Characterization of adenosine deaminase (ADA)-negative B-lymphoblastoid cells cocultured with ADA-positive fibroblasts

Abstract: A cell line, BAD05, derived from B lymphocytes of an adenosine deaminase (ADA; EC 3,5,4,4)-deficient patient could not proliferate in a serum-free medium containing 100 μmol/l deoxyadenosine. When BAD05 was cultured with ADA-positive fibroblasts, the proliferation of BAD05 was improved. BAD05 cell density increased when the initially mixed ratio of fibroblasts/BAD05 was 1/10 or higher, but decreased when the ratio was 1/20 or lower. Deoxyadenosine concentrations in the medium and ATP and deoxyATP (dATP) levels in the BAD05 were measured after 4 hours of coculture at initial BAD05 cell densities of 1 × 10⁵and 1 × 10⁶cells/ml. Deoxyadenosine concentrations in the medium decreased as the density of fibroblasts increased. The dATP level decreased as the mixed ratio rose. The ratio of fibroblasts/BAD05 rather than the cell density of fibroblasts had a larger effect on the dATP levels in BAD05. Under our experimental conditions, ADA-negative cells proliferated well when the ratio of ADA-positive cells/ADA-negative cells was over 1/10.     

Effects of NQO1 deficiency on levels of cyclopurines and other oxidative DNA lesions in liver and kidney of young mice

I-compounds are bulky indigenous DNA adducts that can be detected by (32)P-postlabeling. A subgroup, termed type II I-compounds, represents DNA lesions induced by oxidative stress. Several major type II I-compounds have been identified as dinucleotides containing 3'-terminal 8,5'-cyclo-2'-deoxyadenosine (cA). Levels of type II I-compounds depend on the pro-oxidant status of the cell. For example, enhanced formation of such oxidative DNA lesions in newborn rodents appears to be a consequence of incomplete development of neonatal antioxidant defense systems. We tested the hypothesis that young mice deficient in NAD(P)H:quinone oxidoreductase 1 (NQO1), an antioxidant enzyme catalyzing the detoxification of quinones and their derivatives, show increased formation of these oxidative DNA lesions. Type II I-compound levels were determined by (32)P-postlabeling in liver and kidney DNA of untreated male wild-type or NQO1-null C57BL/6 mice of different ages. NQO1 catalytic activities and contents were measured by spectrophotometric and Western blotting techniques, respectively. Elevated oxidative adduct levels including those containing cA were detected in NQO1-null compared to wild-type mice at 10, 30 and 90 days in liver and at 30 and 90 days in kidney DNA. Furthermore, there were statistically significant inverse relationships between type II I-compound levels and NQO1 activities in wild-type mice up to 30 days of age. Taken together, the results suggest that NQO1 plays an important role in attenuating endogenous oxidative DNA damage in vivo. Our results show also that type II I-compounds represent useful and sensitive biomarkers with utility in studies of oxidative DNA damage and its consequences.     

The effects of the sperm motility activators 2-deoxyadenosine and pentoxifylline used for sperm micro-injection on mouse and human embryo development

The sperm motility stimulants 2-deoxyadenosine (DOA) and pentoxifylline(PTF), used to improve the success of insemination and spermmicro-injection for low motility sperm samples, were studiedfor their effects on the developmental capacity of mouse andhuman oocytes. When human oocytes were micro-injected with spermatozoain 3 mM DOA 80% of them became blocked at the 1-cell pronuclearstage. However, when spermatozoa in 3 mM PTF were used for micro-injectionor when spermatozoa were washed to remove DOA before micro-injectiononly a few oocytes (9–10%) were blocked. Pregnancies occurredin five of 14 patients into whom cleaving embryos from all threetreatments had been transferred, indicating that once cleavagewas initiated, development of embryos occurred at expected rates.Exposure of mouse oocytes to DOA for a short period during insemination(4–6 h) or a longer period during the pronuclear cellcycle (18 – 20 h) significantly reduced cleavage beyondthe 2-cell stage, resulting in a dramatic reduction in blastocystformation. PTF did not significantly reduce mouse embryo development.Similar results were obtained for oocytes inseminated in vitroor micro-injected with a spermatozoon into the perivitellinespace. Neither DOA nor PTF increased fertilization of mouseoocytes. PTF reduced fertilization, particularly in cumulus-enclosedoocytes and oocytes micro-injected with spermatozoa in PTF.We conclude that DOA is a potent inhibitor of embryo developmentand oocytes should not be exposed to DOA. Exposure of oocytesto PTF had little effect on their subsequent development buttreatment of cauda epididymal mouse spermatozoa can reduce theirfertilizing capacity.     

Persistence of benzo[a]pyrenediolepoxide DNA adduct in mouse skin

The covalently bound products of [³H] benzo[a]pyrene (BP) were determined in the DNA isolated from the skin of mice 3 weeks after application of the carcinogen. In the relatively repidly proliferating skin the persistence of BP-DNA adducts was observed, 50% of which were unmodified nucleosides. Small amounts of (7R) BPDE I-dG adduct (5% from the initial formation) were found after 3 weeks. The minor adducts observed at 18 h after treatment were excised.     

Protection by various deoxynucleosides against deoxyadenosine-induced DNA damage in adenosine deaminase-inactivated lymphocytes

Adenosine deaminase deficiency is an inborn error resulting in immunodeficiency. The pathogenesis of the lymphopenia is not fully understood. Intracellular increases in dATP in the absence of deamination retard DNA repair in human resting lymphocytes and results in the slow accumulation of DNA strand breaks. We focused on the relationship between DNA damage and DNA precursor pools in cultures of deoxycoformycin-treated, ADA-inhibited resting lymphocytes. The addition of 10 microM deoxyadenosine led to a substantial number of DNA strand breaks within 12 h, breaks equivalent to those which occur with about 190 rad irradiation. Addition of any of the other deoxynucleosides used partially prevented this dAdo-induced DNA damage and promoted DNA repair. However, the preventive effects did not correlate inversely with intracellular dATP levels. Resting lymphocytes have very small dNTP pools. Treatment with dAdo slightly reduced dTTP and dCTP. Three kinds of deoxynucleosides, other than dAdo, restored or raised the corresponding dNTP level but the pool imbalance was only minimally corrected. Regarding the toxic effects of dAdo in ADA deficiency, not only dATP levels but also dNTP pool balance has a crucial role in the pathogenesis. Pool sizes of dTTP, dCTP, and possibly dGTP must be maintained at normal levels, if dAdo-induced DNA damage is to be avoided.     

Specificity of anti-DNA antibodies in SLE-I. Definition and gross specificity of antibody populations in human SLE plasma

Populations of anti-DNA antibodies in two SLE plasma were defined based on their patterns of reactivity in inhibition assays with single and double-stranded DNA as well as mono- and oligonucleotides. Two populations of anti-DNA antibodies were seen in both plasma tested. The first population reacted specifically with ssDNA and was inhibited by relatively low concentrations of free nucleotides indicating that it recognized the nucleotide bases in ssDNA. The second population bound both ss and ds calf thymus DNA with apparent equal affinity. The cross-reactive anti-DNA antibodies were inhibited by mononucleotides (at high concentrations) and by single-stranded oligonucleotides (average length tetranucleotides). For one of the plasma tested (PS), pBR322 plasmid DNA (54% G + C) was a significantly more effective inhibitor than calf thymus DNA (39% G + C). These results suggested that nucleotide bases contributed to dsDNA binding by cross-reactive anti-DNA antibodies.     

A Novel Therapeutic Strategy Against Monocytic Leukemia with Deoxyadenosine Analogs and Adenosine Deaminase Inhibitors

The adenosine deaminase inhibitor 2'-deoxycoformycin (dCF) significantly inhibits the proliferation of leukemia and lymphoma cells in the presence of either 2'-deoxyadenosine (dAdo) or its analog adenine arabinoside (araA). The concentration of dCF required to induce apoptosis of monocytoid leukemia cells is much lower than that required for myeloid, erythroid, or lymphoma cells. dATP effectively induces caspase-3 activation in cytosol from monocytoid cells, but not in that from non-monocytoid cells, suggesting that dATP-dependent caspase-3 activation is involved in the preferential induction of apoptosis in monocytoid leukemia cells. Athymic nude mice inoculated with human monocytoid leukemia U937 cells show significantly prolonged survival following combined treatment with dCF and araA. The clinical usefulness of the combination of adenosine deaminase inhibitor and dAdo analog is discussed.     

Long‐term safety and activity of cladribine in patients with extranodal B‐cell marginal zone lymphoma of the mucosa‐associated lymphoid tissue (MALT) lymphoma

The purine analogue 2‐chloro‐deoxyadenosine (2‐CDA, cladribine) +/? rituximab has been successfully tested in mucosa‐associated lymphoid tissue lymphoma (MALT lymphoma) patients. However, studies using cladribine in other indications have reported the potential for prolonged hematological side effects and secondary hematologic and non‐hematologic malignancies. To date, there have been no data on long‐term effects of cladribine in MALT lymphoma patients. We have analyzed a large number of 49 patients treated with cladribine at our institution 1997–2011. All patients were treated within clinical trials and had undergone a standardized follow‐up protocol minimizing a potential bias in the detection of late sequels and relapses. After a median follow‐up time of 61 months (interquartile range: 43–72) for 49 analyzed patients, 35 (71%) are alive, while 14 (29%) have died. In the entire collective, three cases (6%) of prolonged pancytopenia including manifest myelodysplastic syndrome in one patient (2%), three cases (6%) of secondary lymphoid malignancies, and five cases (10%) of non‐hematologic cancers were documented. In terms of outcome, 42/49 (86%) patients responded to cladribine‐containing treatment, and only 10/42 (24%) responding patients needed further treatment after a median time to progression of 14 months (interquartile range, 8–34). Currently, 25/35 (71%) patients being alive are in ongoing complete remission and 2/35 (6%) in ongoing stable disease, respectively. Eight patients (23%) are free of lymphoma after second‐line therapy, with the median overall survival not having been reached. Our data suggest that cladribine might be safely applied in patients with MALT lymphoma, also in terms of long‐term toxicities. These data also confirm the potential of cladribine to induce durable remissions. Copyright © 2015 John Wiley & Sons, Ltd.     

纸色谱-分光光度法测定生物转化体系中脱氧腺苷含量

目的建立纸色谱-分光光度法测定生物转化液中脱氧腺苷(dAR)含量。方法用不同极性的展开剂进行纸色谱从混合物中分离dAR,分光光度法测定分离的dAR浓度。结果测试了4种展开剂对dAR的分离效果,发现用展开剂正丁醇-异丙醇-氨水-水(3∶3∶2∶2)一次色谱可将dAR与其它成分分离。将分离并重新溶解的dAR在258 nm处测定其吸光度值,根据标准曲线即可计算出转化反应液中的产物浓度。结论该法测定结果与高效液相色谱法测定结果一致,是一种简易有效的快速测定生物转化体系中dAR含量的方法。