The correlation of salivary telomere length and single nucleotide polymorphisms of the ADIPOQ, SIRT1 and FOXO3A genes with lifestyle-related diseases in a Japanese population.

BACKGROUND: It has been reported that genetic factors are associated with risk factors and onset of lifestyle-related diseases, but this finding is still the subject of much debate. OBJECTIVE: The aim of the present study was to investigate the correlation of genetic factors, including salivary telomere length and three single nucleotide polymorphisms (SNPs) that may influence lifestyle-related diseases, with lifestyle-related diseases themselves. METHODS: In one year at a single facility, relative telomere length and SNPs were determined by using monochrome multiplex quantitative polymerase chain reaction and TaqMan SNP Genotyping Assays, respectively, and were compared with lifestyle-related diseases in 120 Japanese individuals near our university. RESULTS: In men and all participants, age was inversely correlated with relative telomere length with respective p values of 0.049 and 0.034. In men, the frequency of hypertension was significantly higher in the short relative telomere length group than in the long group with unadjusted p value of 0.039, and the difference in the frequency of hypertension between the two groups was of borderline statistical significance after adjustment for age (p = 0.057). Furthermore, in men and all participants, the sum of the number of affected lifestyle-related diseases, including hypertension, was significantly higher in the short relative telomere length group than in the long group, with p values of 0.004 and 0.029, respectively. For ADIPOQ rs1501299, men's ankle brachial index was higher in the T/T genotype than in the G/G and G/T genotypes, with p values of 0.001 and 0.000, respectively. For SIRT1 rs7895833, men's body mass index and waist circumference and all participants' brachial-ankle pulse wave velocity were higher in the A/G genotype than in the G/G genotype, with respective p values of 0.048, 0.032 and 0.035. For FOXO3A rs2802292, women's body temperature and all participants' saturation of peripheral oxygen were lower in the G/T genotype than in the T/T genotype, with respective p values of 0.039 and 0.032. However, relative telomere length was not associated with physiological or anthropometric measurements except for height in men (p = 0.016). ADIPOQ rs1501299 in men, but not the other two SNPs, was significantly associated with the sum of the number of affected lifestyle-related diseases (p = 0.013), by genotype. For each SNPs, there was no significant difference in the frequency of hypertension or relative telomere length by genotype. CONCLUSION: Relative telomere length and the three types of SNPs determined using saliva have been shown to be differentially associated with onset of and measured risk factors for lifestyle-related diseases consisting mainly of cardiovascular diseases and cancer.

Chloroquine Inhibits Self-Renewal of Blast Progenitors Synergistically With Phytochemicals or Nonsteroidal Anti-inflammatory Drugs in Hematological Malignant Cell Lines.

BACKGROUND: This study examined whether and how chloroquine inhibits blast progenitor self-renewal (SR) synergistically with phytochemicals or nonsteroidal anti-inflammatory drugs in seven hematological malignant cell lines. MATERIALS AND METHODS: Vitamin C, resveratrol, cyclo-oxygenase inhibitor NS-398 and indomethacin heptyl ester (Ind) were added to cell culture with or without 3 µM chloroquine. RESULTS: Chloroquine synergistically inhibited blast colony formation in methylcellulose with vitamin C, resveratrol, NS-398 and Ind in one, two, none and one cell lines, respectively, in a total of four out of 28 conditions. Chloroquine synergistically inhibited blast progenitor SR in suspension with vitamin C, resveratrol, NS-398 and Ind in four, six, one and five cell lines, respectively, in a total of 16 out of 28 conditions. In contrast, chloroquine abolished SR inhibition by another agent in four out of 28 conditions. CONCLUSION: Chloroquine exerted a marked synergistic inhibition of blast progenitor SR, but not blast colony formation.

Effects of Nonsteroidal Anti-inflammatory Drugs on the Self-renewal Capacity of Blast Progenitors in Hematological Malignancies.

BACKGROUND: With our newly established long-term suspension culture, we investigated the effects of nonsteroidal anti-inflammatory drugs (NSAIDs) on the self-renewal capacity of blast progenitors in seven hematological malignant cell lines. MATERIALS AND METHODS: Cyclo-oxygenase inhibitors NS-398 (NS) or indomethacin heptyl ester (indomethacin) at 30 µM was added to the cell culture. In U-937 cells, indomethacin was added at 0.3 or 3 µM. RESULTS: In all cell lines, the agents significantly and markedly inhibited blast colony formation (BCF) and telomerase activity, respectively. Significant exponential clonogenic cell growth was noted under all 23 conditions with or without the agent. The relative slope (SLP) of the line (SLPagent/SLPcontrol) reflects the level of self-renewal induced by the agent and self-renewal was inhibited (relative SLP at 0.9 or below) in four out of 16 conditions, including in U-937 cells treated with 0.3 or 3 µM indomethacin, in Daudi cells treated with indomethacin and in U-266 cells treated with NS. Indomethacin enhanced senescence, necrosis and apoptosis in U-937 and Daudi cells, whereas NS reduced apoptosis in U-937 cells and had no effect on senescence, necrosis and apoptosis in Daudi cells. In U-266 cells, NS enhanced senescence and necrosis, whereas indomethacin reduced apoptosis. There was no significant correlation between the control of BCF and relative SLP. CONCLUSION: NSAIDs enhance or reduce stress responses, inhibit telomerase activity and differentially regulate BCF and self-renewal.

Establishment of a quenching probe method for detection of NPM1 mutations in acute myeloid leukemia cells.

Nucleophosmin (NPM1) mutations, generally consisting of a four base-pair insertion, are present in ~60% of all cytogenetically normal acute myeloid leukemia (AML) cases. The mutation is clinically significant as an important prognostic factor. Direct sequencing is the current standard method of mutation detection, however, it is quite costly and time consuming. The present study aimed to establish a highly sensitive quenching probe (QP) method to detect NPM1 mutations efficiently. Melting curve analysis was performed using a QP, following polymerase chain reaction for amplification of the involved region of the gene. The curve derived from the fluorescent intensity with respect to the temperature of OCI/AML3, a heterozygous NPM1 mutant AML cell line, was W-shaped with melting peaks at 61°C and 68°C. That of M-07e, the homozygous wild type cell line, was V-shaped with a melting peak at 68°C. Thus, the curve derived from the mutant allele was easily discriminated from that of the wild-type allele. The mutant allele was detected in concentrations as low as 3% as determined by a subsequent sensitivity study. With a short testing time and a high sensitivity, this assay was applicable for NPM1-mutated AML patient samples and is appropriate for screening NPM1 mutations. It does require further examination as to whether it would be useful as a detection method for other mutant alleles since NPM1 mutations may consist of 61 known types of mutant sequences. To the best of our knowledge, this is the first report describing the QP method for the detection of NPM1 mutations.

Promotion of the self-renewal capacity of human leukemia cells by sonic hedgehog protein.

BACKGROUND: Hedgehog (Hh) signaling is involved in cancer cell growth. However, the effects of Hh stimulation on leukemia cells are unknown. MATERIALS AND METHODS: The effects of recombinant sonic Hedgehog (Shh) protein on the in vitro growth of one B-lymphoma and four myeloid leukemia cell lines were examined. RESULTS: Shh stimulation had no significant effect on the short-term growth of whole cell populations in any of the five cell lines. However, Shh promoted clonogenic cell recovery after suspension culture, suggesting promotion of leukemia stem or progenitor cell amplification in three cell lines. The lack of Hh receptors in one cell line and endogenous Shh expression in another were possible reasons for the lack of effects of Shh in these cases. CONCLUSION: These results suggest that Shh stimulation promotes the self-renewal capacity of leukemia stem cells in some cell lines. Inhibition of Hh signaling could represent a novel therapeutic approach in leukemia.

Promotion of the self-renewal capacity of human acute leukemia cells by Wnt3A.

BACKGROUND: Wnt/beta-catenin signaling is involved in the growth of various types of cancer cells. Wnt3A has been reported to promote the self-renewal of hematopoietic stem cells. MATERIALS AND METHODS: The effects of recombinant Wnt3A protein on the in vitro growth of four acute myeloid leukemia (AML) and four acute T-lymphoblastic leukemia (T-ALL) cell lines was examined. RESULTS: Wnt3A stimulation either had no effect on, or slightly suppressed, the short-term growth of these cell lines. In three cell lines, Wnt3A promoted clonogenic cell recovery after suspension culture, suggesting the promotion of the self-renewal capacity of leukemic stem or progenitor cells. Immunoblot analysis showed that Wnt3A stimulation reduced phosphorylated beta-catenin and increased beta-catenin in these cells, indicating that Wnt3A stimulation activated Wnt/beta-catenin signaling. CONCLUSION: Wnt3A stimulation did not promote the growth of whole cell populations, but did promote the self-renewal of leukemic stem/progenitor cells in some AML and T-ALL cell lines.

Lactate dehydrogenase-linked immunoglobulin Akappa in a patient with nonimmune chronic idiopathic neutropenia.

A 39-year-old patient with cervical cancer, stage Ia, was successfully treated by total hysterectomy. Then, after sustained neutropenia for more than 4 years and coincident with its exacerbation, the serum lactate dehydrogenase (LD) level started to elevate and reached a plateau. A test for antineutrophil antibody was negative and LD-3-linked IgAkappa, which may be responsible for high LD activity, was confirmed. The absolute number of blood NK cells was reduced, and a diagnosis of nonimmune chronic idiopathic neutropenia of adult was made. The successive occurrence of these 3 disorders may be based on interrelated immunological abnormalities.

Serum levels of Thl/Th2 cytokines, angiogenic growth factors, and other prognostic factors in young adult patients with hemophagocytic syndrome.

Serum levels of T helper 1 (Th1)/T helper 2 (Th2) cytokines, angiogenic growth factors, and other prognostic factors were measured in 5 young adult patients with virus-associated hemophagocytic syndrome (HPS). Levels of 2 Th1 cytokines (interleukin [IL]-18 and tumor necrosis factor-alpha), 2 Th2 cytokines (IL-10 and IL-6), and 2 angiogenic growth factors (soluble intercellular adhesion molecule-1 and hepatocyte growth factor) were high in all of the patients examined, whereas those of Th1 cytokines such as IL-12 and macrophage inflammatory protein-1a were normal or low. Levels of IL-18 and IL-10 were highest in case 2, with a fatal outcome, and were lowest in case 4, with rapid recovery within 1 month. Although IFN-gamma levels were not elevated in 2 patients (cases 3 and 5), IL-18 levels were markedly high in both of these cases and the IL-6 level was highest in case 3. In contrast with the marked increase in the level of IL-10, the levels of IL-6, sIL-2R, and ferritin decreased rapidly and returned to normal within 2 months after therapy in case 3. The IL-18 level decreased somewhat, but remained elevated for 6 months, and the patient achieved a complete response within 11 months. Taken together, our findings suggest that both IL-18 and IL-10, but not IL-12, may play important roles in young adult patients with HPS via enhancing and suppressing Th1 immune responses, respectively.

Soluble transferrin receptor and its ratio to erythroblasts in bone marrow may be a new diagnostic tool to distinguish between aplastic and refractory anemia.

Myelodysplastic syndromes (MDS), especially refractory anemia (RA) are very heterogeneous diseases regarding their morphological, biological and clinical features. One important clinical problem is the difficulty of diagnosis. Soluble transferrin receptors (sTfRs) reflect the erythropoietic activity in the bone marrow (BM). To establish whether determination of serum sTfR could be useful for the differential diagnosis between RA and aplastic anemia (AA), we measured the serum sTfR concentrations, BM cellularity and BM erythroblast percentages in 14 untreated AA and 7 untreated RA patients. The serum sTfR levels of the RA patients (820.1 +/- 402.8 ng/ml) were significantly higher than those of the AA patients (491.1 +/- 195.2 ng/ml; p = 0.0207). However, the serum sTfR values of RA and AA patients also overlapped. A new index, the 'sTfR-E index' [the ratio of serum sTfR level (ng/ml) to BM cellularity (%) x BM erythroblasts (%)] is proposed, which is expected to reflect the number of transferrin receptors (TfR) on the cell membrane per BM erythroblast. The sTfR-E index values of the 7 RA patients (0.395 +/- 0.234) were significantly lower than those of the 14 AA patients (2.669 +/- 1.633; p = 0.0003). The sTfR-E index values of AA and RA patients overlapped only marginally. In conclusion, the sTfR-E index may be a useful new diagnostic tool to distinguish between AA and RA patients.

Differential response to stem cell factor and Flt3 ligand by the FAB subtype in acute myeloid leukemia clonogenic cells.

Proliferative response of blast clonogenic cells to various hematopoietic growth factors (HGF), including stem cell factor (SCF) and flt3 ligand (FL) was investigated in 100 patients with acute myeloid leukemia (AML) and chronic myelogenous leukemia (CML) in myeloid crisis (MC). The frequency of spontaneous colony formation was significantly high in CML in MC (55%) and AML French-American-British (FAB) subtype M4 (48%) compared with M2 (16%). No spontaneous colony was formed in any of the patients with M1 and M3. The frequency of proliferative response to various HGF alone and in combination according to FAB subtype and CML in MC was as follows: that to granulocyte colony-stimulating factor (G-CSF) was lowest in M1 and CML in MC (50%) compared with other FAB subtypes (>or=86%), that to granulocyte-macrophage CSF (GM-CSF) was lowest in CML in MC (44%) compared with FAB subtypes (>or=74%), and that to interleukin-3 (IL-3) was lowest in CML in MC (30%) compared with FAB subtypes (>or=78%). SCF and FL stimulated blast colony formation in 11% and 17% of patients with M3, respectively, but there was no response to both, and in 60% and 57% of patients with CML in MC, respectively, with 14% showing a response to both. The frequency of proliferative response to both SCF and FL increased in the order of M1 (33%), M2 (63%), M4-5 (95%), and M6 (100%). The results are summarized as follows: absence of spontaneous colony formation and response to HGF other than SCF and FL, designated as HGF-dependent growth (M3); spontaneous colony formation and lowest response to HGF, designated as autonomous growth (CML in MC); and spontaneous colony formation and highest response to HGF including SCF and FL, designated as autocrine growth (M4-6). M1 and M2 were intermediate between CML in MC and M4-6. The relation between in vitro growth pattern of blast clonogenic cells and prognosis in AML FAB subtype and CML in MC is discussed.

Efficacy of carboplatin with an MEP (mitoxantrone, etoposide and prednisone) regimen for relapsed and CHOP-resistant diffuse large B-cell lymphomas.

Mitoxantrone, etoposide and prednisone (MEP)-based regimens using granulocyte colony-stimulating factor (G-CSF) were designed for relapsed and CHOP-resistant diffuse large B-cell lymphomas in a single institution, and the therapeutic effects and adverse reactions were studied. In a total of 49 patients, the MEP regimen had a 41% (9/22) overall response rate compared with 48% (13/27) for the MEP plus carboplatin (C-MEP) regimen (Chi-squared test, P=0.602). Among 38 CHOP-resistant patients, however, the overall response rate to C-MEP [42% (10/24)] was significantly superior compared with MEP [7% (1/14)] (P=0.023), and the overall survival to C-MEP was superior compared with MEP (P=0.088). Taken together, our results, although non-randomized, suggest that a combination of MEP with carboplatin is better than MEP alone in CHOP-resistant diffuse large B-cell lymphomas.