contaminated with RNA in the form of hybrid structures or primers (17), ["HIFdUrd detectability was unaltered after treating the DNA with RNase A or alkali which would remove any RNA contamination. The tritium on position 6 of ["HIFdUrd is alkali labile (13); however, under appropriate conditions, alkali treatment can be used to digest any contaminating RNA without removing significant amounts of radioactivity from the labeled DNA strand.
Further analysis of the labeled DNA fraction by digestion to 5"nucleotides resulted in tritium radioactivity co-migrating with 5'-FdUMP. The subsequent shift of this radioactivity to co-migration with FdUrd after BAP treatment lends further support to the presence of FdUrd residues in DNA. A similar analysis of the labeled DNA by digestion to 3'-nucleotides also demonstrates the presence of FdUrd in DNA and identifies their internucleotide position.
Although FdUrd residues have been detected in bacterial DNA (14), this finding has never been established for eukaryotic DNA. Previous attempts to identify fluorinated derivatives of uracil in mammalian DNA have employed low specific activity [I4C]FUra (9). It is possible that using labeled compounds of low specific activity might have prevented the identification of FdUrd in DNA, while our studies have employed higher specific activity tritium-labeled derivatives. Furthermore, studies showing that FUra is excised from DNA by uracil DNA-glycosylase and that fluorodeoxyuridine-triphosphate is hydrolyzed by nucleotide hydrolase suggest that cell lines may vary in their ability to incorporate FdUrd in DNA.
The significance of misincorporating FdUrd in DNA presently remains unclear; however, incorporation of uracil in DNA leads to the production of small DNA fragments (17) and a similar effect with FdUrd might produce lethal cellular events. The relationship between FdUrd incorporation in DNA and cytotoxicity will be explored in subsequent studies. It will also now be of interest to determine the rate of excision of FdUrd residues from DNA in different cell lines. The comparison of tumor and normal cell repair of FdUrd misincorporation might provide a basis for the selectivity of this agent against malignant cells.