A series of monoclonal antibodies has been prepared against the base excision repair enzyme uracit DNA glycosylase isolated from human placenta. Spleen cells from BALB/ c mice immunized with purified human placentaluracil DNA glycosylase were fused with either P3X63 Ag8.653 or SP2/0 myeloma cells. Hybridomas producing antibodies directed against the placental glycosylase were identified in an enzyme-linked immunosorbent assay. Each positive hybridoma was cloned twice by limit dilution and tested for anti-glycosylase activity in an enzyme immunoprecipitation assay. Each of the four clones examined in detail precipitated enzyme activity in an immunoprecipitation reaction only in the presence of rabbit anti-mouse IgG as a second antibody. No anti-uracil DNA glycosylase activity was observed in a spontaneous hybridoma used as a control. Each monoclonal antibody immunoprecipitated uracil DNA glycosylases isolated from several human tissues. Partial crossreactivity was observed with rat liver glycosylase and with a hamster enzyme. In contrast, no crossreactivity was observed with yeast or Escherichia coli glycosylase. Glycerol gradient sedimentation analysis demonstrated that one of the antibodies bound to the glycosylase at a site that did not diminish its catalytic activity. A second monoclonal antibody bound at a determinant that affected catalytic activity. Analysis of antibody-glycosylase interactions suggests that human cells contain antigenically distinct glycosylase species that may be encoded by individual uracil DNA glycosylase genes. The potential use of these monoclonal antibodies in studies examining the regulation of.glycosylase isoenzymes during cell proliferation in normal human cells and in cells from cancer-prone individuals is considered.Recent studies have indicated that there is a correlation between the proliferative state of a eukarvotic cell and the capacity of that cell to repair DNA after chemical or physical insult. Increases in the.repair capacity of proliferating cells above basal levels present in quiescent cells have been observed for all DNA repair pathways that have been examined. These have included nueleotide excision repair after UV irradiation (1-6) or after exposure to N-acetoxyacetylaminofluorene (2, 6), base excision repair after exposure to methyl methanesulfonate (5, 6) or to sodium bisulfite (5), and repair after exposure to ionizing radiation (7). This enhancement of repair capacity has been observed with both asynchronous and synchronous cell populations, using various protocols to quantitate DNA repair. The in vitro quantitation of individual DNA repair enzyme activities during cell proliferation demonstrated that increases in the spe-

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Macrocyclic polyethers (crown ethers) are a family of compounds that possess the ability to complex with and transport metal ions across membranes. Because of their unique ionophoric characteristic, they have wide application in industry and research, chemistry and biology. In the current investigation the relationship between heavy metal mutagenesis and crown ether co-mutagenicity and/or antimutagenicity in mammalian cells has been examined using sister chromatid exchange (SCE) as the cytogenetic end point. Chinese hamster ovary cells were treated with lead or cadmium, with and without selected crown ethers. Several genotoxic end points, including SCEs were scored and statistically compared. We report here that most of the crown ethers studied had little or no influence on lead- or cadmium-induced SCEs or chromosome aberrations. On the other hand, the substituted crown ether dicyclohexyl 21-crown-7 significantly decreased both spontaneous and metal-induced SCE frequencies, suggesting that this crown ether may possess antimutagenic activity.

Apurinic/apyrimidinic endonuclease (APE), an essential DNA repair enzyme, initiates the base excision repair pathway by creating a nick 5′ to an abasic site in double‐stranded DNA. Although the Chinese hamster ovary cells remain an important model for DNA repair studies, the Chinese hamster APE (chAPE1) has not been studied in vitro in respect to its kinetic characteristics. Here we report the results of a kinetic study performed on cloned and overexpressed enzyme in sf9 cells. The kinetic parameters were fully compatible with the broad range of kinetic parameters reported for the human enzyme. However, the activity measures depended on the time point of the culture. We applied inductivity coupled plasma spectrometry to measure the phosphorylation level of chAPE1. Our data showed that a higher phosphorylation of chAPE1 in the expression host was correlated to a lower endonuclease activity. The phosphorylation of a higher activity batch of chAPE1 by casein kinase II decreased the endonuclease activity, and the dephosphorylation of chAPE1 by lambda phosphatase increased the endonuclease activity. The exonuclease activity of chAPE1 was not observed in our kinetic analysis. The results suggest that noticeable divergence in reported activity levels for the human APE1 endonuclease might be caused by unaccounted phosphorylation. Our data also demonstrate that only selected kinases and phosphatases exert regulatory effects on chAPE1 endonuclease activity, suggesting further that this regulatory mechanism may function in vivo to turn on and off the function of this important enzyme in different organisms.

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