A 3'-5' exonuclease in human leukemia cells: implications for resistance to 1-beta -D-arabinofuranosylcytosine and 9-beta -D-arabinofuranosyl-2-fluoroadenine 5'-monophosphate. (1/101)

A 3'-5' exonuclease that excises the nucleotide analogs 1-beta-d-arabinofuranosylcytosine monophosphate and 9-beta-d-arabinofuranosyl-2-fluoroadenine 5'-monophosphate incorporated at 3' ends of DNA was purified from the nuclei of: 1) primary human chronic lymphocytic leukemia cells, 2) primary and established human acute myeloblastic leukemia cells, and 3) lymphocytes obtained from healthy individuals. The activity of this nuclear exonuclease (exoN) is elevated approximately 6-fold in 1-beta-d-arabinofuranosylcytosine-resistant leukemia cells as compared with drug-sensitive cells, and it differs between two healthy individuals and among three leukemia patients. exoN is a 46-kDa monomer, requires 50 mm KCl and 1 mm magnesium for optimal activity, and shows a preference for single-stranded over duplex DNA. Its physical and enzymatic properties indicate that exoN is a previously uncharacterized enzyme whose activity may confer resistance to clinical nucleoside analogs in leukemia cells.  (+info)

The role of c-Jun kinase in the apoptotic response to nucleoside analogue-induced DNA damage. (2/101)

Activation of the c-Jun NH2-terminal kinase type 1 (JNK1) signaling pathway is often associated with apoptosis. In this report, we elucidated the role of this kinase in the programmed cell death induced by the nucleoside analogue 9-beta-D-arabinosyl-2-fluoroadenine (F-ara-A). Treatment of ML-1 cells with 3 or 10 microM F-ara-A specifically killed cells in the S-phase of the population. Incorporation of F-ara-ATP, the nucleoside triphosphate of F-ara-A, into DNA resulted in the activation of JNK1 in a time- and dose-dependent fashion. Activation of JNK1 temporally preceded DNA fragmentation. When incorporation of F-ara-A into DNA was blocked by pretreatment of the cells with aphidicolin to inhibit DNA synthesis, neither JNK1 signaling nor apoptosis was evident. Furthermore, inhibition of JNK1 by treatment of the cells with forskolin or by pretreatment with an antisense oligonucleotide directed against JNK1 mRNA resulted in a decrease in F-ara-A-induced apoptosis. Finally, the JNK1 signaling pathway appeared to be upstream to that of the effector caspases in nucleoside analogue-induced apoptosis. Thus, our data strongly suggest that JNK1 is involved in transduction of F-ara-A-induced distress signals into an apoptotic response.  (+info)

Interactions between 2-fluoroadenine 9-beta-D-arabinofuranoside and the kinase inhibitor UCN-01 in human leukemia and lymphoma cells. (3/101)

Interactions between the purine analogue 2-fluoroadenine 9-beta-D-arabinofuranoside (F-ara-A) and the kinase inhibitor UCN-01 have been examined in human leukemia cells (U937 and HL-60) with respect to induction of mitochondrial damage, caspase activation, apoptosis, and loss of clonogenic survival. Simultaneous or subsequent exposure of F-ara-A-treated cells (2 microM) to UCN-01 (100 nM) resulted in a marked potentiation of apoptosis, manifested by loss of mitochondrial membrane potential (delta psi(m)), cleavage/activation of procaspase-9 and procaspase-3, DNA fragmentation, and degradation of poly-ADP(ribosyl) polymerase. Coadministration of UCN-01 with F-ara-A was also associated with diminished phosphorylation of the cdc25 phosphatase. In contrast, exposure of cells to the sequence UCN-01, followed by F-ara-A, resulted in only a modest increase in apoptotic cells. The ability of UCN-01 to potentiate F-ara-A-mediated lethality was not mimicked by the selective PKC inhibitor bisindolylmaleimide, nor did treatment of cells with UCN-01 enhance formation of F-ara-ATP or increase incorporation of [3H]F-ara-A into DNA. Enhanced apoptosis in cells exposed sequentially or simultaneously to F-ara-A and UCN-01 was accompanied by a substantial reduction in colony formation (e.g., to 0.01% of control values). Cotreatment with UCN-01 also increased F-ara-A-mediated apoptosis and loss of delta psi(m) in U937 cells ectopically expressing Bcl-2, although not to the same extent as that observed in empty-vector controls. Finally, simultaneous exposure (24 h) of malignant B lymphocytes from the pleural effusion of a patient with indolent non-Hodgkin's lymphoma to F-ara-A and UCN-01 ex vivo resulted in a striking increase in apoptosis, as determined by terminal deoxynucleotidyltransferase-mediated nick end labeling assay. These findings indicate that UCN-01 increases F-ara-A-induced mitochondrial damage and apoptosis in human leukemia cells in a sequence-dependent manner, and that these events occur in at least some primary human lymphoma cells.  (+info)

Evaluation of the combination of nelarabine and fludarabine in leukemias: clinical response, pharmacokinetics, and pharmacodynamics in leukemia cells. (4/101)

PURPOSE: A pilot protocol was designed to evaluate the efficacy of fludarabine with nelarabine (the prodrug of arabinosylguanine [ara-G]) in patients with hematologic malignancies. The cellular pharmacokinetics was investigated to seek a relationship between response and accumulation of ara-G triphosphate (ara-GTP) in circulating leukemia cells and to evaluate biochemical modulation of cellular ara-GTP metabolism by fludarabine triphosphate. PATIENTS AND METHODS: Nine of the 13 total patients had indolent leukemias, including six whose disease failed prior fludarabine therapy. Two patients had T-acute lymphoblastic leukemia, one had chronic myelogenous leukemia, and one had mycosis fungoides. Nelarabine (1.2 g/m(2)) was infused on days 1, 3, and 5. On days 3 and 5, fludarabine (30 mg/m(2)) was administered 4 hours before the nelarabine infusion. Plasma and cellular pharmacokinetic measurements were conducted during the first 5 days. RESULTS: Seven patients had a partial or complete response, six of whom had indolent leukemias. The disease in four responders had failed prior fludarabine therapy. The median peak intracellular concentrations of ara-GTP were significantly different (P =.001) in responders (890 micromol/L, n = 6) and nonresponders (30 micromol/L, n = 6). Also, there was a direct relationship between the peak fludarabine triphosphate and ara-GTP in each patient (r = 0.85). The cellular elimination of ara-GTP was slow (median, 35 hours; range, 18 to > 48 hours). The ratio of ara-GTP to its normal counterpart, deoxyguanosine triphosphate, was higher in each patient (median, 42; range, 14 to 1,092) than that of fludarabine triphosphate to its normal counterpart, deoxyadenosine triphosphate (median, 2.2; range, 0.2 to 27). CONCLUSION: Fludarabine plus nelarabine is an effective, well-tolerated regimen against leukemias. Clinical responses suggest the need for further exploration of nelarabine against fludarabine-refractory diseases. Determination of ara-GTP levels in the target tumor population may provide a prognostic test for the activity of nelarabine.  (+info)

Solution structure of an arabinonucleic acid (ANA)/RNA duplex in a chimeric hairpin: comparison with 2'-fluoro-ANA/RNA and DNA/RNA hybrids. (5/101)

Hybrids of RNA and arabinonucleic acid (ANA) as well as the 2'-fluoro-ANA analog (2'F-ANA) were recently shown to be substrates of the enzyme RNase H. Although RNase H binds to double-stranded RNA, no cleavage occurs with such duplexes. Therefore, knowledge of the structure of ANA/RNA hybrids may prove helpful in the design of future antisense oligonucleotide analogs. In this study, we have determined the NMR solution structures of ANA/RNA and DNA/RNA hairpin duplexes and compared them to the recently published structure of a 2'F-ANA/RNA hairpin duplex. We demonstrate here that the sugars of RNA nucleotides of the ANA/RNA hairpin stem adopt the C3'-endo (north, A-form) conformation, whereas those of the ANA strand adopt a 'rigid' O4'-endo (east) sugar pucker. The DNA strand of the DNA/RNA hairpin stem is flexible, but the average DNA/RNA hairpin structural parameters are close to the ANA/RNA and 2'F-ANA/RNA hairpin parameters. The minor groove width of ANA/RNA, 2'F-ANA/RNA and DNA/RNA helices is 9.0 +/- 0.5 A, a value that is intermediate between that of A- and B-form duplexes. These results rationalize the ability of ANA/RNA and 2'F-ANA/RNA hybrids to elicit RNase H activity.  (+info)

DNA repair initiated in chronic lymphocytic leukemia lymphocytes by 4-hydroperoxycyclophosphamide is inhibited by fludarabine and clofarabine. (6/101)

PURPOSE: Chronic lymphocytic leukemia (CLL) lymphocytes respond to DNA alkylation by excision repair, with the extent of repair increasing as the cells acquire resistance to alkylating agents. Because incorporation of nucleotide analogues into the repair patches elicits death signals in quiescent cells, the increased capacity for excision repair in alkylator-resistant cells could facilitate incorporation of nucleotide analogues. We hypothesized that the mechanism-based interaction of nucleoside analogues with alkylating agents could elicit greater than additive killing of CLL cells. EXPERIMENTAL DESIGN: Lymphocytes from 50 patients with CLL that were not refractory to alkylators were treated in vitro with 4-hydroperoxycyclophosphamide (4-HC) with or without prior incubation with fludarabine nucleoside (F-ara-A) or with clofarabine (Cl-F-ara-A). DNA damage repair kinetics were determined by the single-cell gel electrophoresis (comet) assay. Cytotoxicity was assessed by staining with annexin V. RESULTS: CLL lymphocytes promptly initiated and completed excision repair in response to 4-HC. A 2-h preincubation with 10 microM F-ara-A or 10 microM Cl-F-ara-A inhibited the repair initiated by 4-HC, with inhibition peaking at the intracellular concentrations of 50 microM F-ara-ATP or 5 microM Cl-F-ara-ATP. Combining 4-HC with either F-ara-A or Cl-F-ara-A produced more than additive apoptotic cell death than the sum of each alone. The increase in cytotoxicity was proportional to the initial magnitude of the DNA incision and to the extent of repair inhibition by the nucleoside analogues, suggesting close correlation between the repair inhibition and induction of cell death. CONCLUSIONS: DNA repair, which is active in CLL lymphocytes, may be a biological target for facilitating the incorporation of nucleoside analogues and increasing their cytotoxicity. Thus, the increased repair capacity associated with resistant disease may be manipulated to therapeutic advantage.  (+info)

Interferon alpha plus intermittent oral Ara-C ocfosfate (YNK-01) in chronic myeloid leukemia primarily resistant or with minimal cytogenetic response to interferon. (7/101)

BACKGROUND AND OBJECTIVES: Subcutaneous Ara-C plus interferon (IFN) produces more cytogenetic responses than IFN in chronic myeloid leukemia (CML) but a greater toxicity. The objective of this study was to determine the efficacy and tolerance of IFN plus oral Ara-C ocfosfate (YNK-01) in IFN-resistant CML patients. DESIGN AND METHODS: A phase II pilot study was conducted in 19 CML patients primarily resistant or with minimal cytogenetic response to IFN. Patients were scheduled to receive 6 monthly 14-day cycles of YNK-01 (500 mg/day), with progressive escalation if tolerated, in addition to IFN. Cytogenetic assessment was performed thereafter. RESULTS: Of the first 7 patients, 5 had severe hematologic and 5 moderate gastrointestinal toxicity; IFN was reduced in 6, YNK-01 in 5, and treatment discontinued in 2; hematologic response was achieved in 2 of the 5 evaluable patients. In the following 4 patients the Ara-C was reduced to 300 mg: 2 had severe hematologic and 2 moderate gastrointestinal toxicity; IFN and Ara-C were reduced in 2 patients and treatment discontinued in 2 due to progression or toxicity; the other 2 achieved a minor cytogenetic response, progressing in one to a major response after 6 more cycles. In 8 patients the starting Ara-C dose was 200 mg: 5 had moderate-severe hematologic and 5 mild gastrointestinal toxicity; IFN was reduced in 5, Ara-C in 1, and treatment discontinued in 1; Ara-C was increased in 7 cases; hematologic response was obtained in 4 patients, 2 of whom attained a minor and 1 a major cytogenetic response. INTERPRETATION AND CONCLUSIONS: These results provide background for future studies aimed at ascertaining the role of oral Ara-C combined with IFN or STI571 in newly diagnosed CML.  (+info)

Results of a phase II trial of a combination of oral cytarabine ocfosfate (YNK01) and interferon alpha-2b for the treatment of chronic myelogenous leukemia patients in chronic phase. (8/101)

Cytarabine ocfosfate (YNK01) is a prodrug analogue of cytarabine which is resistant to systemic deamination after oral administration. Following initial studies indicating significant anti-tumour activity of YNK01 a phase II trial was initiated in order to assess the tolerability and efficacy of a combination of this agent with interferon alpha-2b (IFN-alpha2b) in recently diagnosed chronic phase CML patients (n = 98). The treatment was subdivided into cycles consisting of 4 weeks of continuous administration of IFN-alpha-2b (3 MU/m(2)/day 1st week and then 5 MU/m(2)/day) and 14 days of oral YNK01 (600 mg/day 1st cycle). At the end of each cycle the dose of YNK01 was adjusted according to the blood count observed during the previous 4 weeks. The median time from diagnosis to inclusion in the trial was 2 months (range 6 days to 7.5 months). At 12 weeks, 62 patients (63%; 95% CI, 54-73) achieved a complete hematological response. At 24 weeks, of 98 patients, two achieved a complete cytogenetic response, 14 a partial response (16% major cytogenetic response rate; 95% CI, 9-24) and 34 a minor response; 19 patients were not evaluable for cytogenetic response. During the trial, 20 patients progressed to accelerated (6) or blastic phases (14). The median time to progression was 15 months (range 2-38 months). At 3 years the overall survival was 79% (95% CI, 70-88). Although the complete hematological response rate compared favorably with the 40% response rate previously obtained with the subcutaneous formulation of Ara-c, the cytogenetic response rate was less than expected. Most of the patients experienced side-effects and all permanently stopped YNK01. Although the combination seems attractive the initial dose of 600 mg per day is probably too high and should be reconsidered in further trials.  (+info)

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