Accumulating evidence suggests an important role of interleukin-8 (IL-8) in Helicobacter pylori infectionassociated chronic atrophic gastritis and peptic ulcer. We observed in this study that a gastric cancer-derived cell line, MKN45, produced a massive amount of IL-8 upon coculture with live H. pylori but not with killed H. pylori, H. pylori culture supernatants, or live H. pylori separated by a permeable membrane, indicating that IL-8 production requires a direct contact between the cells and live bacteria. Moreover, the tyrosine kinase inhibitor herbimycin but neither a protein kinase C inhibitor (staurosporine) nor a protein kinase A inhibitor (H89) inhibited IL-8 production by MKN45 cells cocultured with live bacteria, suggesting the involvement of a tyrosine kinase(s) in H. pylori-induced IL-8 production. In addition, coculture of H. pylori induced IL-8 mRNA expression in MKN45 cells and an increase in luciferase activity in cells which were transfected with a luciferase expression vector linked with a 5-flanking region of the IL-8 gene (bp ؊133 to ؉44), indicating that the induction of IL-8 production occurred at the transcriptional level. This region contain three cis elements important for induction of IL-8 gene expression: AP-1 (؊126 to ؊120 bp), NF-IL6 (؊94 to ؊81 bp), and NF-B (؊80 to ؊70 bp) binding sites. Mutation of the NF-B binding site abrogated completely the induction of luciferase activity, whereas that of the AP-1 site partially reduced the induction. However, mutation of the NF-IL6 binding site resulted in no decrease in the induction of luciferase activity. Moreover, specific NF-B complexes were detected in the nuclear proteins extracted from MKN45 cells which were infected with H. pylori. Collectively, these results suggest that H. pylori induced the activation of NF-B as well as AP-1, leading to IL-8 gene transcription.

These findings suggest that the differences observed among ELISA test results may be due principally to differences between the profiles of antigen coated on plates for the assays, rather than to differences between antibodies in serum and urine. The urine-based ELISA (URINELISA H. pylori) developed in this study is very accurate and would be useful for screening H. pylori infection as an alternative to serum ELISAs.

The mechanism of 2Ј-C-cyano-2Ј-deoxy-1-␤-D-arabino-pentofuranosylcytosine (CNDAC) action was investigated in human lymphoblastoid CEM cells and myeloblastic leukemia ML-1 cells. CNDAC was metabolized to its 5Ј-triphosphate and incorporated into DNA, which was associated with inhibition of DNA synthesis. After incubation of cells with [ 3 H]CNDAC, metabolites were detected in 3Ј35Ј phosphodiester linkage and at the 3Ј terminus of cellular DNA. Specific enzymatic hydrolysis of DNA demonstrated that the parent nucleoside and its 2Ј-epimer 2Ј-C-cyano-2Ј-deoxy-2-ribo-pentofuranosylcytosine accounted for approximately 65% of the total analogs incorporated into DNA and essentially all of the drug in the 3Ј35Ј phosphodiester linkage. In contrast, all detectable radioactivity at 3Ј termini was associated with 2Ј-C-cyano-2Ј,3Ј-didehydro-2Ј,3Ј-dideoxycytidine. This de facto DNA chain-terminating nucleotide arises from an electronic characteristic and cleavage of the 3Ј-phosphodiester bond subsequent to the addition of a nucleotide to the incorporated CNDAC moiety by ␤-elimination, a process that generates a single strand break in DNA. Investigation of the biological consequences of these actions indicated that, after incubation with cytostatic concentrations of CNDAC, cell cycle progression was delayed during S phase, but that cells arrested predominantly in the G 2 phase. This differed from the S phase-arresting actions of ara-C and gemcitabine, other deoxycytidine analogs that inhibit DNA replication but do not cause strand breaks. Thus, once incorporated into DNA, the CNDAC molecule appears to act by a dual mechanism that 1) delays the progress of further DNA replication, but 2) upon addition of a deoxynucleotide results in the conversion of the incorporated analog to a de facto DNA chain terminator at the 3Ј terminus of a single strand break. It is likely that DNA strand breaks trigger cell cycle arrest in G 2 .

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Abstract2V -C -cyano-2V -deoxy-1-B-D-arabino -pentofuranosylcytosine (CNDAC) is a nucleoside analogue with a novel mechanism of action that is currently being evaluated in clinical trials. Incorporation of CNDAC triphosphate into DNA and extension during replication leads to single-strand breaks directly caused by B-elimination. These breaks, or the lesions that arise from further processing, cause cells to arrest in G 2 . The purpose of this investigation was to define the molecular basis for G 2 checkpoint activation and to delineate the sequelae of its abrogation. Cell lines derived from diverse human tissues underwent G 2 arrest after CNDAC treatment, suggesting a common mechanism of response to the damage created. CNDAC-induced G 2 arrest was instituted by activation of the Chk1-Cdc25C-Cdk1/cyclin B checkpoint pathway. Neither Chk2, p38, nor p53 was required for checkpoint activation. Inhibition of Chk1 kinase with 7-hydroxystaurosporine (UCN-01) abrogated the checkpoint pathway as indicated by dephosphorylation of checkpoint proteins and progression of cells through mitosis and into G 1 . Cell death was first evident in hematologic cell lines after G 1 entry. As indicated by histone H2AX phosphorylation, DNA damage initiated by CNDAC incorporation was transformed into double-strand breaks when ML-1 cells arrested in G 2 . Some breaks were manifested as chromosomal aberrations when the G 2 checkpoint of CNDAC-arrested cells was abrogated by UCN-01 but also in a minor population of cells that escaped to mitosis during treatment with CNDAC alone. These findings provide a mechanistic rationale for the design of new strategies, combining CNDAC with inhibitors of cell cycle checkpoint regulation in the therapy of hematologic malignancies. (Cancer Res 2005; 65(15): 6874-81)

Design, synthesis, and tumor cell growth inhibitory effects of 2'-C-cyano-2'-deoxy-1-beta-D-arabinofuranosyl derivatives of cytosine (1i, CNDAC), thymine (6a), uracil (6c), and adenine (6d) have been described. The synthesis of the target compounds was achieved from the corresponding 2'-keto nucleosides 2a-d. Cyanohydrins of 2a-d were converted to thionocarbonates, which were deoxygenated to give the desired 2'-beta-cyano-2'-deoxy derivatives 5a-d, followed by deprotection to furnish the target nucleosides. Of these nucleosides, CNDAC was the most potent inhibitor of cell growth with an IC50 value of 0.53 microM against L1210 cells. In vitro cytotoxicity of CNDAC against human tumor cell lines was also examined; compared with that of 1-beta-D-arabinofuranosylcytosine (ara-C) and 5-fluorouracil (5-FU), CNDAC was more cytotoxic to several cell lines refractory to ara-C. The in vivo effect of CNDAC on M5076 mouse reticulum cell sarcoma was very strong; 99% tumor volume inhibition on day 20 was achieved when it was administrated orally on days 1, 4, 7, 10, 13, and 16 at a dose of 400 mg/kg/day, while 5'-deoxy-5-fluorouridine (5'-DFUR) and 5-FU caused only 50% inhibition at a dose of 500 mg/kg/day and 28% inhibition at a dose of 50 mg/kg/day, respectively, on the same schedule. These results indicated that CNDAC may have potential as a new antineoplastic agent with a broad antitumor spectrum.

We have designed 2'-C-cyano-2'-deoxy-1-beta-D-arabino- pentofuranosylcytosine (CNDAC) as a potential mechanism-based DNA-strand-breaking nucleoside, which showed potent tumor cell growth inhibitory activity against various human tumor cell lines in vitro and in vivo. When measuring the pKa of the 2' alpha-proton of CNDAC, we found that CNDAC epimerized to 2'-C-cyano-2'-deoxy-1-beta-D-ribo-pentofuranosylcytosine (CNDC) with concomitant degradation of both CNDAC and CNDC to cytosine and 1,4-anhydro-2-C-cyano-2-deoxy-D-erythro-pent-1- enitol. Kinetic analysis of these reactions showed that abstraction of the acidic 2'-proton of CNDAC and CNDC initiated the reactions, which quickly reached an equilibrium. In the equilibrium, a concentration ratio of CNDAC and CNDC was about 3:5. Concomitant degradation of these nucleosides was found to be rather slow. Deuterium incorporation experiments with CNDAC in a D2O buffer suggested the mechanism of the beta-elimination reactions is an E1cB type. These epimerization and degradation reactions were found even in neutral conditions (pH 7.5) and also occurred in RPMI 1640 cell culture medium. The discovery of which nucleoside possesses the predominate tumor cell growth inhibitory activity was important. While both nucleosides showed potent tumor cell growth inhibitory activity against three human tumor cell lines (colon carcinoma WiDr, small cell lung carcinoma SBC-5, and stomach carcinoma MKN-74 cells) in 48 h of incubation, in 20 min of incubation, CNDAC was 11-50 times more effective than CNDC. In vivo antileukemic activity of these nucleosides against a mouse P388 model, CNDAC was obviously superior to CNDC.(ABSTRACT TRUNCATED AT 250 WORDS)

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