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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

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

The molecular mechanisms by which tumor cells metastasize to bone are likely to involve invasion,cell adhesion to bone,and the release of soluble mediators from tumor cells that stimulate osteoclast-mediated bone resorption. Bisphosphonates (BPs) are powerful inhibitors of the osteoclast activity and are,therefore,used in the treatment of patients with osteolytic metastases. However,an added beneficial effect of BPs may be direct antitumor activity. We previously reported that BPs inhibit breast and prostate carcinoma cell adhesion to bone (Boissier et al.,Cancer Res.,57: 3890-3894,1997). Here,we provided evidence that BP pretreatment of breast and prostate carcinoma cells inhibited tumor cell invasion in a dose-dependent manner. The order of potency for four BPs in inhibiting tumor cell invasion was: zoledronate textgreater ibandronate textgreater NE-10244 (active pyridinium analogue of risedronate) textgreater clodronate. In addition,NE-58051 (the inactive pyridylpropylidene analogue of risedronate) had no inhibitory effect,whereas NE-10790 (a phosphonocarboxylate analogue of risedronate in which one of the phosphonate groups is substituted by a carboxyl group) inhibited tumor cell invasion to an extent similar to that observed with NE-10244,indicating that the inhibitory activity of BPs on tumor cells involved the R2 chain of the molecule. BPs did not induce apoptosis in tumor cells,nor did they inhibit tumor cell migration at concentrations that did inhibit tumor cell invasion. However,although BPs did not interfere with the production of matrix metalloproteinases (MMPs) by tumor cells,they inhibited their proteolytic activity. The inhibitory effect of BPs on MMP activity was completely reversed in the presence of an excess of zinc. In addition,NE-10790 did not inhibit MMP activity,suggesting that phosphonate groups of BPs are responsible for the chelation of zinc and the subsequent inhibition of MMP activity. In conclusion,our results provide evidence for a direct cellular effect of BPs in preventing tumor cell invasion and an inhibitory effect of BPs on the proteolytic activity of MMPs through zinc chelation. These results suggest,therefore,that BPs may be useful agents for the prophylactic treatment of patients with cancers that are known to preferentially metastasize to bone. View Publication

Breast cancer resistance protein (BCRP) is a novel member of the adenosine triphosphate-binding cassette superfamily of transport proteins. Transfection and enforced expression of BCRP in drug-sensitive cells confer resistance to mitoxantrone,doxorubicin,daunorubicin,and topotecan. We studied blast cells from 21 acute leukemia patients (20 acute myeloid leukemia,1 acute lymphocytic leukemia) for the expression of BCRP mRNA using a quantitative reverse-transcription polymerase chain reaction assay. BCRP mRNA expression varied more than 1000-fold among the samples tested,with low or barely detectable expression in half of the samples. Seven samples (33%) had relatively high expression of BCRP mRNA. High expression of BCRP did not correlate strongly with high expression of P-glycoprotein,suggesting that BCRP may cause resistance to certain antileukemic drugs in P-glycoprotein-negative cases. High expression of BCRP mRNA is sufficiently frequent in AML to warrant more extensive investigations to determine the relation of disease subtype and treatment outcome to BCRP expression and function. View Publication

There is no known treatment for fatty liver,a ubiquitous cause of chronic liver disease. However,because it is associated with hyperinsulinemia and insulin-resistance,insulin-sensitizing agents might be beneficial. To evaluate this possibility,insulin-resistant ob/ob mice with fatty livers were treated with metformin,an agent that improves hepatic insulin-resistance. Metformin improved fatty liver disease,reversing hepatomegaly,steatosis and aminotransferase abnormalities. The therapeutic mechanism likely involves inhibited hepatic expression of tumor necrosis factor (TNF) alpha and TNF-inducible factors that promote hepatic lipid accumulation and ATP depletion. These findings suggest a mechanism of action for metformin and identify novel therapeutic targets in insulin-resistant states. View Publication

The specificities of 28 commercially available compounds reported to be relatively selective inhibitors of particular serine/threonine-specific protein kinases have been examined against a large panel of protein kinases. The compounds KT 5720,Rottlerin and quercetin were found to inhibit many protein kinases,sometimes much more potently than their presumed targets,and conclusions drawn from their use in cell-based experiments are likely to be erroneous. Ro 318220 and related bisindoylmaleimides,as well as H89,HA1077 and Y 27632,were more selective inhibitors,but still inhibited two or more protein kinases with similar potency. LY 294002 was found to inhibit casein kinase-2 with similar potency to phosphoinositide (phosphatidylinositol) 3-kinase. The compounds with the most impressive selectivity profiles were KN62,PD 98059,U0126,PD 184352,rapamycin,wortmannin,SB 203580 and SB 202190. U0126 and PD 184352,like PD 98059,were found to block the mitogen-activated protein kinase (MAPK) cascade in cell-based assays by preventing the activation of MAPK kinase (MKK1),and not by inhibiting MKK1 activity directly. Apart from rapamycin and PD 184352,even the most selective inhibitors affected at least one additional protein kinase. Our results demonstrate that the specificities of protein kinase inhibitors cannot be assessed simply by studying their effect on kinases that are closely related in primary structure. We propose guidelines for the use of protein kinase inhibitors in cell-based assays. View Publication

There is evidence that the total cellular content of placental cord blood (PCB) grafts is related to the speed of engraftment,though the total nucleated cell (TNC) dose is not a precise predictor of the time of neutrophil or platelet engraftment. It is important to understand the reasons for the quantitative association and to improve the criteria for selecting PCB grafts by using indices more precisely predictive of engraftment. The posttransplant course of 204 patients who received grafts evaluated for hematopoietic colony-forming cell (CFC) content among 562 patients reported previously were analyzed using univariate and multivariate life-table techniques to determine whether CFC doses predicted hematopoietic engraftment speed and risk for transplant-related events more accurately than the TNC dose. Actuarial times to neutrophil and platelet engraftment were shown to correlate with the cell dose,whether estimated as TNC or CFC per kilogram of recipient's weight. CFC association with the day of recovery of 500 neutrophils/microL,measured as the coefficient of correlation,was stronger than that of the TNC (R = -0.46 and -0.413,respectively). In multivariate tests of speed of platelet and neutrophil engraftment and of probability of posttransplantation events,the inclusion of CFC in the model displaced the significance of the high relative risks associated with TNC. The CFC content of PCB units is associated more rigorously with the major covariates of posttransplantation survival than is the TNC and is,therefore,a better index of the hematopoietic content of PCB grafts. (Blood. 2000;96:2717-2722) View Publication

BACKGROUND Glycogen synthase kinase-3 (GSK-3) is a serine/threonine protein kinase,the activity of which is inhibited by a variety of extracellular stimuli including insulin,growth factors,cell specification factors and cell adhesion. Consequently,inhibition of GSK-3 activity has been proposed to play a role in the regulation of numerous signalling pathways that elicit pleiotropic cellular responses. This report describes the identification and characterisation of potent and selective small molecule inhibitors of GSK-3. RESULTS SB-216763 and SB-415286 are structurally distinct maleimides that inhibit GSK-3alpha in vitro,with K(i)s of 9 nM and 31 nM respectively,in an ATP competitive manner. These compounds inhibited GSK-3beta with similar potency. However,neither compound significantly inhibited any member of a panel of 24 other protein kinases. Furthermore,treatment of cells with either compound stimulated responses characteristic of extracellular stimuli that are known to inhibit GSK-3 activity. Thus,SB-216763 and SB-415286 stimulated glycogen synthesis in human liver cells and induced expression of a beta-catenin-LEF/TCF regulated reporter gene in HEK293 cells. In both cases,compound treatment was demonstrated to inhibit cellular GSK-3 activity as assessed by activation of glycogen synthase,which is a direct target of this kinase. CONCLUSIONS SB-216763 and SB-415286 are novel,potent and selective cell permeable inhibitors of GSK-3. Therefore,these compounds represent valuable pharmacological tools with which the role of GSK-3 in cellular signalling can be further elucidated. Furthermore,development of similar compounds may be of use therapeutically in disease states associated with elevated GSK-3 activity such as non-insulin dependent diabetes mellitus and neurodegenerative disease. View Publication

The imidazoquinoline R-848,originally identified as a highly effective antiviral agent,has recently been shown to be capable of potent B lymphocyte activation. The B cell-activating properties of R-848 are strikingly similar to the effects of the CD40 ligand CD154. The present study demonstrates that this similarity extends to the intracellular signaling pathways triggered by the compound,although both overlapping and distinct mechanisms of signaling were seen. Like CD40 ligation,R-848 stimulated activation of the stress-activated protein kinases c-Jun kinase and p38 and activated the NF-kappaB family of transcription factors. Both R-848- and CD40-mediated B cell differentiation were dependent upon NF-kappaB activation,although the relative importance of individual NF-kappaB family members appeared to differ between R-848- and CD40-mediated signals. Both signals were partially dependent upon induction of TNF-alpha and IL-6,and the cytoplasmic adaptor molecule TNF receptor-associated factor 2 is involved in both R-848- and CD40-mediated differentiation. View Publication

This study analyzes the gene repertoire coding for antibodies to an evolutionary novel immunogenic carbohydrate antigen in mice. The alpha-gal epitope (Gal alpha 1-3Gal beta 1-4GlcNAc-R) is an autoantigen,abundantly expressed in wild type mice,but absent in alpha 1,3galactosyltransferase knock-out (KO) mice,where it can induce the production of the anti-Gal antibody. Hybridoma clones secreting anti-Gal were isolated from different mice and their immunoglobulin genes were analyzed. All anti-Gal clones were found to be encoded by the heavy chain gene VH22.1 and light chain gene VK5.1. Moreover,one 'forbidden' anti-Gal clone,produced in a wild type mouse,was also encoded by VH 22.1 and VK 5.1. The genes coding for the different anti-Gal clones were found to contain somatic mutations and different CDR3 domains. These data imply that a highly restricted gene usage combined with junctional diversity and somatic mutations can generate new antibodies that have not been produced in the course of the evolution of a species. View Publication

It has long been known that small changes to the structure of the R(2) side chain of nitrogen-containing bisphosphonates can dramatically affect their potency for inhibiting bone resorption in vitro and in vivo,although the reason for these differences in antiresorptive potency have not been explained at the level of a pharmacological target. Recently,several nitrogen-containing bisphosphonates were found to inhibit osteoclast-mediated bone resorption in vitro by inhibiting farnesyl diphosphate synthase,thereby preventing protein prenylation in osteoclasts. In this study,we examined the potency of a wider range of nitrogen-containing bisphosphonates,including the highly potent,heterocycle-containing zoledronic acid and minodronate (YM-529). We found a clear correlation between the ability to inhibit farnesyl diphosphate synthase in vitro,to inhibit protein prenylation in cell-free extracts and in purified osteoclasts in vitro,and to inhibit bone resorption in vivo. The activity of recombinant human farnesyl diphosphate synthase was inhibited at concentrations textgreater or = 1 nM zoledronic acid or minodronate,the order of potency (zoledronic acid approximately equal to minodronate textgreater risedronate textgreater ibandronate textgreater incadronate textgreater alendronate textgreater pamidronate) closely matching the order of antiresorptive potency. Furthermore,minor changes to the structure of the R(2) side chain of heterocycle-containing bisphosphonates,giving rise to less potent inhibitors of bone resorption in vivo,also caused a reduction in potency up to approximately 300-fold for inhibition of farnesyl diphosphate synthase in vitro. These data indicate that farnesyl diphosphate synthase is the major pharmacological target of these drugs in vivo,and that small changes to the structure of the R(2) side chain alter antiresorptive potency by affecting the ability to inhibit farnesyl diphosphate synthase. View Publication

Reinfusion of ex vivo-expanded autologous megakaryocytes together with a stem cell transplantation may be useful to prevent or reduce the period of chemotherapy-induced thrombocytopenia. In this study,we analyzed several serum-containing and serum-free media to identify the most suitable medium for megakaryocyte expansion. Moreover,two thrombopoietin (Tpo)-mimetic peptides were tested to evaluate whether they could replace Tpo in an expansion protocol. To analyze the effects of different media on megakaryocyte expansion,we used an in vitro liquid culture system. For this purpose,CD34(+) cells were isolated from peripheral blood and cultured for 8 days in the presence of Tpo and interleukin-3 (IL-3). The presence of megakaryocytes was analyzed by flow cytometric analysis after staining for CD41 expression. For our standard culture procedure,megakaryocyte medium (MK medium) supplemented with 10% AB plasma was used. Addition of 5% or 2.5% AB plasma yielded higher numbers of megakaryocytes,implying the presence of inhibitory factors in plasma. However,some plasma components are required for optimal megakaryocyte expansion because addition of less than 1% AB plasma or addition of human serum albumin instead of AB plasma resulted in the formation of lower numbers of megakaryocytes. Two commercially available serum-free media were also tested: Cellgro and Stemspan. If CD34(+) cells were cultured in Cellgro medium similar numbers of megakaryocytes were obtained as when CD34(+) cells were cultured in MK medium supplemented with 10% AB plasma. In MK medium with 2.5% AB plasma,higher numbers of megakaryocytes were cultured than in MK medium supplemented with 10% AB plasma. Therefore,Cellgro medium is not the best alternative medium. In cultures with Stemspan medium,higher numbers of megakaryocytes were obtained compared to MK medium with 10% AB plasma. Stemspan is thus a good alternative for MK medium. Two Tpo-mimetic peptides,AF13948 and PK1M,were tested for their ability to replace Tpo. In cultures with AF13948,comparable numbers of megakaryocytes were obtained as in the presence of Tpo,but in cultures with PK1M the number of megakaryocytes was lower. This study shows that high concentrations of plasma in medium inhibits megakaryocyte formation,but some plasma components are required for optimal megakaryocyte expansion. For an ex vivo expansion protocol,it is worthwhile to test several media,because the number of megakaryocytes differs widely with the medium used. View Publication