Substrate competition methods that were previously used to quantitate the involvement of free Cl 2 in the chloride-dependent peroxidatic reactions catalyzed by chloroperoxidase (CPO) (Libby, R. D., Shedd, A. L., Phipps, K. A., Beachy, T. M., and Gerstberger, S. M., (1992) J. Biol. Chem. 267, 1769 -1775) are extended to CPO-catalyzed halogenation reactions. Relative substrate specificities of halogen acceptor substrates (RHs) antipyrine (ap), NADH, 2-chlorodimedone (2cd), and barbituric acid (ba) are compared with previously studied peroxidatic substrates catechol (cat) and 2,4,6-trimethylphenol (tmp) in their reactions with the CPO-H 2 O 2 -Cl system versus the hypochlorite-Cl system. Studies were carried out at pH 2.75 over a chloride concentration range of 1-100 mM and at pH 4.80 over a chloride concentration range of 100 -400 mM. Competition studies involved successive pairwise comparisons of substrates of increasing enzyme specificity. The orders of specificities, ba > 2cd > ap > cat > tmp at pH 2.75 and ba > 2cd > NADH > ap > cat > tmp at pH 4.80, are the same for both the CPO-H 2 O 2 -Cl and hypochlorite-Cl systems. However, the magnitudes of the specificities are different between the two systems. In all comparisons except ap versus cat, the specificity of the CPO-H 2 O 2 -Cl system toward the preferred substrate is higher than that of the hypochlorite-Cl system. Quantitative comparisons between specificities of CPO-H 2 O 2 -Cl and hypochlorite-Cl systems indicate that at least 98% of the CPO-catalyzed halogenation reactions of ba, 2cd, NADH, and ap occur by mechanisms in which the substrate reacts directly with the enzyme. Thus, less than 2% of any of the CPO reactions could possibly involve a free oxidized halogen intermediate. All data are consistent with a mechanism in which RH binds to the CPO chlorinating intermediate (EOCl), and the chlorine atom is transferred directly from EOCl to RH. Further, the results indicate that any halogenation substrate with a higher CPO specificity than ap must also undergo direct chlorine transfer from the enzyme.These results underscore the critical need for quantitative kinetic evidence in establishing the extent of involvement of any potential reaction intermediate. 269, 7950 -7956).Recently we reported the first direct kinetic evidence for the involvement of a free oxidized chlorine intermediate, Cl 2 , in a reaction catalyzed by chloroperoxidase (chloride:hydrogen peroxide oxidoreductase, EC 1.11.1.10) (1). CPO 1 is a very versatile enzyme. In addition to catalyzing reactions characteristic of peroxidases, hydrogen peroxide-supported oxidation, and iodi-* This work was supported by NIGMS, National Institutes of Health, Grant 7 R15 GM42078-02. 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.‡ To whom correspondence should be addressed.1 The abbreviations used are: CPO, chloroperoxidase; t...

Mitomycin C (MC) is a powerful antitumor agent that targets the DNA sequence CpG. Because it is likely that this dinucleotide will contain 5-methylcytosine in vivo, we have compared the cross-linking efficiency of MC for DNA containing either 5-methylcytosine or normal cytosine embedded in random-sequence DNA oligomers. We have found that mitomycin C displays a small but significant preference for methylated DNA. Recognition of an abnormal methylation pattern in the DNA of transformed cells may therefore be one mechanism by which MC exerts its chemotherapeutic effects.

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Primary cells in cultures naturally lose replicative potential with successive subculturing until the end point of division, senescence, is reached (reviewed in references 5 and 68). The expression of simian virus 40 (SV40) T antigen allows cells to bypass natural senescence and thereby become capable of an indefinite number of cell divisions (immortalized). The expression of a ras oncogene alters the process of senescence. Transient expression of ras is mitogenic. ras expression increases the level of cyclin D in cells (13,41). Cyclin D in concert with cyclin E and their associated kinases is responsible for phosphorylating the tumor suppressor protein Rb (reviewed in reference 36). In its hypophosphorylated form, Rb binds to E2F transcription factors and represses the E2F-dependent transcription of S-phase genes (reviewed in reference 22). Rb phosphorylation releases E2F from complexes with Rb, promoting entry into S phase. However, prolonged expression of ras results in transactivation of the E2F-responsive p16 and p19ARF genes (48, 50) and acceleration of the senescence process (29, 50). Both p16 and p19ARF proteins contribute to senescence. p16 is one of a class of cyclin-dependent kinase (CDK) inhibitors which act specifically on cyclin D-dependent kinases (reviewed in reference 49). High levels of p16 prevent Rb phosphorylation and the consequent release of E2F. The p19ARF protein is an inhibitor of the oncoprotein MDM2. MDM2 forms complexes with p53 and targets p53 for degradation (18,27). The formation of complexes between p19ARF and MDM2, however, promotes the rapid degradation of MDM2 instead (73). As a result, p53 accumulates and is available to transactivate the p21 WAF1,CIP1 gene. p21 is one of a class of CDK inhibitors with broader activity and inhibits several CDKs, including cyclin D-and E-dependent kinases (69). This protein further limits Rb phosphorylation and promotes the accumulation of the hypophosphorylated, growth-suppressing form of the protein. While both p53 and p16 are essential for ras-induced senescence, genetic interruption of either pathway is sufficient to prevent the permanent G 1 arrest characteristic of senescence (29, 50) in rat cells. The requirement for p53 and p16 in ras-induced senescence is consistent with the demonstrated involvement of p53-inducible p21 and p16 in the early and late stages of natural senescence, respectively (59).The expression of the SV40 large T antigen allows primary rodent cells to overcome ras-induced senescence (reviewed in references 3, 33, and 53). Two regions of the large T antigen, amino acids 1 to 147 (T1-147) and amino acids 251 to 708 (T251-708), were previously shown to independently cooperate with a ras oncogene to produce dense foci on monolayers of rat embryo fibroblasts (REF) (6). Each of these regions contains activities that could be involved in bypassing ras-induced senescence. N-terminal segments of T antigen that retain the capacity to bind and inactivate hypophosphorylated Rb (21,29,30) would be expected to abrogate ras-induced...