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OBJECTIVE: +HOX genes are expressed in the hematopoietic system and increasing data point to their involvement in the control of proliferation and/or differentiation. Genes belonging to the C cluster are preferentially expressed in developing and differentiated lymphoid lineages. However,recent studies demonstrated,by RT-PCR,that the HOXC4 gene is also actively transcribed in the most undifferentiated hematopoietic cells (CD34(+)38(low)) and in more mature myeloid and erythroid progenitors. We evaluated the expression of HOXC4 protein on human CD34(+) cells and the in vitro effect of its overexpression on proliferation and differentiation. MATERIALS AND METHODS: We assessed the expression of HOXC4 on human CD34(+) cells using a polyclonal antibody raised against the C-terminal portion of the protein expressed using the baculovirus system. Overexpression of HOXC4 in human CD34(+) cells was obtained by retroviral gene transfer; its effect on clonogenic (CFU-GM,BFU-E,and CFU-GEMM) and early progenitors (LTC-IC) was evaluated. RESULTS: The HOXC4 protein is indeed expressed in human CD34(+) cells,and its overexpression in human CD34(+) cells increases the proliferation potential of clonogenic and early progenitors. CFU-GM showed a median threefold expansion (range: 1.1-19.4; p textless 0.002) compared with control transduced with the vector alone. The increment of BFU-E was higher (median ninefold,range 2.5-35; p textless 0. 0009) and erythroid colonies presented a larger size with normal morphology. An even more marked effect was observed on LTC-IC (median 13,onefold; range 4.1-102.1,p textless 0.0001). CONCLUSION: We demonstrate that HOXC4 is expressed in CD34(+) cells and that its overexpression induces an in vitro expansion of committed as well as very early hematopoietic progenitors. The most striking effect was obtained on LTC-IC with an expansion of 13.1-fold. The enforced expression of HOXC4 induced a significant increase (p textless 0.009) in the number of erythroid colonies compared with CFU-GM,although without perturbing,at least in vitro,the maturation program of the cells. On the other hand,the effect of the gene overexpression did not induce any skewing in the colony types derived from the myeloid lineage. View Publication

BACKGROUND: Transforming growth factor beta (TGF-beta) plays an important role in skeletal remodelling. However,few studies have examined its effects on cultured human osteoblasts. Our aim is to characterise the biological effects of TGF-beta1 on human osteoblasts and to examine the interaction between TGF-beta1 and calcitriol. DESIGN: In vitro study employing two models of normal human osteoblasts: human bone marrow stromal cells [hMS/(OB)] containing osteoprogenitor cells and trabecular bone osteoblasts (hOB),which are mature osteoblasts. A reverse-transcriptase-polymerase-chain-reaction assay was employed to measure steady state mRNA levels of TGF-beta(s) isoforms and receptors. Effects of short-term treatment of TGF-beta1 on osteoblast proliferation and differentiation markers were assessed. The effect of cotreatment of calcitriol (10-8 M) and TGF-beta1 on osteoblast differentiation was also determined. RESULTS: Both hMS(OB) and hOB cells expressed mRNA transcripts of TGF-beta1,TGF-beta2,TGF-beta 3,TGF-beta type I and type II receptors. TGF-beta 1 stimulated osteoblast proliferation in hMS(OB) and in hOB cultures. In hOB cultures,TGF-beta1 stimulated AP production and cotreatment with calcitriol induced a synergistic increase in AP levels to 250 +/- 61% of calcitriol-treated controls. Effects of TGF-beta1 and calcitriol were less pronounced in hMS(OB) cultures. TGF-beta1 inhibited collagen type I production in hMS(OB) cells and these effects were abolished in presence of calcitriol. In presence of calcitriol,TGF-beta1 increased collagen type I production in hOB cells. In both hOB and hMS(OB) cultures,TGF-beta1 inhibited osteocalcin production. CONCLUSIONS: TGF-beta increases osteoblastic cell proliferation irrespective of the differentiation state. In presence of calcitriol,it initiates osteoblast cell differentiation and matrix formation. As TGF-beta inhibits osteocalcin production,other factors are necessary for inducing terminal differentiation of osteoblasts. The observed effects of TGF-beta on human osteoblasts in vitro may represent important regulatory steps in controlling osteoblast cell proliferation and differentiation in vivo. View Publication

Protein kinase CK2 (formerly casein kinase II) is a serine/threonine kinase overexpressed in many human tumors,transformed cell lines,and rapidly proliferating tissues. Recent data have shown that many cancers involve inappropriate reactivation of Wnt signaling through ectopic expression of Wnts themselves,as has been seen in a number of human breast cancers,or through mutation of intermediates in the Wnt pathway,such as adenomatous polyposis coli or beta-catenin,as described in colon and other cancers. Wnts are secreted factors that are important in embryonic development,but overexpression of certain Wnts,such as Wnt-1,leads to proliferation and transformation of cells. We report that upon stable transfection of Wnt-1 into the mouse mammary epithelial cell line C57MG,morphological changes and increased proliferation are accompanied by increased levels of CK2,as well as of beta-catenin. CK2 and beta-catenin co-precipitate with the Dvl proteins,which are Wnt signaling intermediates. A major phosphoprotein of the size of beta-catenin appears in in vitro kinase reactions performed on the Dvl immunoprecipitates. In vitro translated beta-catenin,Dvl-2,and Dvl-3 are phosphorylated by CK2. The selective CK2 inhibitor apigenin blocks proliferation of Wnt-1-transfected cells,abrogates phosphorylation of beta-catenin,and reduces beta-catenin and Dvl protein levels. These results demonstrate that endogenous CK2 is a positive regulator of Wnt signaling and growth of mammary epithelial cells. View Publication

Y-27632 [(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide++ + dihydrochloride] is widely used as a specific inhibitor of the Rho-associated coiled-coil forming protein serine/threonine kinase (ROCK) family of protein kinases. This study examined the inhibition mechanism and profile of actions of Y-27632 and a related compound,Y-30141 [(+)-(R)-trans- 4-(1-aminoethyl)-N-(1H-pyrrolo[2,3-b]pyridin-4-yl)cyclohexan-ecarboxamide dihydrochloride]. Y-27632 and Y-30141 inhibited the kinase activity of both ROCK-I and ROCK-II in vitro,and this inhibition was reversed by ATP in a competitive manner. This suggests that these compounds inhibit the kinases by binding to the catalytic site. Their affinities for ROCK kinases as determined by K(i) values were at least 20 to 30 times higher than those for two other Rho effector kinases,citron kinase and protein kinase PKN. [(3)H]Y-30141 was taken up by cells in a temperature- and time-dependent and saturable manner,and this uptake was competed with unlabeled Y-27632. No concentrated accumulation was found,suggesting that the uptake is a carrier-mediated facilitated diffusion. Y-27632 abolished stress fibers in Swiss 3T3 cells at 10 microM,but the G(1)-S phase transition of the cell cycle and cytokinesis were little affected at this concentration. Y-30141 was 10 times more potent than Y-27632 in inhibiting the kinase activity and stress fiber formation,and it caused significant delay in the G(1)-S transition and inhibition of cytokinesis at 10 microM. View Publication

Green tea has shown remarkable anti-inflammatory and cancer chemopreventive effects in many animal tumor bioassays,cell culture systems,and epidemiological studies. Many of these biological effects of green tea are mediated by epigallocatechin 3-gallate (EGCG),the major polyphenol present therein. We have earlier shown that EGCG treatment results in apoptosis of several cancer cells,but not of normal cells (J. Natl. Cancer Inst. 89,1881-1886 (1997)). The mechanism of this differential response of EGCG is not known. In this study,we investigated the involvement of NF-kappaB during these differential responses of EGCG. EGCG treatment resulted in a dose-dependent (i) inhibition of cell growth,(ii) G0/G1-phase arrest of the cell cycle,and (iii) induction of apoptosis in human epidermoid carcinoma (A431) cells,but not in normal human epidermal keratinocytes (NHEK). Electromobility shift assay revealed that EGCG (10-80 microM) treatment results in lowering of NF-kappaB levels in both the cytoplasm and nucleus in a dose-dependent manner in both A431 cells and NHEK,albeit at different concentrations. EGCG treatment was found to result in a dose-based differential inhibition of TNF-alpha- and LPS-mediated activation of NF-kappaB in these cells. The inhibition of NF-kappaB constitutive expression and activation in NHEK was observed only at high concentrations. The immunoblot analysis also demonstrated a similar pattern of inhibition of the constitutive expression as well as activation of NF-kappaB/p65 nuclear protein. This inhibition of TNF-alpha-caused NF-kappaB activation was mediated via the phosphorylative degradation of its inhibitory protein IkappaBalpha. Taken together,EGCG was found to impart differential dose-based NF-kappaB inhibitory response in cancer cells vs normal cells; i.e.,EGCG-mediated inhibition of NF-kappaB constitutive expression and activation was found to occur at much higher dose of EGCG in NHEK as compared to A431 cells. This study suggests that EGCG-caused cell cycle deregulation and apoptosis of cancer cells may be mediated through NF-kappaB inhibition. View Publication

U937 monocytic cells are shown here to express a range of PDE4,cAMP-specific phosphodiesterase (PDE) isoenzymes: the long isoenzymes,PDE4A4,PDE4D5 and PDE4D3,plus the short isoenzyme,PDE4B2. These isoenzymes provide around 76% of the total cAMP PDE activity of U937 cells. The specific activities of the total PDE4A,PDE4B and PDE4D activities were 0.63+/-0.09,8.8+/-0.2 and 34.4+/-2.9 pmol/min per mg of protein respectively. The PDE4 selective inhibitor,rolipram,inhibited immunopurified PDE4B and PDE4D activities similarly,with IC(50) values of approx. 130 nM and 240 nM respectively. In contrast,rolipram inhibited immunopurified PDE4A activity with a dramatically lower IC(50) value of around 3 nM. Rolipram increased phosphorylation of cAMP-response-element-binding protein (CREB) in U937 cells in a dose-dependent fashion,which implied the presence of both high affinity (IC(50) value approx. 1 nM) and low affinity (IC(50) value approx. 120 nM) components. Rolipram dose-dependently inhibited the interferon-gamma (IFN-gamma)-stimulated phosphorylation of p38 mitogen-activated protein (MAP) kinase in a simple monotonic fashion with an IC(50) value of approx. 290 nM. On this basis,it is suggested that rolipram inhibition of PDE4A4 is involved in regulating CREB phosphorylation but not IFN-gamma-stimulated p38 MAP kinase phosphorylation. PDE4A4 was also selectively activated by challenge of U937 cells with either bacterial lipopolysaccharide (LPS) or IFN-gamma through a process which was attenuated by both wortmannin and rapamycin. It is proposed that the PDE4A4 isoform is involved in compartmentalized cAMP signalling responses in U937 monocytes. View Publication

Cell fate during development is defined by transcription factors that act as molecular switches to activate or repress specific gene expression programmes. The POU transcription factor Oct-3/4 (encoded by Pou5f1) is a candidate regulator in pluripotent and germline cells and is essential for the initial formation of a pluripotent founder cell population in the mammalian embryo. Here we use conditional expression and repression in embryonic stem (ES) cells to determine requirements for Oct-3/4 in the maintenance of developmental potency. Although transcriptional determination has usually been considered as a binary on-off control system,we found that the precise level of Oct-3/4 governs three distinct fates of ES cells. A less than twofold increase in expression causes differentiation into primitive endoderm and mesoderm. In contrast,repression of Oct-3/4 induces loss of pluripotency and dedifferentiation to trophectoderm. Thus a critical amount of Oct-3/4 is required to sustain stem-cell self-renewal,and up- or downregulation induce divergent developmental programmes. Our findings establish a role for Oct-3/4 as a master regulator of pluripotency that controls lineage commitment and illustrate the sophistication of critical transcriptional regulators and the consequent importance of quantitative analyses. View Publication

Potent inhibition of Janus kinase 3 was found for a series of naphthyl(beta-aminoethyl)ketones (e.g. 7,pIC50 = 7.1+/-0.3). Further studies indicated that these compounds fragment in less than 1 h by retro-Michael reaction in the Jak3 in vitro ELISA assay procedure. The breakdown product of 7,2-naphthylvinyl ketone (22,pIC50 = 6.8+/-0.3) showed very similar inhibitory activity to 7. Compounds 7 (in neutral buffer) and 22 will be useful pharmacological tools for the investigation of the Janus tyrosine kinase Jak3. View Publication

Adult human mesenchymal stem cells are primary,multipotent cells capable of differentiating to osteocytic,chondrocytic,and adipocytic lineages when stimulated under appropriate conditions. To characterize the molecular mechanisms that regulate osteogenic differentiation,we examined the contribution of mitogen-activated protein kinase family members,ERK,JNK,and p38. Treatment of these stem cells with osteogenic supplements resulted in a sustained phase of ERK activation from day 7 to day 11 that coincided with differentiation,before decreasing to basal levels. Activation of JNK occurred much later (day 13 to day 17) in the osteogenic differentiation process. This JNK activation was associated with extracellular matrix synthesis and increased calcium deposition,the two hallmarks of bone formation. Inhibition of ERK activation by PD98059,a specific inhibitor of the ERK signaling pathway,blocked the osteogenic differentiation in a dose-dependent manner,as did transfection with a dominant negative form of MAP kinase kinase (MEK-1). Significantly,the blockage of osteogenic differentiation resulted in the adipogenic differentiation of the stem cells and the expression of adipose-specific mRNAs peroxisome proliferator-activated receptor gamma2,aP2,and lipoprotein lipase. These observations provide a potential mechanism involving MAP kinase activation in osteogenic differentiation of adult stem cells and suggest that commitment of hMSCs into osteogenic or adipogenic lineages is governed by activation or inhibition of ERK,respectively. View Publication

Hematologic malignancies such as acute and chronic myeloid leukemia are characterized by the malignant transformation of immature CD34(+) progenitor cells. Transformation is associated with elevated expression of the Wilm's tumor gene encoded transcription factor (WT1). Here we demonstrate that WT1 can serve as a target for cytotoxic T lymphocytes (CTL) with exquisite specificity for leukemic progenitor cells. HLA-A0201- restricted CTL specific for WT1 kill leukemia cell lines and inhibit colony formation by transformed CD34(+) progenitor cells isolated from patients with chronic myeloid leukemia (CML),whereas colony formation by normal CD34(+) progenitor cells is unaffected. Thus,the tissue-specific transcription factor WT1 is an ideal target for CTL-mediated purging of leukemic progenitor cells in vitro and for antigen-specific therapy of leukemia and other WT1-expressing malignancies in vivo. View Publication