Shuichi Ueno

Characterization of stage progression in chronic myeloid leukemia by DNA microarray with purified hematopoietic stem cells.

Chronic myeloid leukemia (CML) is characterized by the clonal expansion of hematopoietic stem cells (HSCs). Without effective treatment, individuals in the indolent, chronic phase (CP) of CML undergo blast crisis (BC), the prognosis for which is poor. It is therefore important to clarify the mechanism underlying stage progression in CML. DNA microarray is a versatile tool for such a purpose. However, simple comparison of bone marrow mononuclear cells from individuals at different disease stages is likely to result in the identification of pseudo-positive genes whose change in expression only reflects the different proportions of leukemic blasts in bone marrow. We have therefore compared with DNA microarray the expression profiles of 3456 genes in the purified HSC-like fractions that had been isolated from 13 CML patients and healthy volunteers. Interestingly, expression of the gene for PIASy, a potential inhibitor of STAT (signal transducer and activator of transcription) proteins, was down-regulated in association with stage progression in CML. Furthermore, forced expression of PIASy has induced apoptosis in a CML cell line. These data suggest that microarray analysis with background-matched samples is an efficient approach to identify molecular events underlying the stage progression in CML.

Macrophage migration inhibitory factor as a redox-sensitive cytokine in cardiac myocytes

OBJECTIVE Macrophage migration inhibitory factor (MIF), which plays a pivotal role in the control of inflammatory responses, was first characterized as a T-cell cytokine, but later was also found as a pituitary peptide released in response to infection and stress. However, MIFs role and expression in the myocardium has never been reported. The goal of this study is to examine MIF in the myocardium. METHODS AND RESULTS MIF protein and mRNA levels were assayed using enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR), respectively. Increased MIF concentrations were detected in the sera of patients with acute myocardial infarction (AMI). In cultured rat cardiac myocytes, significant amounts of MIF were produced in response to hypoxia and hydrogen peroxide (H(2)O(2)), but not to angiotensin II, endothelin-1, interleukin-1beta (IL-1beta) or tumor necrosis factor alpha (TNFalpha). H(2)O(2)-induced MIF production increased in a time- and dose-dependent manner and was completely abolished in the presence of catalase. H(2)O(2) also induced MIF mRNA expression. The H(2)O(2)-induced MIF production was completely inhibited by the protein kinase C (PKC) inhibitor GF109203X, partially inhibited by the tyrosine kinase inhibitor herbimycin A, and uninhibited by calcium chelation or phorbol ester-sensitive PKC down-regulation. This suggests that H(2)O(2)-induced MIF production is mediated by an atypical PKC isoform. DNA microarray analysis revealed that 52 genes were preferentially expressed in response to MIF. Of these, the MIF-induced expression of both glutathione S-transferase (GST) and lipopolysaccharide-induced CXC chemokine (LIX) mRNAs was confirmed using RT-PCR analysis. CONCLUSION The present results suggest that MIF is expressed by the myocardium in response to redox stress and may play a role in the pathogenesis of myocardial ischemia.

Sak serine-threonine kinase acts as an effector of Tec tyrosine kinase.

The murine sak gene encodes a putative serine-threonine kinase which is homologous to the members of the Plk/Polo family. Although Sak protein is presumed to be involved in cell growth mechanism, efforts have failed to demonstrate its kinase activity. Little has been, therefore, elucidated how Sak is regulated and how Sak contributes to cell proliferation. Tec is a cytoplasmic protein-tyrosine kinase (PTK) which becomes activated by the stimulation of cytokine receptors, lymphocyte surface antigens, heterotrimeric G protein-linked receptors, and integrins. To clarify the in vivo function of Tec, we have tried to isolate the second messengers of Tec by using the yeast two-hybrid screening. One of such Tec-binding proteins turned out to be Sak. In human kidney 293 cells, Sak became tyrosine-phosphorylated by Tec, and the serine-threonine kinase activity of Sak was detected only under the presence of Tec, suggesting Sak to be an effector molecule of Tec. In addition, Tec activity efficiently protects Sak from the “PEST” sequence-dependent proteolysis. Internal deletion of the PEST sequences led to the stabilization of Sak proteins, and expression of these mutants acted suppressive to cell growth. Our data collectively supports a novel role of Sak acting in the PTK-mediated signaling pathway.

DNA microarray analysis of T cell-type lymphoproliferative disease of granular lymphocytes

Summary. Lymphoproliferative disease of granular lympho‐ cytes (LDGL) is characterized by the clonal proliferationoflarge granular lymphocytes of either T‐ or natural killer cell origin. To better understand the nature of T cell‐type LDGL, we purified the CD4–CD8+ proliferative fractions from LDGL patients (n=4) and the surface marker‐matched T cells isolated from healthy volunteers (n=4), and compared the expression profiles of 3456 genes using DNA microarray. Through this analysis, we identified a total of six genes whose expression was active in the LDGL T cells, but silent in the normal ones. Interestingly, expression of the gene for interleukin (IL) 1β was specific to LDGL T cells, which was further confirmed by the examination of the serum level of IL‐1β protein. Given its important role in inflammatory reactions, the disease‐specific expression of IL‐1β may have a causative relationship with the LDGL‐ associated rheumatoidarthritis. Spectratyping analysis of the T‐cell receptor repertoire also proved the monoclonal or oligoclonal natureof LDGL cells. These data have shown that microarray analysis with a purified T‐cell subset is an efficient approach to investigate the pathological condition of Tcell‐type LDGL.

Adeno-Associated Virus-Mediated Transfer of Endothelial Nitric Oxide Synthase Gene Inhibits Protein Synthesis of Rat Ventricular Cardiomyocytes

We investigated whether nitric oxide (NO) synthase gene transfer could attenuate growth of cultured cardiac myocytes. First, we investigated the effects of exogenous NO and cGMP analog on protein synthesis of cultured neonatal rat cardiac myocytes. The NO donor 3-morpholino-sydnonimine-hydrochloride (SIN-1) and 8-bromo-cGMP caused concentration-dependent decreases in phenylephrine-stimulated incorporation of 3H-leucine into cardiac myocytes. We then transferred endothelial constitutive NO synthase (ecNOS) gene into cultured neonatal rat cardiac myocytes using adeno-associated virus (AAV) vectors. ecNOS gene transfer into cardiac myocytes induced 140 kD ecNOS protein expression and significantly increased cGMP contents of myocytes compared with control cells. ecNOS gene transfer inhibited 3H-leucine incorporation into cardiac myocytes in response to phenylephrine, which was significantly recovered in the presence of the NOS inhibitor NG-monomethyl-L-arginine acetate. These results indicate that endogenously generated NO by ecNOS gene transfer using AAV vectors inhibits the α-adrenergic agonist-induced cardiac protein synthesis at least partially via cGMP production.

Gene Transfer into Rat Renal Cells Using Adeno-Associated Virus Vectors

Adeno-associated virus (AAV) vectors have a number of attractive features, including lack of cytotoxicity, ability to transduce nondividing cells, and long-term transgene expression. We investigated whether rat renal cells could be efficiently transduced with AAV vectors. Rat glomerular mesangial cells were transduced with AAV-lacZ vector containing β-galactosidase gene in vitro, and the expression of β-galactosidase was evaluated by X-gal staining and ELISA. For ex vivo experiments, sections of rat kidneys were incubated with AAV-lacZ, and then evaluated by X-gal histochemical staining. The level of β-galactosidase expression in cultured rat mesangial cells increased in a dose-dependent manner (ranging from 1 × 105 to 5 × 106 particles/cell). When transduced with 5 × 106 vector particles/cell of AAV-lacZ, about 50% of mesangial cells were stained positively with X-gal, and the level of β-galactosidase expression reached 9.9 ± 1.5 ng/mg protein. Expression was detectable during the culture period for at least 7 days. X-gal histochemical examination of the ex vivo transduced renal tissue revealed tubular cell and interstitial tissue staining. However, gene transfer was not clearly observed in glomeruli. These findings suggest that AAV vectors have the potential for gene therapy of renal diseases.

Nitric Oxide Synthase Gene Transfer Inhibits Protein Synthesis of Rat Cardiac Mycocytes

We investigated whether nitric oxide (NO) synthase gene transfer could attenuate α-adrenergic agonist-induced growth of cardiac myocytes. First, we investigated the effects of exogenous NO and a cGMP analogue on protein synthesis of cultured neonatal rat cardiac myocytes. The NO donor, morpholinosydnonimine (SIN-1), and 8-bromo-cGMP caused concentration-dependent decreases in phenylephrine (Phe)-induced 3H-leucine incorporation into myocytes. We then transferred endothelial NO synthase (eNOS) gene into cardiac myocytes using adeno-associated virus (AAV) vectors. eNOS gene transfer into cardiac myocytes induced 140 kDa eNOS protein expression and significantly increased cGMP contents of myocytes compared with control cells. eNOS gene transfer also inhibited 3H-leucine incorporation into cardiac myocytes in response to Phe, which was significantly recovered in the presence of the NOS inhibitor NG-monometyl-L-arginine acetate. These results indicate that authentic NO attenuates the effects of the α-adrenergic agonist-induced cardiac hypertrophy at least partially via cGMP production, suggesting that eNOS gene transfer using AAV vectors is promising for the gene therapy of cardiac hypertrophy.