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We have reported the vaccine effectiveness of inactivated influenza vaccine in children aged 6 months to 15 years between the 2013/14 and 2018/19 seasons. Younger (6-11 months) and older (6-15 years old) children tended to have lower vaccine effectiveness. The purpose of this study is to investigate whether the recent vaccine can be recommended to all age groups.The overall adjusted vaccine effectiveness was assessed from the 2013/14 until the 2020/21 season using a test-negative case-control design based on rapid influenza diagnostic test results. Vaccine effectiveness was calculated by influenza type and by age group (6-11 months, 1-2, 3-5, 6-12, and 13-15 years old) with adjustments including influenza seasons.A total of 29,400 children (9347, 4435, and 15,618 for influenza A and B, and test-negatives, respectively) were enrolled. The overall vaccine effectiveness against influenza A, A(H1N1)pdm09, and B was significant (44% [95% confidence interval (CI), 41-47], 63% [95 %CI, 51-72], and 37% [95 %CI, 32-42], respectively). The vaccine was significantly effective against influenza A and B, except among children 6 to 11 months against influenza B. The age group with the highest vaccine effectiveness was 1 to 2 years old with both influenza A and B (60% [95 %CI, 55-65] and 52% [95 %CI, 41-61], respectively). Analysis for the 2020/21 season was not performed because no cases were reported.This is the first report showing influenza vaccine effectiveness by age group in children for several seasons, including immediately before the coronavirus disease (COVID-19) era. The fact that significant vaccine effectiveness was observed in nearly every age group and every season shows that the recent vaccine can still be recommended to children for the upcoming influenza seasons, during and after the COVID-19 era.

‘Minomusume’ strawberries were stored in high CO2 atmospheres (20%, 30%, and 40%) by means of a controlled atmosphere (CA) and active modified atmosphere packaging (MAP) for 10 days at 5 °C. The CA of 20% to 40% CO2 was effective in delaying an increase in fungal count and preventing the external formation of mold mycelia, but a CA of >30% CO2 induced black discoloration on the surface of strawberry due to CO2 injury. When strawberry fruit were stored in a MAP flushed with either air or high CO2, all packages approached an equilibrium of ≈20% CO2 and 2% O2 by the end of storage. Fungal counts of strawberry fruit stored in a MAP remained constant throughout the storage period and the diversity of fungal flora was partially similar regardless of the difference in the MAP method. Visual quality (mold incidence and severity of black discoloration) and physicochemical quality (weight loss, firmness, pH, and total ascorbic acid content) were unaffected by CO2 atmospheres as the flushing gas during active MAP storage, except that the fruit in a MAP flushed with 20% and 30% CO2 were firmer than those with air and 40% CO2. After transfer to ambient conditions for 6 days at 10 °C, however, external formation of mold mycelia identified as Botrytis cinerea and surface black discoloration were induced in strawberry fruit in MAP flushed with 30% and 40% CO2.

During influenza epidemics, Japanese clinicians routinely conduct rapid influenza diagnostic tests (RIDTs) in patients with influenza-like illness, and patients with positive test results are treated with anti-influenza drugs within 48 h after the onset of illness. We assessed the vaccine effectiveness (VE) of inactivated influenza vaccine (IIV) in children (6 months–15 years old, N = 4243), using a test-negative case-control design based on the results of RIDTs in the 2018/19 season. The VE against influenza A(H1N1)pdm and A(H3N2) was analyzed separately using an RIDT kit specifically for detecting A(H1N1)pdm09. The adjusted VE against combined influenza A (H1N1pdm and H3N2) and against A(H1N1)pdm09 was 39% (95% confidence interval [CI], 30%–46%) and 74% (95% CI, 39%–89%), respectively. By contrast, the VE against non-A(H1N1)pdm09 influenza A (presumed to be H3N2) was very low at 7%. The adjusted VE for preventing hospitalization was 56% (95% CI, 16%–77%) against influenza A. The VE against A(H1N1)pdm09 was consistently high in our studies. By contrast, the VE against A(H3N2) was low not only in adults but also in children in the 2018/19 season.

Objectives We assessed the vaccine effectiveness (VE) of inactivated influenza vaccine (IIV) in children 6 months to 15 years of age during the 2016/17 season. In addition, we estimated the impact of repeated vaccination in children on VE. Methods Our study for VEs in preventing influenza and admission due to influenza were conducted according to a test-negative case-control design (TNCC) based on influenza rapid diagnostic test results. We also analyzed the VE by vaccine status in the current and previous seasons for the impact of repeated vaccination. Results During the 2016/17 season, the quadrivalent IIV was used in Japan. The adjusted VE in preventing influenza illness was 38% (95% CI, 29–46) against influenza A and 39% (95% CI, 18–54) against influenza B. Infants showed no significant VE. The VE in preventing hospitalization was not demonstrated. For the analysis of repeated vaccination, the vaccine was effective only when immunization occurred in the current season. The children who were immunized in two consecutive seasons were more likely to develop influenza compared to those immunized in the current season only (odds ratio, 1.58 [95% CI, 1.05–2.38], adjusted odds ratio, 1.53 [95% CI, 0.99–2.35]). However, the odds ratio of repeated vaccination was not significant when the analysis excluded those who developed influenza in the previous season. Conclusions VE in children in the 2016/17 season was similar to values previously reported. Repeated vaccination interfered with the VE against any influenza infection in the 2016/17 season. The results of our study suggest that decreased VE by repeat vaccination phenomenon was associated with immunity by influenza infection in the previous season. However, the influenza vaccine should be recommended every season for children.

Objectives We assessed the vaccine effectiveness (VE) of inactivated influenza vaccine (IIV) by vaccine dose in children aged 6 months to 12 years for whom two doses are recommended in Japan to ascertain the appropriate vaccine doses. Methods VE was assessed according to a test-negative case-control design based on rapid influenza diagnostic test (RIDT) results. Children aged 6 months to 12 years with a fever ≥38 °C who had received an RIDT in outpatient clinics of 24 hospitals were enrolled for all five seasons since 2013/14. VE by vaccine dose (none vs. once or twice, and once vs. twice) was analyzed. Results In the dose analysis, 20,033 children were enrolled. Both one- and two-dose regimens significantly reduced cases in preventing any influenza, influenza A, and influenza B, but there was no significant difference in adjusted VE between one- and two-dose regimens overall (adjusted OR, 0.560 [95% CI, 0.505–0.621], 0.550 [95% CI, 0.516–0.586]), 0.549 [95% CI, 0.517–0.583], and 1.014 [95% CI, 0.907–1.135], for none vs. once, none vs. twice, none vs. once or twice, and once vs. twice for any influenza, respectively). Both one- and two-dose regimens significantly reduced cases with any influenza and influenza A every season. Also, both regimens significantly reduced cases of any influenza, influenza A, and influenza B among children aged 1–12 years, especially among those aged 1–5 years. In the 2013/14, 2015/16, and 2016/17 seasons, however, only the two-dose regimen was significantly effective in preventing influenza B. Both one- and two-dose regimens significantly reduced cases involving hospitalization due to any influenza and influenza A. Conclusions Both one- and two-doses regimens of IIV were effective in preventing influenza for children aged 6 months to 12 years. The two-dose regimen was more effective against influenza B in some seasons.

In Japan, it is forecasted that massive amounts of concrete waste material will be generated in the future as a result of demolition of many buildings, and expansion of the use of recycled aggregate is expected. In this study, it was verified the effect when relatively large amount of admixture is mixed, a combination of recycled fine aggregate of different quality and various admixtures, combination of each admixture in order to realize high strength and high durability by using recycled aggregate. The increase in the drying shrinkage ratio due to the deterioration of the recycled fine aggregate quality was larger than the fluctuation due to the admixture mixing ratio and the drying shrinkage ratio was distributed by forming a group for each quality of recycled fine aggregate. In the relationship between the pore volume and the compressive strength, when evaluated with pore volume of 2 μm or less in both cases, a good linear relationship could be confirmed. The relationship between the pore volume and the drying shrinkage rate was similar. Therefore, it was suggested that compressive strength and drying shrinkage ratio of mortar contained composite recycled fine aggregate and admixture could be predicted by evaluating with the pore volume of 2 μm or less.

Objectives We assessed the vaccine effectiveness (VE) of inactivated influenza vaccine (IIV) in children 6 months to 15 years of age in 2015/16 season. In addition, based on the data obtained during the three seasons from 2013 to 2016, we estimated the three-season VE in preventing influenza illness and hospitalization. Methods Our study was conducted according to a test-negative case-control design (TNCC) and as a case-control study based on influenza rapid diagnostic test results. Results During 2015/16 season, the quadrivalent IIV was first used in Japan. The adjusted VE in preventing influenza illness was 49% (95% confidence interval [CI]: 42–55%) against any type of influenza, 57% (95% CI: 50–63%) against influenza A and 34% (95% CI: 23–44%) against influenza B. The 3-season adjusted VE was 45% (95% CI: 41–49%) against influenza virus infection overall (N = 12,888), 51% (95% CI: 47–55%) against influenza A (N = 10,410), and 32% (95% CI: 24–38%) against influenza B (N = 9232). An analysis by age groups showed low or no significant VE in infants or adolescents. By contrast, VE was highest in the young group (1–5 years old) and declined with age thereafter. The 3-season adjusted VE in preventing hospitalization as determined in a case-control study was 52% (95% CI: 42–60%) for influenza A and 28% (95% CI: 4–46%) for influenza B, and by TNCC design, it was 54% (95% CI: 41–65%) for influenza A and 34% (95% CI: 6–54%) for influenza B. Conclusion We demonstrated not only VE in preventing illness, but also VE in preventing hospitalization based on much larger numbers of children than previous studies.

Chemical sanitizers may induce no injury (bacteria survive), sublethal injury (bacteria are injured), or lethal injury (bacteria die). The proportion of coliform bacteria that were injured sublethally by chlorine and fungicide mixed with agricultural water (pond water), which was used to dilute the pesticide solution, was evaluated using the thin agar layer (TAL) method. In pure cultures of Enterobacter cloacae , Escherichia coli , and E. coli O157:H7 (representing a human pathogen), the percentage of chlorine-injured cells was 69 to 77% for dilute electrolyzed water containing an available chlorine level of 2 ppm. When agricultural water was mixed with electrolyzed water, the percentage of injured coliforms in agricultural water was 75%. The isolation and identification of bacteria on TAL and selective media suggested that the chlorine stress caused injury to Enterobacter kobei . Of the four fungicide products tested, diluted to their recommended concentrations, Topsin-M, Sumilex, and Oxirane caused injury to coliform bacteria in pure cultures and in agricultural water following their mixture with each pesticide, whereas Streptomycin did not induce any injury to the bacteria. The percentage of injury was 45 to 97% for Topsin-M, 80 to 87% for Sumilex, and 50 to 97% for Oxirane. A comparison of the coliforms isolated from the pesticide solutions and then grown on either TAL or selective media indicated the possibility of fungicide-injured Rahnella aquatilis , Yersinia mollaretii , and E. coli . These results suggest the importance of selecting a suitable sanitizer and the necessity of adjusting the sanitizer concentration to a level that will kill the coliforms rather than cause sanitizer-induced cell injury that can result in the recovery of the coliforms.

Enumeration and Identification of Ethanol-Injured Coliform Bacteria Found on Harvest Equipment and its Cross-Contamination with Cabbage Although disinfection by sanitizers used to produce packing sheds which helps in preventing cross-contamination between farm equipment and produces, sanitizing agents like ethanol are capable of causing sanitizer-injured bacteria. Using the thin agar layer (TAL) method, the proportion of ethanol-injured coliform bacteria resulting from the use of an alcohol agent containing 1.6% and 47% ethanol was evaluated in pure cultures and in a harvest environment of cabbage, respectively. In pure cultures of Enterobacter cloacae, Escherichia coli, and E. coli O157:H7, ethanol-injured cells were observed at a range of 68-95% by treatment with an alcohol agent. When an alcohol agent was sprayed on harvest equipment including knife, container and gloves, 60% of the total coliforms detected on the knife were injured, but no injured coliforms were detected on the container and gloves. The isolation and identification of coliforms on selective and TAL media from the knife suggested that the ethanol stress caused injury to Enterobacter amnigenus, E. asburiae, and E. kobei. Injured bacteria were not detected on cabbage after harvest using the alcohol spray-treated knife because the species of bacteria found in cabbage (14 species belonging to 11 genera) were different from those on the knife. These results indicate that the sanitizer-injured coliforms would not be present on cabbage when transfer of the bacteria from harvest equipment to the cabbage is prevented or when a suitable sanitizer is used that causes death of the bacteria rather than sanitizer-induced cell injury.

The c-Myb and GATA-3 transcription factors play important roles in T cell development. We recently reported that c-Myb, GATA-3, and Menin form a core transcription complex that regulates GATA-3 expression and ultimately Th2 cell development in human peripheral blood T cells. However, c-Myb roles for Th2 cytokine expression were not demonstrated. In this article, we report that c-Myb and GATA-3 cooperatively play an essential role in IL-13 expression though direct binding to a conserved GATA-3 response element (CGRE), an enhancer for IL-13 expression. c-Myb and GATA-3 were shown to activate the CGRE–IL-13 promoter by ∼160-fold, and mutation of the canonical Myb binding site completely abrogated CGRE enhancer activity. In contrast, mutation of the GATA binding site partially decreased CGRE enhancer activity. GATA-3 did not bind to CGRE when c-myb expression was silenced. c-Myb, GATA-3, Menin, and mixed lineage leukemia (MLL) bound to CGRE in human primary CD4+ effector/memory cells. Moreover, c-myb silencing significantly decreased both methylation of histone H3K4 and acetylation of histone H3K9 at the IL-13 locus in CD4+ effector/memory cells. Therefore, in addition to the strong enhancer effect for the transcription of IL-13, the c-Myb/GATA-3 complex recruits MLL to the CGRE for histone modification of the IL-13 locus during the differentiation of memory Th2 cells.

GATA-3 and c-Myb are core elements of a transcriptionally active complex essential for human Th2 cell development and maintenance. We report herein mechanistic details concerning the role of these transcription factors in human peripheral blood Th2 cell development. Silencing c-Myb in normal human naive CD4+ cells under Th2 cell-promoting conditions blocked up-regulation of GATA-3 and interleukin-4, and in effector/memory CD4+ T cells, decreased expression of GATA-3 and Th2 cytokines. In primary T cells, c-Myb allows GATA-3 to autoactivate its own expression, an event that requires the direct interaction of c-Myb and GATA-3 on their respective binding sites in promoter of GATA-3. Immunoprecipitation revealed that the c-Myb/GATA-3 complex contained Menin and mixed lineage leukemia (MLL). MLL recruitment into the c-Myb-GATA-3-Menin complex was associated with the formation Th2 memory cells. That MLL-driven epigenetic changes were mechanistically important for this transition was suggested by the fact that silencing c-Myb significantly decreased the methylation of histone H3K4 and the acetylation of histone H3K9 at the GATA-3 locus in developing Th2 and CD4+ effector/memory cells. Therefore, c-Myb, GATA-3, and Menin form a core transcription complex that regulates GATA-3 expression and, with the recruitment of MLL, Th2 cell maturation in primary human peripheral blood T cells.

Mixed-lineage leukemia (MLL) is a proto-oncogene frequently involved in chromosomal translocations associated with acute leukemia. These chromosomal translocations commonly result in MLL fusion proteins that dysregulate transcription. Recent data suggest that the MYB proto-oncogene, which is an important regulator of hematopoietic cell development, has a role in leukemogenesis driven by the MLL-ENL fusion protein, but exactly how is unclear. Here we have demonstrated that c-Myb is recruited to the MLL histone methyl transferase complex by menin, a protein important for MLL-associated leukemic transformation, and that it contributes substantially to MLL-mediated methylation of histone H3 at lysine 4 (H3K4). Silencing MYB in human leukemic cell lines and primary patient material evoked a global decrease in H3K4 methylation, an unexpected decrease in HOXA9 and MEIS1 gene expression, and decreased MLL and menin occupancy in the HOXA9 gene locus. This decreased occupancy was associated with a diminished ability of an MLL-ENL fusion protein to transform normal mouse hematopoietic cells. Previous studies have shown that MYB expression is regulated by Hoxa9 and Meis1, indicating the existence of an autoregulatory feedback loop. The finding that c-Myb has the ability to direct epigenetic marks, along with its participation in an autoregulatory feedback loop with genes known to transform hematopoietic cells, lends mechanistic and translationally relevant insight into its role in MLL-associated leukemogenesis.

A fast, innovative matching method for the spot power market, considering network constraints, has been developed. In this method, buy and sell order bids are respectively divided into the aggregated volume of several band prices. Then, the aggregated volume and the center of each band price are used to calculate an index band clearing price, which contains the real clearing price. The division and calculation process is iterated until the band price is less than the tick size of the bidding price. This method is applied to a real problem in the Japan power market, with 9 bidding areas, 10 area-connecting lines, and 9000 orders (volume/price pairs). Numerical simulation results show that the new method is ten times faster than conventional linear programming, demonstrating the effectiveness of this method. © 2008 Wiley Periodicals, Inc. Electr Eng Jpn, 165(4): 21–29, 2008; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/eej.20694

D4-GDI, a Rho guanosine diphosphate (GDP) dissociation inhibitor, is preferentially expressed in hematopoietic tissues and binds to a small GTP-binding protein, Rho, and inhibits GDP dissociation from Rho. We identified point mutations in the D4-GDI gene in human leukemic cells. We therefore investigated the functions of D4-GDI and mutated D4-GDI in T cells. Transgenic mice (Tg) harboring human wild-type and mutant D4-GDI transgenes driven by the lck promoter were generated. Cellular immunity responses against cytozoic pathogens were examined. The cytoskeletal organization in the CD3+T cells and the proliferation of splenocytes by Con A were investigated in both Tg and littermates (LMs). Granuloma formation by bacille Calmette-Guerin was impaired in the wild-type D4-GDI Tg. On the other hand, the number of granulomas of the mutated D4-Tg was significantly higher. Infection with Listeria was more rapidly fatal to wild-type D4-GDI Tg than to LMs, while the survival of mutated D4-GDI Tg was prolonged. The CD3+T cells in wild-type D4-GDI Tg showed an impairment in the formation of stress fibers on anti-CD3 antibody-coated plates, whereas the cytoskeletal organization in CD3+T cells of the mutated D4-GDI Tg was augmented. The proliferation of splenocytes after Con A stimulation was higher in the mutated D4-GDI Tg than in the LMs. D4-GDI may have important functions, such as induction of T cell migration, adhesion and/or proliferation in inflammatory foci, in cellular immunity responses to cytozoic pathogens.

The 2014/15 influenza season in Japan was characterised by predominant influenza A(H3N2) activity; 99% of influenza A viruses detected were A(H3N2). Subclade 3C.2a viruses were the major epidemic A(H3N2) viruses, and were genetically distinct from A/New York/39/2012(H3N2) of 2014/15 vaccine strain in Japan, which was classified as clade 3C.1. We assessed vaccine effectiveness (VE) of inactivated influenza vaccine (IIV) in children aged 6 months to 15 years by test-negative case–control design based on influenza rapid diagnostic test. Between November 2014 and March 2015, a total of 3,752 children were enrolled: 1,633 tested positive for influenza A and 42 for influenza B, and 2,077 tested negative. Adjusted VE was 38% (95% confidence intervals (CI): 28 to 46) against influenza virus infection overall, 37% (95% CI: 27 to 45) against influenza A, and 47% (95% CI: -2 to 73) against influenza B. However, IIV was not statistically significantly effective against influenza A in infants aged 6 to 11 months or adolescents aged 13 to 15 years. VE in preventing hospitalisation for influenza A infection was 55% (95% CI: 42 to 64). Trivalent IIV that included A/New York/39/2012(H3N2) was effective against drifted influenza A(H3N2) virus, although vaccine mismatch resulted in low VE.

For superior Au/Al ball-bond reliability, we have developed the 3N (>99.9 wt% Au), 2N5 (>99.5 wt% Au) and 2N (>99 wt% Au) Au alloy bonding wires which contain less than 0.1, 0.5 and 1 wt% Pt, respectively. In the thermal aging test of squashed balls (40 μm in diameter), which consists of storage in air at 250°C and evaluation with wire pull and ball shear, it has been found that there are interesting differences in the ball-bond degradation as a function of wire type. Even the samples bonded using the 3N wire with a very small amount of Pt show superior ball-bond reliability compared with those using a 4N (>99.99 wt% Au) wire without any addition of Pt. The ball-bond degradation in the thermal aging test is reduced significantly with increasing Pt content of a wire. From microstructural analysis of the aged 2N ball bonds, the Pt-containing layer, which seems to play a crucial role in improvement of the ball-bond reliability, have been found in the upper part of intermetallic compounds.

Myb family proteins are ubiquitously expressed transcription factors. In mammalian cells, they play a critical role in regulating the G(1)/S cell cycle transition but their role in regulating other cell cycle checkpoints is incompletely defined. Herein, we report experiments which demonstrate that c-Myb upregulates cyclin B1 expression in normal and malignant human hematopoietic cells. As a result, it contributes directly to G(2)/M cell cycle progression. In cell lines and primary cells, cyclin B1 levels varied directly with c-Myb expression. Chromatin immunoprecipitation assays, mutation analysis, and luciferase reporter assays revealed that c-Myb bound the cyclin B1 promoter preferentially at a site just downstream of the transcriptional start site. The biological significance of c-Myb, versus B-Myb, binding the cyclin B1 promoter was demonstrated by the fact that expression of inducible dominant negative c-Myb in K562 cells accelerated their exit from M phase. In addition, expression of c-Myb in HCT116 cells rescued cyclin B1 expression after B-myb expression was silenced with small interfering RNA. These results suggest that c-Myb protein plays a previously unappreciated role in the G(2)/M cell cycle transition of normal and malignant human hematopoietic cells and expands the known repertoire of c-myb functions in regulating human hematopoiesis.

We have developed a fast, innovative matching method for the spot power market, considering network constraints. In this method, buy and sell order bids are respectively divided into the aggregated volume of several band prices. Then the aggregated volume and the center of each band price are used to calculate a band clearing price, which contains the real clearing price. The dividing and calculating process is iterated until the band price is less than the tick size of the bidding price. We applied this method to a real problem in the Japanese power market with 9 areas, 10 area-connecting lines, and 9000 orders (volume/price pairs). Our simulation results show that the new method is ten times faster than conventional linear programming. This demonstrates the effectiveness of the developed method.

The c-myb proto-oncogene has been implicated in leukemogenesis, but possible mechanisms remain ill defined. To gain further insight to this process, we used transcript profiling in K562 cells expressing a dominant-negative Myb (MERT) protein. A total of 105 potential Myb gene targets were identified. Neuromedin U (NmU), a peptide affecting calcium transport, underwent the greatest expression change (∼ 5-fold decrease). To verify a linkage between c-myb and NmU, their mRNA levels were quantitated using real-time polymerase chain reaction in primary acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL), as well as normal hematopoietic cells. We found that c-myb was elevated in AML and ALL samples, but NmU expression was increased only in AML cells. Significantly, only AML cells expressed the cognate receptor of NmU, NMU1R, suggesting the presence of a novel autocrine loop. We examined this possibility in detail. Exogenous NmU “rescued” growth suppression in K562-MERT cells and stimulated the growth of primary AML cells. Short interfering RNA “knockdown” of NmU in K562 cells arrested cell growth. Exposing Indo-1–labeled K562 cells to NmU induced an intracellular Ca++ flux consistent with engagement of the NMU1R. Combined, these results suggest that NmU expression is related to Myb and that the NmU/NMU1R axis constitutes a previously unknown growth-promoting autocrine loop in myeloid leukemia cells.

A chimeric gene, AML1-MTG16, showing high homology to AML1-MTG8, was recently identified in adult leukemic patients with the abnormal karyotype t(16;21)(q24;q22). We recently saw a child patient of 11 years of age who developed acute myelogenous leukemia with the karyotype t(16;21)(q24;q22), 11 months after autologous peripheral blood stem-cell transplantation (PBSCT) for acute promyelocytic leukemia with karyotype t(15;17)(q22;q11). The reciprocal translocation was localized by fluorescence in situ hybridization (FISH) analysis, reverse transcription polymerase chain reaction (RT-PCR), and Southern blot analysis of bone marrow blood cells and peripheral blood cells. FISH analysis identified a reciprocal translocation between chromosomes 16 and 21. RT-PCR analysis identified expression of the chimeric gene AML1-MTG16. Southern blot analysis revealed a breakpoint occurring at a 1.4 kb Eco RI fragment between exons 3 and 4 of MTG16. The breakpoint is within the same region as that of secondary leukemias, which has been reported previously. This case suggests the possibility that the region of the breakpoint of MTG16 is a characteristic of secondary leukemia.

Objective The functions of a small GTP-binding protein, Rho, in human leukemic cell invasion was investigated in vivo and in vitro. Materials and Methods Human leukemic KM3 and Reh cells (derived from B-cell-type common acute lymphoid leukemias) were inoculated into severe combined immundeficiency (SCID) mice. Alteration of invasion in SCID mice inoculated with KM3 cells that were introduced with the expression vector for Rho Val14 (Rho V14), an activated mutant form of Rho, was observed. Results SCID mice inoculated with KM3 and Reh cells developed paraplegia 21 days after inoculation. All died by day 26–27. The leukemic cells were localized to bone marrow and around the spinal cord, with no infiltration into peripheral blood, spleen, liver, thymus, or lymph nodes. SCID mice inoculated with Rho V14-transfected KM3 cells showed a 5-day reduction in the time to paraplegia and death compared with SCID mice inoculated with hygromycin-resistance gene-transfected KM3 (hyg r ) cells. In addition, the mice inoculated with Rho V14 cells showed leukemic cell infiltration, not only into bone marrow and around the spinal cord but also into peripheral blood, liver, and spleen. There were no in vitro or in vivo differences in growth rates of Rho V14 and hyg r cells. However, the Rho V14 cells showed markedly increased cell adhesion compared to the hyg r cells. Conclusion Results suggest that Rho activation accelerates human leukemic cell invasion via augmentation of cell adhesion.

This report describes a unique case of acute myeloid leukemia with hypergranular cytoplasm and t(X;11)(q24;q23). The breakpoint on 11q23 was identified within the MLL gene. The hypergranular cytoplasm of leukemic cells and the associated coagulopathy resembled a characteristic of acute promyelocytic leukemia, despite the absence of RARalpha gene rearrangement in this case. The Xq24 site possibly played a role in this atypical blast phenotype.

To reduce boiler fuel cost, a fuel cost model is developed and applied to economic load dispatch (ELD). The purpose of ELD is to determine the power demands of individual generators so that total fuel cost of those generators may be minimized. In evaluating the fuel cost of each generator, an approximate model, which describes the fuel cost as a function of the generation, is used. Therefore, the accuracy of the model has a great influence on the preciseness of the minimization. So far, one of the most prevailing models has been a quadratic model, which describes the fuel cost as a quadratic function of the generation. However, this model often deviates from the actual data, especially during the time while the generation is dynamically varying. This fact implies that, if a model, which is accurate even when the generation dynamically varies, is developed and the model is applied to ELD, the total fuel cost can be reduced. First, taking into account that the deviation might be due to the dynamics of the automatic boiler controller (ABC), we employed as a supplementary model the autoregressive moving average (ARMA) model, which is capable of describing dynamics and is suitable for an on-line ELD calculation. Second, the ELD problem based on the ARMA-model-supplemented quadratic model is formulated as a quadratic programming (QP) problem, which can be solved by the standard QP method. Finally, the cost reduction effect is evaluated by computer simulations on Kansai Electric Power System.

This paper presents an economic dispatching method, BEST method , where BEST is short for Boiler-dynamics-based Economic load diSpaTch. Because this method is based on dynamics models more accurate than the conventional quadratic ones, it can reduce boiler fuel cost. First, to reduce the model deviations from the actual data, the autoregressive moving average (ARMA) model is employed. Second, an economic dispatching problem based on this model is formulated as a quadratic programming (QP) problem. Finally, the cost reduction is evaluated by computer simulations.

We studied the effects of Lyn ablation on the survival of drug-resistant chronic myelogenous leukemia (CML) blast crisis cells using siRNA. Lyn siRNA reduced Lyn protein in both normal hematopoietic cells and BCR-ABL1-expressing (BCR-ABL1(+)) blasts by 80-95%. Within 48 h, siRNA-treated BCR-ABL1(+) blasts underwent apoptosis, whereas normal cells remained viable. This increased dependence on Lyn signaling for BCR-ABL1(+) blast survival provides the basis for rational treatment of drug-resistant CML blast crisis, particularly when lymphoid in nature.

A new practical method is proposed which gives a quasi-optimal solution of the dynamic load-dispatching problem formulated by a multiconstraint multiobjective optimization problem. Here, the multiple constraints are rate reserve constraints and power flow ones, and the multiple objectives are fuel cost and CO2 emission. The proposed method is an integration of the following three techniques: (1) “Group Dispatch Scheme,” which is developed by the authors, is used to satisfy a violated constraint easily. This scheme consists of three steps. The first step is to classify all generators into two groups. One is the group which should be more loaded to satisfy the violated constraint, and the other is the should-be less loaded group. The second step is to add some load to the former group, and to subtract the same load from the latter. The third step is to dispatch each group load to each groups generators, respectively; (2) the dynamic load-dispatching algorithm is used which provides, in practical time, quasi-optimal generation trajectories; (3) the conventional weighting parametric method is used to obtain a Pareto optimal solution of multiobjective problems. The effectiveness of the method is clarified by a computer simulation on the actual power system of Kansai Electric Power Company.

This paper introduces a protective relay setting system capable of handling a large network up to 10,000 buses. The remarkable features are such useful functions as network analysis (fault analysis, load flow calculation, zero-sequence current calculation, etc.), relay operation simulation, and automated setting coordination tasks. Fault currents in the maximum and the minimum operation networks are calculated to examine the setting values. The protection zones are searched automatically along the network connection. Protection coordination tasks among the primary and the backup relays composed of different types of relays can be performed by the relay operation simulation. The system is built on an engineering workstation, which works in cooperation with other network operation support systems. Its graphical user interface helps every engineer to examine the relay setting using a variety of databases.

The requirement of c-Myb during erythropoiesis spurred an interest in identifying c-Myb target genes that are important for erythroid development. Here, we determined that the neuropeptide neuromedin U (NmU) is a c-Myb target gene. Silencing NmU, c-myb, or NmU's cognate receptor NMUR1 expression in human CD34+ cells impaired burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) formation compared with control. Exogenous addition of NmU peptide to NmU or c-myb siRNA-treated CD34+ cells rescued BFU-E and yielded a greater number of CFU-E than observed with control. No rescue of BFU-E and CFU-E growth was observed when NmU peptide was exogenously added to NMUR1 siRNA-treated cells compared with NMUR1 siRNA-treated cells cultured without NmU peptide. In K562 and CD34+ cells, NmU activated protein kinase C-βII, a factor associated with hematopoietic differentiation-proliferation. CD34+ cells cultured under erythroid-inducing conditions, with NmU peptide and erythropoietin added at day 6, revealed an increase in endogenous NmU and c-myb gene expression at day 8 and a 16% expansion of early erythroblasts at day 10 compared to cultures without NmU peptide. Combined, these data strongly support that the c-Myb target gene NmU functions as a novel cofactor for erythropoiesis and expands early erythroblasts.

Objective Rho GTPase may be involved in human cancer invasion via the augmentation of cell motility and adhesion. We report on two point mutations of the D4-guanine diphosphate (GDP)–dissociation inhibitor (GDI) gene, one of the Rho-GDIs, which were found in a human leukemic cell line, Reh, and the mutated D4-GDI functions as an accelerator of leukemic cell invasion. Material and Methods We investigated the altered activity of GDP dissociation by mutated (mt) D4-GDI and the functions of this mt and wild-type (wt) D4-GDI in invasion. The mice inoculated with wt or mt D4-GDI vector–transfected Raji cells were observed and examined pathologically. Adhesiveness and cell motility of wt or mt D4-GDI vector–transfected Raji cells were examined. Finally, it was examined whether Rho activation was changed by mutation of D4-GDI under the condition of Rho-GDI knockdown. Results Two point mutations of the D4-GDI gene were found in Reh cells. The region of mutations is conserved among members of the Rho-GDI family at the amino acid level. D4-GDI with two mutations (V68L and V69A) functioned in a dominant negative manner in the inhibition of GDP dissociation from Rho. Severe combined immune-deficient mice inoculated with Raji cells developed hemiparalysis. The Raji cells were present in bone marrow and peripheral blood, and hepatic invasion was observed in 20% of the mice. Mice inoculated with wt D4-GDI vector–transfected Raji cells (wt D4) showed later paralysis and none developed hepatic invasion. Mice inoculated with mt D4-GDI–transfected Raji cells (mt D4) showed a 5-day reduction in the time to paraplegia and death. In addition, hepatic invasion was evident in 80% of mice transplanted with mt D4 cells. There were no differences in growth rates and amounts of guanine triphosphate (GTP)–bound Rho, cdc42, or Rac among all clones, however, GTP-bound Rho in mt D4 clone with short hairpin RNA (shRNA) vector for Rho-GDI knockdown was increased compared with wt D4 clone with shRNA vector for Rho-GDI knockdown. The mt D4 cells showed an augmentation of adhesiveness and cell motility. On the other hand, wt D4 cells showed a decreased ability of cell motility. Conclusion These results suggest the mutated D4-GDI functions as a dominant negative molecule against the wt D4-GDI and accelerates invasion via regulation of cytoskeletal machinery.

We studied the effects of Lyn ablation on CXCR4 receptor-mediated migration and adhesion of hematopoietic precursors. Down-regulation of Lyn expression with siRNA greatly reduced CXCR4-dependent hematopoietic cell movement, and increased cell adherence to stroma. This increase was associated with the up-regulated expression of activation-dependent epitopes of the β2 integrin LFA-1 and was prevented by antibodies that selectively block cell adhesion mediated by ICAM-1. Attachment to surfaces coated with ICAM-1 was also enhanced in Lyn-depleted hematopoietic cells, as compared with Lyn-expressing cells. Functional rescue experiments with Lyn siRNA targeting the 3′ UTR indicated that the observed effects can be attributed directly to specific inhibition of Lyn. Our results show that in chemokine-stimulated hematopoietic cells Lyn kinase is a positive regulator of cell movement while negatively regulating adhesion to stromal cells by inhibiting the ICAM-1–binding activity of β2 integrins. These results provide a molecular mechanism for cross talk between the chemokine receptor CXCR4 and β2 integrins. This cross talk may allow chemokine receptors to modulate the arrest of rolling hematopoietic precursors on the surface of bone marrow stromal cells.

Most cancers are characterized by abnormal gene expression, which is thought to contribute to the pathogenesis and maintenance of the malignant phenotype; abnormal proliferation, maturation, and apoptosis. Silencing such genes would appear to be a rational approach to the therapy of cancer, and some preliminary clinical studies support this concept. Of the strategies available, the anti-mRNA gene silencing approach has attracted much attention and is the focus of this review. This strategy includes three types of agents: (1) single-stranded antisense oligonucleotides; (2) catalytically active oligonucleotides, such as ribozymes, and DNAzymes that possess inherent RNA cleaving activity; and (3) small interfering RNA (siRNA) molecules that induce RNA interference (RNAi). Among these agents, antisense oligonucleotides, especially phosphorothioate (PS) oligonucleotides, have been the most frequently used in clinical trials. In this article, we provide an overview of anti-mRNA gene silencing agents and their development for use as cancer therapeutics.

We report 2 patients with necrotizing tracheobronchitis (NTB) associated with patent ductus arteriosus-dependent cyanotic congenital heart disease. The pathologic findings suggest that hypotension and decreased tracheo-bronchial perfusion were the major contributing factors in the development of NTB. Necrotizing tracheobronchitis developed in infants with pulmonary atresia and Ebstein's anomaly with pulmonary stenosis. Both infants required prostaglandin E1 infusion from early infancy, and presented with sudden onset of dyspnea and hypercapnea. In one infant, NTB developed prior to mechanical ventilation. In the other infant, NTB developed after 4 days of mechanical ventilation. Care of both infants involved minimal pressures and FiO2, adequate humidification, and optimal temperature of inspired gases; these factors probably did not play a role in the development or worsening of NTB. Both infants had hypotension and hypoxemia. These factors could have contributed to the development of NTB because of decreased perfusion pressure and tissue hypoxia. As the area of necrosis and its severity correlated with the area of blood supply served by the specific feeding arteries, we speculate that tissue hypoperfusion was the major cause of NTB. Pediatr Pulmonol. 2001; 32:480–483. © 2001 Wiley-Liss, Inc.

Jumping translocations (JTs) are unbalanced chromosomal translocations in which an identical chromosomal region is translocated to the telomeric region of different chromosomes. JTs are rare in hematological malignancies where they are second translocations and may be an indicator of poor prognosis. We report a case of acute myeloid leukemia with t(16;21) and a JT in which the long arm of chromosome 1 distal to q21 is translocated to the terminal region of chromosome 10. The leukemic cells exhibit high expression of EAT/mcl1, an anti-apoptotic Bcl-2 related gene. Since EAT/mcl1 is mapped to 1q21 near the breakpoint in the JTs, high level expression of EAT/mcl1 may be associated with the poor prognosis of leukemia with JTs.

Abstract 3669 Poster Board III-605 The c-myb proto-oncogene encodes a transcription factor, c-Myb, which regulates genes important for early myeloid, B, and T lymphoid cell development. Our group has recently shown that c-Myb binds a conserved site in the GATA-3 promoter, upregulating its expression and forming a complex that plays an important role in human Th2 cell development. We have also observed that silencing c-Myb in primary human peripheral blood CD4+ cells under Th2 cell promoting conditions decreases the expression of the Th2 cytokines, IL-4, IL-5 and IL-13. We wondered if this was a direct effect of c-Myb alone, or was also due to an effect of the c-Myb/GATA-3 complex? The human IL-4, IL-5 and IL-13 genes are located within a 150kb locus that is called the “Th2 cytokine gene” on Chromosome 5. The Th2 cytokine locus has numerous DNase I hypersensitive sites (DHSs) which act as regulatory elements for Th2 cytokine gene expression. Among these DHSs, the conserved GATA-3 response element (CGRE), the conserved noncoding sequence 1 (CNS-1), the intronic enhancer (IE) and the hypersensitive site V (HSV) are all known to be activated by GATA-3 directly. Interestingly, sequence analysis identified c-Myb binding sites in close proximity to GATA binding sites within all 4 DHSs. To determine if c-Myb, and GATA-3, occupied these putative binding sites, we performed ChIP assays and found that anti-c-Myb antibody, for example, enriched the CGRE locus 30-fold compared to control IgG. Similar experiments revealed that both c-Myb and GATA-3 bound to all 4 of the elements in human naïve CD4+ T-cells under Th2 promoting conditions. To determine the role of c-Myb and GATA-3 in Th2 cytokine gene expression, we performed reporter assays in 293T, Jurkat and human CD4+ T-cells using IL-4, IL-13 and IL-5 promoter constructs with, or without, the 4 DHSs. c-Myb weakly (2-2.5-fold), but significantly, activated both the IL-4 and IL-5 promoters in 293T cells in the absence of DHSs. In contrast, GATA-3 did not activate the IL-4 and IL-5 promoters without the enhancer elements, though it weakly activated the IL-4 promoter through CNS-1. GATA-3 activated the minimal IL-13 promoter 5-fold, and the addition of c-Myb increased promoter activation 10-fold. In marked contrast to these results, an IL-13 promoter construct containing the CGRE enhancer was activated 65-fold by GATA-3 alone, and 160-fold by the addition of c-Myb. The CGRE locus has only one canonical Myb binding site, but Myb's strong enhancer effect on IL-13 expression was completely lost when the CGRE element's Myb binding site was mutated. Mutating the GATA binding sites decreased IL-13 promoter activity by ∼50%. These results suggest that c-Myb and GATA-3 regulate IL-13 expression directly, and in a cooperative manner. These results lead us to hypothesize that the binding of a c-Myb and GATA-3 complex to the CGRE element changes the chromatin structure of the IL-13 locus which acts as a switch to promote IL-13 expression. Though the most dramatic effects of c-Myb were on IL-13 expression, because c-Myb binds to the other three enhancers, and activates both the IL-4 and IL-5 promoters as well, we hypothesize that c-Myb directly contributes to all Th2 cytokine gene expressions, including IL-13. Finally, a ChIP assay showed that MLL also bound to the locus of CGRE. Since we have already shown that c-Myb can bind to MLL through Menin those results suggest, in aggregate, the possibility of histone modification in Th2 cytokine locus by MLL, GATA-3 and c-Myb. This possibility is under active investigation in our laboratory. Disclosures: No relevant conflicts of interest to declare.

Abstract 448 The c-myb proto-oncogene was first identified as the cellular homologue of the v-myb oncogene carried by the avian leukemia viruses AMV, and E26. c-myb encodes a transcription factor, c-Myb, that is highly expressed in immature hematopoietic cells. In such primitive cells, c-Myb has been found to exert an important role in lineage fate selection, cell cycle progression, and differentiation of both myeloid, B, and T lymphoid progenitor cells. c-Myb is also highly expressed in many leukemia cells and on this basis has been implicated in leukemic transformation. Despite intensive study, a mechanisms based understanding for c-Myb's myriad effects on blood cell development has yet to be fully achieved though c-Myb's ability to interact with a variety of transcriptionally active co-factors, such as p300, CBP, and FLASH, as well as to modulate its own expression, have all been reported to contribute to its activities. Therefore, we undertook a series of biochemical, molecular, and clinical studies to further address c-Myb's role in leukemic hematopoiesis. Using in vitro translated proteins and nuclear extracts from leukemic cells in immunoprecipitation (IP) assays, we found that c-Myb is associated with MLL1, the SET1 proteins WDR5, RbBp5, and Ash2L, and menin, all of which form a complex with histone methyltransferase (HMT) activity. c-Myb associated with the MLL1 and SET1 proteins through menin, which served as an adapter protein by interacting (as previously shown) with the extreme amino terminus of the MLL1 protein, and, as we show, with a region around the c-Myb transactivation domain (aa 194-325). We demonstrated in vitro with purified proteins and an H3 peptide, that c-Myb contributed to the HMT activity of the MLL1 complex. In leukemia patients being treated with a c-myb targeted antisense oligodeoxynucleotide (ASODN), and in leukemic cell lines, silencing c-myb evoked a significant decrease in H3K4 methylation demonstrating biological relevance of this observation. The decrease in H3K4 methylation is the direct result of silencing c-myb and is not due to changes in cell proliferation, and could not be reproduced by silencing B-myb. Also, we confirmed that c-Myb is a downstream target of HoxA9, and Meis 1, but showed unexpectedly that leukemic blasts derived from the c-myb ASODN treated patients, and c-myb siRNA treated cell lines, decrease c-myb expression also led to a decrease in Hoxa9 and Meis1 expression. This suggested the presence of an autoregulatory feedback loop between c-Myb and HoxA9. This finding too was specific for c-myb and not associated with a block in proliferation or silencing B-myb. Finally, disrupting the c-Myb-MLL1 interaction impairs localization of MLL1 and menin on the Hoxa9 gene promoter, as well as the MLL-ENL induced transformation of normal murine bone marrow cells. In summary, our results bring new insights regarding c-Myb function in human hematopoietic cells, suggest new mechanisms whereby c-Myb may contribute to cell transformation, and suggest new therapeutic targets for the treatment of acute leukemia. Disclosures: No relevant conflicts of interest to declare.

The c-myb protooncogene encodes a transcription factor, c-Myb, which is highly expressed in immature hematopoietic cells. c-Myb is required for many critical aspects of blood cell development including lineage fate selection, proliferation, and at multiple time points during early myeloid, and B and T lymphoid cell development. GATA-3, which belongs to a family of zinc finger transcription factors, is also required at several steps in early T cell development, and specifically in regard to this communication, for the development of T helper type 2 (Th2) cells. A recent study by Maurice et al (EMBO2007, 26:3629–3640) reported that c-myb regulates T helper cell lineage commitment in developing mouse thymocytes via regulation of GATA-3 expression. As we were unaware of any studies that have addressed the role of c-Myb and GATA-3 in normal human peripheral blood lymphocytes (PBL), we explored the potential regulatory relationship between these transcription factors in cells of this type. Proceeding from the murine studies, we performed a chromatin immunoprecipitation assay (ChIP) which showed that c-Myb bound the GATA-3 downstream promoter in naïve CD4+ T cells under conditions designed to promote Th2 growth. Such binding was not observed in cells stimulated under Th1 promoting conditions. The interaction of c-Myb and GATA-3 proteins was also detected in cell lysates under Th2 cell promoting conditions by immunoprecipitation with both anti-c-Myb, and anti-GATA-3 polyclonal antibodies. Of note, immunoprecipitation with these same antibodies did not show binding of either protein to STAT6. Additional studies revealed that c-Myb activated a GATA-3 minimal promoter by direct binding to a conserved c-Myb binding site in peripheral blood T cells. Of even greater interest, in 293T cells, GATA-3 activated its own promoter ~6 fold when c-Myb was co-expressed in 293T cells. In the absence of c-Myb, GATA-3 did not significantly activate its own promoter in these cells. We have recently shown that c-Myb binds to MLL via menin. A ChIP assay also showed that MLL and Menin bound to the GATA-3 promoter suggesting that c-Myb and GATA-3 form a co-activator complex on the GATA-3 promoter with MLL. Finally, to explore the role of c-myb expression in human peripheral blood naive CD4+ T cells, we employed c-Myb targeted, and control, short hairpin RNA (shRNA) expressed from a lentivirus vector. This strategy yielded a sequence specific 80–90% knockdown of c-Myb expression in our hands. Stimulation of naive peripheral blood CD4+ T cells expressing the c-Myb directed shRNA with cytokines promoting Th2 cell formation (IL-4, IL-2, and anti-IL-12 antibody) blocked the up-regulation of GATA-3 mRNA expression ~90% compared to cells in which a control shRNA had been expressed. Flow cytometric analysis revealed that intracellular IL-4 expression also was diminished. In contrast, silencing c-myb had no effect on T-bet mRNA expression, or intracellular interferon-expression in the cells induced to undergo Th1 cell formation with IL-12, IL-2 and anti-IL-4 antibody. We conclude from these studies that c-Myb regulates developmental programs specific for Th2, as opposed to Th1, cell development. We hypothesize that such control is exerted in peripheral blood T lymphocytes, at least in part, through direct control of GATA-3, whose expression is auto-regulated with the assistance of c-Myb, and perhaps MLL, acting as transcriptional co-factors.

c-Myb is an obligate hematopoietic transcription factor which is highly expressed in immature hematopoietic cells. It plays a critical role in both myeloid and lymphoid cell development, and specifically in regard to this communication, at multiple points during early T cell development. While the role of c-Myb in developing cells has been intensively studied, we noted that there is a relative paucity of investigations focused on c-myb function in peripheral blood T cells. This situation exists despite the relatively high level of c-myb expression we observe in unstimulated cells, and the increase that occurs when such cells are stimulated. Very recently (Embo J, Aug 2007), Maurice et al demonstrated that c-myb regulates T helper cell lineage commitment in developing mouse thymocytes, at the same time that it appears to block development of cytotoxic T cells, via regulation of GATA-3. However, the role of c-Myb and GATA-3 in normal human peripheral blood lymphocytes was not explored. Here we show that c-myb regulates GATA-3 expression directly in peripheral blood CD4+ cells and has a critical role in human Th2 cell development. To explore the role of c-myb expression in human peripheral blood naive CD4+ cells we employed c-Myb targeted, and control, short hairpinRNA (shRNA) expressed from a lentivirus vector. This strategy yielded a sequence specific ~ 80–90% knockdown of c-Myb expression. Stimulation of naive peripheral blood CD4+ T cells in which the c-Myb directed shRNA was expressed, with a cocktail designed to promote Th2 cell formation (IL-4, IL-2, and anti-IL-12 antibody) blocked the up-regulation of GATA-3 mRNA expression ~90% compared to cells in which a control shRNA had been expressed. Flow cytometric analysis showed that intracellular interleukin-4 expression was also diminished in CD4+ cells stimulated under Th2 promoting conditions. In contrast, silencing c-myb did not affect T-beta mRNA expression, or intracellular interferon-γ expression in the cells induced to undergo Th1 cell formation with IL-12, IL-2 and anti-IL-4 antibody. A ChIP assay showed that c-myb bound to the GATA-3 promoter in human primary CD4+ cells stimulated under Th2 cell promoting conditions, but not under Th1 promoting conditions. A reporter assay demonstrated that c-myb over-expression increased GATA-3 promoter activity by ~5 fold in 293T cells, and approximately 3 fold in human primary T cells. Silencing c-myb in primary human T cells with shRNA resulted in an approximately 50% decrease in GATA-3 promoter activity. These results demonstrate that c-myb plays an important role in Th2 cell development at least in part through direct regulation of GATA-3 expression. In primary human effector/memory CD4+ T cells, which includes established Th2 cells, c-myb suppression with shRNA also decreased GATA-3 promoter activity by approximately 85%, but the suppression of IL-4 expression was only moderate (~50%). These results suggest that c-myb may also play a role in the homeostasis of established Th2 cells. Finally, and as might be expected, silencing c-myb suppressed proliferation of naive CD4+ cells. We conclude that c-Myb plays multiple roles in human peripheral blood T lymphocytes, including the generation and maintainence of Th2 cells, in addition to regulation of cell proliferation. It performs these functions, at least in part, through direct regulation of GATA-3.

Myb family transcription factors are ubiquitously expressed, and are known to play a critical role in regulating G1/S cell cycle transition. Recently, Myb-like proteins have been found to regulate G2/M transit in plants, yeast, and Drosophila. A recent study in human T98G ganglioblastoma cells revealed that E2F, together with B-Myb, but not c-Myb, regulated cyclin B1 expression directly. However, in hematopoietic cells Myb’s role in regulating cell cycle check points other than G1/S is less well defined. Herein we report that c-Myb, as well as B-Myb, up-regulates cyclin B1 expression in normal and malignant human hematopoietic cells, thereby contributing to G2/M cell cycle progression. Our initial experiments revealed a direct relationship between Myb and cyclin B1 expression. We then attempted to show causality using a variety of experimental approaches. First, ChIP assays demonstrated that c-Myb protein directly bound the cyclin B1 promoter in K562 and Mo7e cells. Second, a cycle 3 GFP reporter construct, driven by the cyclin B1 promoter, was upregulated in cells co-tranfected with a c-myb expression vector. Third, a conditionally active c-Myb restored cyclin B1 mRNA expression in K562 human leukemia cells in presence of cycloheximide in 6 hours. All these assays strongly suggest that c-myb directly regulates cyclin B1. Finally, cyclin B1 expression decreased by 85–90 % in Mo7e human leukemia cells in which c-myb had been silenced with siRNA. siRNA targeted to B-myb also decreased cyclin B1 expression, while neither siRNA species decreased cdc2 or cyclin A. The biologic significance of this relationship was revealed by two independent lines of experimentation. First, silencing B-myb resulted in a delay of cell cycle progression from S to G2/M, and an accumulation of cells in M phase, in HCT116 cells and K562 cells respectively. These abnormalities could be rescued, at least partially, by expression of exogenous c-myb. This observation conflicts with the report that c-Myb does not regulate cyclin B1 or G2/M progression in T98G cells suggesting that Myb functions could well be cell type specific. Additional analysis using PCR array showed that the absence of B-myb decreased the expression of 19 of 84 cell cycle related genes. Exogenous c-myb expression partially rescued 11 genes including cyclin B1, cyclin B2, cdc2, cdc20, CKS1B, p15INK4b and Ki-67, but not cyclin D1. In another experiment an inducible dominant negative c-Myb protein decreased cyclin B1 expression in K562 human leukemia cells, and the expected consequence of this, accelerated exit from the M phase, was observed. In activated primary human T-lymphocytes with IL-2 and CD34+ bone marrow cells, expression of c-Myb and cyclin B1 increased concordantly and silencing c-myb expression resulted in decreased cyclin B1 expression. We conclude from these studies that c-Myb in addition to B-myb plays a heretofore unappreciated role in G2/M cell cycle transition in normal and malignant human hematopoietic cells by directly regulating cyclin B1 expression.

Myb family transcription factors are found throughout the phyla, and recent studies have demonstrated that Drosophila myb, as well as plant and yeast c-myb-like transcription factors, play an important role in regulating transition though the G1/S and G2/M phases of the cell cycle. Myb’s ability to regulate passage through G2/M is due at least in part to its ability to induce Cyclin B1 expression. A recent study in human T98G ganglioblastoma cells revealed that E2F, together with B-Myb, regulated cyclin B1 expression. Though c-myb was expressed in these cells, it was not found in immunoprecipitated E2F-B-Myb protein complexes and for this reason was felt not to participate in cyclin B1 expression in these cells. Since c-myb plays such a critical role in regulating hematopoietic cell proliferation, and its role in regulating G2/M in blood cells has not previously been explored, we investigated whether c-myb was important is regulating this phase of the cell cycle using K562 and Mo7e cells, as well as PHA stimulated human T lymphocytes. In distinct contrast to findings reported for T98G cells, we now report that in normal and malignant human hematopoietic cells, c-Myb directly upregulates cyclin B1 expression. Several lines of evidence support this claim. First, cyclin B1 expression decreased in Mo7e human leukemia cells in which c-myb had been silenced with siRNA. siRNA targeted to B-myb also decreased cyclin B1 expression, while neither siRNA species decreased cdc2 or cyclin A in these cells. As expected, siRNA targeted against c-myb or B-myb impaired Mo7e cell proliferation. Simultaneous exposure to both siRNA blocked proliferation completely. Second, using an alternative strategy, an inducible dominant negative c-Myb protein also decreased cyclin B1 expression in K562 human leukemia cells. The expected consequence of this, accelerated exit from the M phase, was also observed. Third, we examined c-Myb expression in human T cells by western and Real Time PCR, pre and post PHA stimulation. c-Myb expression began to gradually increase in the G1 phase of cell cycle, continued to increase after S phase, with the maximal protein level being found in G2/M phase, and concordant with cyclin B1 expression. These results indicated a correlation between c-Myb and cyclin B1 expression but did not indicate if c-Myb regulated cyclin B1 expression directly. To address this question, several additional experiments were carried out. A CAT assay showed that overexpressing c-Myb protein could increase activity when driven by a cyclin B1 promoter construct ~5X compared to K562 control cells. Next, examination of the cyclin B1 promoter showed eight potential c-Myb binding sites. Two were canonical [5′-pyrimidine AACG/TG-3′] and located upstream of 6 others which were [5′-AACNG-3′] in type. An in vitro c-Myb binding assay revealed that c-Myb bound the canonical sites. We then performed a Chromatin Immunoprecipitation (ChIP) Assay with anti-c-Myb antibody and specifically enriched cyclin B1 promoter DNA sequences which strongly suggested that c-Myb bound the cyclin B1 promoter in vivo. A control antibody was inactive. Finally, a conditionally active c-Myb restored cyclin B1 mRNA expression in K562 human leukemia cells in presence of cycloheximide within 6 hours. Therefore, in addition to its role in regulating G1/S cell cycle transition, c-Myb also regulates cyclin B1 expression and therefore transition through the G2/M phase in human hematopoietic cells.

To examine the importance of Lyn kinase activity in CML cells, we employed Lyn targeted siRNA in Bcr/Abl-positive cell lines (K562, EM2, EM3), Bcr/Abl-negative cell lines (Mo7e, HL-60), and primary CML blast crisis cells isolated from patients with drug sensitive, and resistant disease. The siRNA were delivered by electroporation using an amaxa nucleoporator (Amaxa, Inc.) at a final working concentration between 1–5 μg/100 μl. By 48 hours after treatment, western blotting showed an 70–95% reduction in Lyn protein. Loss of Lyn protein was associated with an 3 fold inhibition of cell growth and viability at 72–96 hours in Bcr/Abl (+) cell lines. On the contrary, no inhibition in cell growth/viability was observed in Bcr/Abl (−) cell lines. Nucleoporation of control siRNA had no significant effect on cell growth in any of the lines examined. Based upon the differential sensitivity of Bcr/Abl (+) and (−) cell lines to Lyn ablation, we next asked if there were differential requirements for Lyn signaling in primary blast cells derived from Gleevec-resistant (n=3), and Gleevec-sensitive (n=2) patients, and whether normal hematopoietic progenitor cells also relied on Lyn signaling for growth. Anti-Lyn siRNA, delivered as described above, reduced Lyn protein, variably, in both malignant and normal primary cells. Lyn protein decrease ranged from 70 to 90% and was accompanied by a variable decrease in cell viability in CML blasts. Lymphoid blasts derived from Gleevec-resistant patients were particularly sensitive to Lyn ablation undergoing a massive induction of apoptosis by 48–96 hours after exposure to Lyn siRNA (90–100% reduction in cell viability). In contrast, CML cells from non-Gleevec resistant myeloid blast crisis patients appeared less sensitive to Lyn ablation since only 40–60% reduction in the cell viability was observed. Importantly, ablation of Lyn expression had no effect on the viability of normal CD34+ progenitors derived from healthy individuals (n=3). To ensure that growth arrest/apoptotic effects were not due to double stranded (ds) RNA triggering of the interferon (IFN) response pathway, we measured activation of dsRNA-dependent protein kinase, PKR, in K562 cells. While PKR kinase was robustly phosphorylated in long dsRNA treated cells (positive control), we did not detect any activation of PKR in anti-Lyn, or control siRNA-treated K562 cells. Finally, to ensure that “off target” silencing effects were not responsible for the observed decreases in cell viability and growth, we performed functional rescue experiments. To carry out these experiments, K562 cells were transfected with a pool of four individual SMARTselection-designed siRNA (Dharmacon, Inc.) designed to silence endogenous Lyn expression by specifically targeting the 3′UTR. Treated cells were then co-transfected with a Lyn expression construct that was lacking the 3′UTR. We observed that cells treated with siRNA targeting the 3′UTR underwent growth arrest and apoptosis. Cells co-transfected with the Lyn expression construct continued to grow and did not undergo apoptosis. In conclusion, these results demonstrate that Bcr/Abl (+) leukemias are dependent on Lyn kinase for survival and proliferation. Normal cells, and Bcr/Abl (−) leukemic cell lines, are not Lyn dependent and can tolerate its loss without apparent ill effect. Accordingly, Lyn is a legitimate therapeutic target in CML blast crisis and siRNAs targeting Lyn may find utility in the treatment of this disease, especially when Gleevec resistant.