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Runt-related transcription factor 1 (RUNX1) is generally considered to function as a tumor suppressor in the development of leukemia, but a growing body of evidence suggests that it has pro-oncogenic properties in acute myelofd leukemia (AML). Here we have demonstrated that the antileukemic effect mediated by RUNX1 depletion is highly dependent on a functional p53-mediated cell death pathway. Increased expression of other RUNX family members, including RUNX2 and RUNX3, compensated forthe antitumor effect elicited by RUNX1 silencing, and simultaneous attenuation of all RUNX family members as a cluster led to a much stronger antitumor effect relative to suppression of individual RUNX members. Switching off the RUNX cluster using alkylating agent-conjugated pyrrole-imidazole (PI) polyamides, which were designed to specifically bind to consensus RUNX-binding sequences, was highly effective against AML cells and against several poorprognosis solid tumors in a xenograft mouse model of AML without notable adverse events. Taken together, these results identify a crucial role forthe RUNX cluster in the maintenance and progression of cancer cells and suggest that modulation of the RUNX cluster usingthe PI polyamide gene-switch technology is a potential strategy to control malignancies., Introduction Runt-related transcription factor 1 (RUNX1), also known as acute myelofd leukemia 1 protein (AML1), is an essential master transcription factor implicated in the differentiation of hematopofetic stem cells (1). [...]

ABSTRACT In the present study, we report the effects of the ethanol extract from Mallotus philippinensis bark (EMPB) on mesenchymal stem cell (MSC) proliferation, migration, and wound healing in vitro [...]

Diverse histone modifications are catalysed and recognized by various specific proteins, establishing unique modification patterns that act as transcription signals (1,2). In particular, histone H3 trimethylation at lysine 36 (H3K36me3) is associated with actively transcribed regions and has been proposed to provide landmarks for continuing transcription (3,4); however, the control mechanisms and functions of H3K36me3 in higher eukaryotes are unknown. Here we show that the H3K36me3-specific histone methyltransferase (HMTase) Wolf-Hirschhorn syndrome candidate 1 (WHSC1, also known as NSD2 or MMSET) functions in transcriptional regulation together with developmental transcription factors whose defects overlap with the human disease Wolf-Hirschhorn syndrome (WHS) (5,6). We found that mouse Whsc1, one of five putative Set2 homologues (2,7,8), governed H3K36me3 along euchromatin by associating with the cell-type-specific transcription factors Sall1, Sall4 and Nanog in embryonic stem cells, and Nkx2-5 in embryonic hearts, regulating the expression of their target genes. Whsc1-deficient mice showed growth retardation and various WHS-like midline defects, including congenital cardiovascular anomalies. The effects of Whsc1 haploinsufficiency were increased in Nkx2-5 heterozygous mutant hearts, indicating their functional link. We propose that WHSC1 functions together with developmental transcription factors to prevent the inappropriate transcription that can lead to various pathophysiologies., Deletions of human chromosome 4p16.3 cause the dominant disorder WHS, which is characterized by cranio-facial malformations, learning disability, growth delays, heart defects and a diverse array of associated problems, many [...]

Sustained hepatic inflammation induced by various causes can lead to liver fibrosis. Transcription factor NF-[kappa]B is important in regulating inflammatory responses, especially in macrophages. We presently investigated whether an NF-[kappa]B decoy, a synthetic oligodeoxynucleotide (ODN) imitating the NF-[kappa]B binding site, inhibited the inflammatory response after [CCl.sub.4] intoxication to prevent [CCl.sub.4]-induced hepatic injury and fibrosis. The NF-[kappa]B decoy was introduced into livers by injecting the spleens of mice, using a hemagglutinating virus of Japan (HVJ)-liposome method. ODN was transferred mainly to macrophages in normal or fibrotic livers. Increases in serum transaminases and production of inflammatory cytokines after a single challenge with [CCl.sub.4] were inhibited by the NF-[kappa]B decoy, which suppressed nuclear translocation of NF-[kappa]B in liver macrophages. Liver fibrosis induced by [CCl.sub.4] administration for 8 wk was suppressed by the NF-[kappa]B decoy, accompanied by diminished mRNA expression for transforming growth factor (TGF)-[beta], procollagen type 1 [alpha], and [alpha]-smooth muscle actin (SMA). In vitro, isolated liver macrophages showed increased DNA binding activity of NF-[kappa]B and inflammatory cytokine production alter hydrogen peroxide treatment; both increases were inhibited significantly by the NF-[kappa]B decoy. In contrast, NF-[kappa]B decoy transferred to isolated hepatic stellate cells (HSC) had no effect on their morphological activation or [alpha]-SMA expression, although the decoy accelerated tumor necrosis factor (TNF)- [alpha]-induced apoptosis in activated HSC. The effect of NF-[kappa]B decoy suppressing fibrosis probably results mainly from anti-inflammatory effects on liver macrophages, with a possible minor contribution from its direct proapoptotic effect on activated HSC. HVJ liposome; liver macrophage; oligodeoxynucleotide; hydrogen peroxide

Complex transitions in chromatin structure produce changes in genome function during development in metazoa. Linker histones, the last component of nucleosomes to be assembled into chromatin, comprise considerably divergent subtypes as compared with core histones. In all metazoa studied, their composition changes dramatically during early embryogenesis concomitant with zygotic gene activation, leading to distinct functional changes that are still poorly understood. Here, we show that early embryonic linker histone B4, which is maternally expressed, is functionally different from somatic histone H1 in influencing chromatin structure and dynamics. We developed a chromatin assembly system with nucleosome assembly protein-1 as a linker histone chaperone. This assay system revealed that maternal histone B4 allows chromatin to be remodeled by ATP-dependent chromatin remodeling factor, whereas somatic histone H1 prevents this remodeling. Structural analysis shows that histone B4 does not significantly restrict the accessibility of linker DNA. These findings define the functional significance of developmental changes in linker histone variants. We propose a model that holds that maternally expressed linker histones are key molecules specifying nuclear dynamics with respect to embryonic totipotency. ATP-dependent chromatin remodeling | linker histone assembly

Hepatocyte growth factor (HGF), a multifunctional cytokine, accelerates intestinal epithelial proliferation. We studied the effects of HGF in mice with trinitrobenzene sulfonic acid-induced colitis, which shows clinical and molecular resemblance to Crohn's disease. Mice with colitis repeatedly were transfected intramuscularly with human HGF cDNA. Weight, survival, histopathology, proinflammatory cytokine mRNAs, and leukocyte infiltration were assessed. Treatment with HGF cDNA induced tyrosine phosphorylation of intestinal c-Met/HGF receptors, inhibited apoptosis, and promoted mitosis in intestinal epithelial cells, accelerating intestinal epithelial restoration and suppressing inflammation. Transfection with HGF cDNA markedly suppressed intestinal mRNA expression of T-helper 1 cytokines such as interleukin-12 and -1[beta], interferon-[gamma], and tumor necrosis factor-[alpha]. Numbers of total and CD4-positive T cells, neutrophils, and myloperoxidase activity in intestinal epithelium were diminished by HGF gene transfer, which also prevented weight loss, and improved survival. HGF might prove useful for controlling inflammatory bowel disease. 2,4,6-trinitrobenzene sulfonic acid; gene therapy

Heparin-binding EGF-like growth factor (HB-EGF) is first synthesized as a membrane-anchored form (proHB-EGF), and its soluble form (sHB-EGF) is released by ectodomain shedding from proHB-EGF. To examine the significance of proHB-EGF processing in vivo, we generated mutant mice by targeted gene replacement, expressing either an uncleavable form ([HB.sup.uc]) or a transmembrane domain-truncated form ([HB.sup.[DELTA]tm]) of the molecule. [HB.sup.uc/uc] mice developed severe heart failure and enlarged heart valves, phenotypes similar to those in proHB-EGF null mice. On the other hand, mice carrying [HB.sup.[DELTA]tm] exhibited severe hyperplasia in both skin and heart. These results indicate that ectodomain shedding of proHB-EGF is essential for HB-EGF function in vivo, and that this process requires strict control.

Author(s): Takeshi Nishikawa [1, 2]; Diane Edelstein [1]; Xue Liang Du [1]; Sho-ichi Yamagishi [1]; Takeshi Matsumura [1]; Yasufumi Kaneda [3]; Mark A. Yorek [4]; David Beebe; Peter J. Oates; [...]

Byline: Junko Sakuma-Mochizuki (1), Mitsuhiro Yoshida (1), Kin ya Abe (1), Toru Arai (1), Masahide Mori (1), Yasufumi Kaneda (2), Seiji Hayashi (1) Abstract: An inflammatory cytokine, tumor necrosis factor (TNF)-[alpha], has been implicated in the pathogenesis of inflammatory lung diseases such as interstitial pneumonia (IP). To clarify the role of the inflammatory cytokine in the pathogenesis of lung inflammation, we introduced a murine TNF-[alpha] gene into murine lungs by the hemagglutinating virus of Japan (HVJ)-liposome method. Seven days after the TNF-[alpha] gene introduction resulted in marked cellular infiltration of alveoli, and mild histological change was observed 28 days after the gene introduction. Electron microscopic analysis revealed minimal deposition of collagen fibrils. Analysis of the BAL revealed that the total cell number was markedly increased 3 and 7 days after the gene introduction, and more than 90% of the cells were macrophages. The increase in the cell number was returned to below the normal level 28 days after the gene introduction. During the development of IP, TNF-[alpha] may regulate pathologic change of the pulmonary interstitium and alveolar cells. Author Affiliation: (1) Department of Molecular Medicine, Osaka University Medical School, 2--2 Yamada-oka, Suita, Osaka, 565-0871, Japan (2) Division of Gene Therapy Science, Osaka University Medical School, 2--2 Yamada-oka, Suita, Osaka, 565-0871, Japan Article History: Registration Date: 09/10/2004

An approach for promoting the rapid transfer of antisense oligodeoxynucleotides (ODN) into blood vessel endothelium is discussed. This approach utilizes the hemagglutinating virus of Japan-liposome method in the central nervous system. In vivo and in vitro delivery of fluorescein isothiocyanate (FTIC)-labeled ODN into primary cultured granular cells of the rat cerebellum are discussed.

To improve the efficiency of liposome-mediated DNA transfer as a tool for gene therapy, we have developed a fusigenic liposome vector based on principles of viral cell fusion. The fusion proteins of hemagglutinating virus of Japan (HVJ; also Sendai virus) are complexed with liposomes that encapsulate oligodeoxynucleotide or plasmid DNA. Subsequent fusion of HVJ-liposomes with plasma membranes introduces the DNA directly into the cytoplasm. In addition, a DNA-binding nuclear protein is incorporated into the HVJ-liposome particle to enhance plasmid transgene expression. The fusigenic viral liposome vector has proven to be efficient for the intracellular introduction of oligodeoxynucleotide, as well as intact genes up to 100 kbp, both in vitro and in vivo. Many animal tissues have been found to be suitable targets for fusigenic vital liposome DNA transfer. In the cardiovascular system, we have documented successful cytostatic gene therapy in models of vascular proliferative disease using antisense oligodeoxynucleotides against cell cycle genes, double-stranded oligodeoxynucleotides as 'decoys' to trap the transcription factor E2F, and expression of a transgene encoding the constitutive endothelial cell form of nitric oxide synthase. Similar strategies are also effective for the genetic engineering of vein grafts and for the treatment of a mouse model of immune-mediated glomerular disease.

The human interleukin (IL) 6 and IL-6 receptor genes ensure interstitial pneumonia with the characteristic histopathological alterations. The transforming growth factor beta-1 and platelet-derived growth factor B genes cause fibrosis and less of alveolitis, but IL-6 and IL-6 receptor induce effective alveolitis and less of fibrosis. For the experimental observations, expression vectors are incorporated in Wistar rats to the locally overexpression humoral factors in the lungs through the trachea.

A gene therapy strategy using a transcription factor decoy of the E2F binding site may be useful in stopping lesion formation after injury to blood vessels. Transfection with E2F decoy inhibits expression of vascular smooth muscle cell proliferation by preventing the activation of the genes that control cell cycle progression. In mice which sustained experimental carotid injury, neointimal formation was inhibited by E2F decoy two weeks after in vivo transfection and continued up to eight weeks.

Intraoperative gene therapy may be possible in the treatment of occlusive vascular diseases by altering vein graft adaptation and preventing autologous vein graft failure. A genetic engineering technique tested in rabbits used antisense oligodeoxynucleotides to block medial smooth muscle cell proliferation. This decreased the grafts' susceptibility to atherosclerosis.

A study of the factors influencing vascular smooth muscle cell accumulation indicate that endothelial cell nitric oxide synthase (ec-NOS) is an endogenous inhibitor of in vivo formation of vascular lesions. A Sendai virus/liposome in vivo gene transfer technique enables total expression of ec-NOS in vessel walls. The vascular reactivity of the damaged vessel and NO synthesis increases with ec-NOS gene transfection.

Introduction of the interleukin-10 gene into mice inhibited bleomycin-induced lung injury in vivo. Am J Physiol Lung Cell Mol Physiol 278: L914-L922, 2000.--Interleukin (IL)-10 has been shown to reduce many inflammatory reactions. We investigated the in vivo effects of IL-10 on a bleomycin-induced lung injury model. Hemagglutinating virus of Japan (HVJ)-liposomes containing a human IL-10 expression vector (hIL10-HVJ) or a balanced salt solution as a control (Cont-HVJ) was intraperitoneally injected into mice on day -3. This was followed by intratracheal instillation of bleomycin (0.8 mg/kg) on day 0. Myeloperoxidase activity of bronchoalveolar lavage fluid and tumor necrosis factor-[Alpha] mRNA expression in bronchoalveolar lavage fluid cells on day 7 and hydroxyproline content of the whole lung on day 21 were inhibited significantly by hILl0-HVJ treatment. However, Cont-HVJ treatment could not suppress any of these parameters. We also examined the in vitro effects of IL-10 on the human lung fibroblast cell line WI-38. IL-10 significantly reduced constitutive and transforming growth factor-[Beta]-stimulated type I collagen mRNA expression. However, IL-10 did not affect the proliferation of WI-38 cells induced by platelet-derived growth factor. These data suggested that exogenous IL-10 may be useful in the treatment of pulmonary fibrosis. transforming growth factor-[Beta]; pulmonary fibrosis; collagen

The human 15-lipoxygenase (15-LO) gene was transfected into rat kidneys in vivo via intra-renal arterial injection. Three days later, acute (passive) or accelerated forms of antiglomerular basement membrane antibody-mediated glomerulonephritis were induced in transfected and nontransfected or sham-transfected controls. Studies of glomerular functions (filtration and protein excretion) and ex vivo glomerular leukotriene [B.sub.4] biosynthesis at 3 hr, and up to 4 days, after induction of nephritis revealed preservation or normalization of these parameters in transfected kidneys that expressed human 15-LO mRNA and mature protein, but not in contralateral control kidneys or sham-transfected animals. The results provide in vivo-derived data supporting a direct anti-inflammatory role for 15-LO during immune-mediated tissue injury.

Increased Expression of DNA Cointroduced with Nuclear Protein in Adult Rat Liver INTRODUCTION OF FOREIGN GENES into cultured cells by methods such as calcium-phosphate precipitation, electroporation, virus vector, and liposome [...]