Faculty Bibliography

5-Hydroxymethyl-2'-deoxyuridine (HmdUrd), a cytotoxic analogue of thymidine, has been proposed for use as an anticancer agent. HmdUrd is incorporated into DNA and then removed at a rate of 30-40% per day. The removal of HmdUrd from DNA has been attributed to the action of 5-hydroxymethyluracil-DNA glycosylase (HmUra-DNA glycosylase). We demonstrated the release of [3H]HmUra into the growth medium of V79 Chinese hamster cells that had incorporated [3H]HmdUrd into their DNA. The amount of [3H]HmUra recovered from the growth medium was equal to the amount of [3H]HmdUrd lost from DNA. These experiments confirmed that the initial step of repair of HmUra in DNA was mediated by DNA glycosylase activity. A combination of HmUra and HmdUrd resulted in increased uptake of HmdUrd by cells and increased cytotoxicity. The increased incorporation of HmdUrd into DNA was not due to inhibition of repair. 3-Aminobenzamide, an inhibitor of poly(ADP-ribose) synthesis, was cytotoxic to cells which incorporated and repaired HmdUrd. The extent of toxicity was directly related to the number of HmUra residues in DNA. HeLa cells, known to be resistant to the toxic effects of HmdUrd, do not incorporate HmdUrd into their DNA. HeLa cells were resistant to the toxic effects of 3-aminobenzamide, confirming that the absence of HmdUrd in their DNA was not due to an accelerated rate of repair. These experiments indicate that the potential therapeutic antineoplastic properties of HmdUrd may be enhanced by using HmUra to increase the incorporation of HmdUrd into DNA and 3-aminobenzamide to interfere with repair of HmUra in DNA

5-Hydroxymethyluracil (HmUra) is formed from thymine in DNA through the action of ionizing radiation or reactive oxygen species generated by activated leukocytes. HmUra is removed from DNA by a specific DNA glycosylase, suggesting that it is also formed from endogenously generated reactive oxygen species and that its formation in DNA is potentially deleterious. To determine whether HmUra residues in DNA are mutagenic, hamster V79 cells were grown in the presence of 5-hydroxymethyl-2'-deoxyuridine (HmdUrd) which is incorporated into DNA, and mutagenicity at the ouabain- and thioguanine-resistant loci was determined. Levels of substitution ranged from 1/500 to 1/5,000 HmUra residues/thymine residues. There was slight mutagenicity at the thioguanine-resistant locus but none at the ouabain-resistant locus. The mutagenicity of HmdUrd, expressed as a function of HmUra substitution in DNA, was 1/30,000 in the hypoxanthine-guanine-phosphoribosyltransferase target gene. This low frequency indicates that the oxidation of thymine to HmUra in a preexisting AT base pair does not contribute significantly to the mutagenicity of ionizing radiation, because the yield of HmUra formed in DNA at mutagenic doses of radiation is too low. To determine whether repair of HmUra might be inhibited by ionizing radiation, cells were grown in medium containing HmdUrd and exposed to as much as 5 Gy of gamma-irradiation, and the removal of HmUra from DNA was measured. No inhibition of repair was noted. Preirradiation of cells neither accelerated the rate of repair nor raised the level of HmUra-DNA glycosylase activity, indicating that repair of HmUra was not induced by this type of oxidative stress. Although the mutagenicity of HmUra residues in DNA is low, even a rare mutation might be sufficiently deleterious to higher organisms to promote the development of HmUra-DNA glycosylase activity

Many DNA repair enzyme activities are present in both prokaryotic and eukaryotic organisms. Among these are DNA exo- and endonucleases and DNA glycosylases which remove oxidatively damaged portions of the DNA molecule, thereby initiating excision-repair. The existence of these enzymes may be taken as evidence that cellular DNA is continuously subject to endogenous oxidative stress. Many of the lesions introduced by ionizing and ultraviolet radiation are identical to those introduced into DNA by reactive oxygen species generated by activated white cells, and are substrates for the repair enzymes. The chemical nature of the lesions, their biologic effects, and the mechanism of their repairability are described

The enzymatic release of 5-hydroxymethylcytosine from T2 bacteriophage DNA was effected by an extract of calf thymus. Like the previously described 5-hydroxymethyluracil DNA glycosylase, 5-hydroxymethylcytosine DNA glycosylase was not detectable in bacterial extracts. The phylogenetic distribution of these activities indicates that their primary function is the maintenance of methylcytosine residues in differentiated tissue

V79 cells incorporated 5-hydroxymethyl-2'-deoxyuridine (HmdUrd) into their DNA linearly over a wide range of concentrations and time. Cells grew normally when 0.03% of thymidine residues were replaced with HmdUrd. At this level of substitution, 5-hydroxymethyluracil (HmUra) was removed from DNA at a rate of 30-40%/24 h. Concentrations of HmdUrd in the growth medium which produced higher levels of substitution reduced survival and caused cells to delay their transit through S phase. However, the treatment of HmdUrd-containing cells with 3-aminobenzamide caused extensive cell death. At levels of HmdUrd substitution compatible with near 90% survival, the addition of 3-aminobenzamide, an inhibitor of poly (adenosine diphosphoribose) synthesis, killed over 90% of the cells. This toxicity was not due to inhibition of the removal of HmUra from DNA. Cells killed by this combination of agents arrested in the G2 phase of the cell cycle. We conclude that the toxicity of HmdUrd resulted primarily from the repair of the HmUra residue in DNA and not from any intrinsic toxicity of the HmUra residue itself. We also conclude that the cytotoxicity of 3-aminobenzamide resulted from interference with the completion of DNA repair following base (HmUra) excision. Since HmUra is also formed in DNA through the action of ionizing radiation, it may be among the components of radiation-induced DNA damage which sensitizes cells to 3-aminobenzamide

Ionizing radiation causes formation of heterogeneous types of damage to DNA. Among those, 5-hydroxymethyl-2'-deoxyuridine (HMdU) was identified as a major thymidine derivative in gamma-irradiated HeLa cells [G.W. Teebor, K. Frenkel and M.S. Goldstein (1984) Proc. Natl. Acad. Sci. (U.S.A.), 81, 318-321]. We report here that HMdU is a strong inducer of lambda prophage in Escherichia coli WP2s(lambda) and is highy mutagenic in Salmonella typhimurium. HMdU causes his+ revertants in strains TA100, which reverts predominantly by base-pair substitution at G-C sites, and TA97, which reverts mainly by frameshift mutation at G-C sites. It does not cause reversion in TA98, another frameshift-sensitive strain, nor in strains TA1535 and TA1537. Of those tested, only the last two strains do not contain pkM101, a plasmid which enhances mutagenic effects of ionizing radiation. HMdU also causes reversion in strains TA102 and TA104, which detect oxidative damage and can revert by base-pair substitution at A-T base pairs at the hisG428 site. We show that HMdU can be incorporated into DNA of TA100 and that, in addition to causing point mutations, it causes suppressor mutations as well. The ability of HMdU to induce lambda prophage and its strong mutagenicity in Salmonella typhimurium provide evidence that the presence of HMdU in DNA is biologically significant and may play a major role in the genetic consequences of ionizing radiation and other types of oxidative damage

To determine the prevalence of the repair enzyme HMU-DNA glycosylase we assayed its activity in whole cell extracts of several bacterial species, the eukaryotic yeast Saccharomyces cerevisiae, mammalian cell lines and murine tissue. Enzyme activity was constitutively present in murine, hamster and human cell lines. It was not inducible by exposing cells to oxidative stress from ionizing radiation or by incubating cells with the 2'-deoxynucleoside of HMU, HMdU. In murine tissue, enzyme activity was highest in brain and thymus. HMU-DNA glycosylase activity was not detectable in bacteria or yeast nor could activity be detected after exposure of cells to H2O2. These results suggest that, in contrast to other DNA-repair enzymes, HMU-DNA glycosylase is a differentiated function limited to higher eukaryotic organisms

An N-glycosylase activity that released cis-[3H]-5,6-dihydroxy-5,6-dihydrothymine (thymine glycol, TG) from chemically oxidized poly(dA-[3H]dT) was unambiguously characterized both in extracts of HeLa cells and in purified Escherichia coli endonuclease III. This was accomplished by use of microderivatization procedure that quantitatively converted cis-TG to 5-hydroxy-5-methylhydantoin (HMH). The reaction products were analyzed by high-pressure liquid chromatography before and after derivatization by using cis-[14C]TG and [14C]HMH, which had been independently synthesized, as reference compounds. This technique facilitated construction of a v/[E]t plot for the enzyme activity in HeLa cells, permitting estimation of its specific activity. The results obtained prove the existence of both human and bacterial N-glycosylase activities that effect removal of TG from DNA

A technique for determining the relative content of each of the diastereoisomers of cis thymidine glycol (dTG) in DNA exposed to ionizing radiation has been developed. [3H]thymidine DNA was gamma-irradiated, digested to 2'-deoxyribonucleosides, authentic [14C] (+, -) cis dTG added to the digestate and the mixture resolved by HPLC. 3H fractions coeluting with [14C] (+, -) dTG were collected and acetylated. The acetoxy derivatives of (+) and (-) cis dTG were easily resolved by a second HPLC analysis and their absolute configuration determined by NMR and mass spectroscopies. We have constructed a dose-response curve for formation of each isomer in gamma-irradiated DNA and shown that they are formed in equal amounts. This technique may be used to determine the relative formation of cis dTG isomers in DNA resulting from other oxidative stresses and whether repair of these is influenced by their configuration