bluuebunny/crossref_metadata_2025 huggingface dataset:
α‐cardiac actin (ACTC) binds to the band 3 (AE1) cardiac isoform
Authors: Moura Lima Paulo Roberto , Ide Salles Teresa Sueko , Costa Fernando Ferreira , Olalla Saad Sara Teresinha
Published: July 2003

The band 3 protein is the major integral protein present in the erythrocyte membrane. Two tissue‐specific isoforms are also expressed in kidney alpha intercalated cells and in cardiomyocytes. It has been suggested that the cardiac isoform predominantly mediates the anion exchange in cardiomyocytes, but the role of the cytoplasmic domain of the band 3 (CDB3) protein in the cardiac tissue is unknown. In order to characterize novel associations of the CDB3 in the cardiac tissue, we performed the two‐hybrid assay, using a bait comprising the region from leu 258 to leu 311 of the erythrocyte band 3, which must also be present in the cardiac isoform. The assay revealed two clones containing the C‐terminal region of the α‐cardiac actin. Immunoprecipitation of whole rat heart using an anti‐actin antibody, immunoblotted with anti‐human band 3, showed that actin binds to band 3 which was confirmed in the reverse assay. The confocal microscopy showed band 3 in the intercalated discs. Thus, besides the in vivo physical interaction in the Saccharomyces cerevisiae cell, we demonstrated using immunopreciptation that there is a physical association of band 3 with α‐cardiac actin in cardiomyocyte, and we suggest that the binding occur “in situ,” in the intercalated disc, a site of cell–cell contact and attachment of the sarcomere to the plasma membrane. © 2003 Wiley‐Liss, Inc.
TNF‐α induction of lipolysis is mediated through activation of the extracellular signal related kinase pathway in 3T3‐L1 adipocytes
Authors: Souza Sandra C. , Palmer Helen J. , Kang You‐Hou , Yamamoto Mia T. , Muliro Kizito V. , Eric Paulson K. , Greenberg Andrew S.
Published: July 2003

Tumor necrosis factor‐α (TNF‐α) increases adipocyte lipolysis after 6–12 h of incubation. TNF‐α has been demonstrated to activate mitogen‐activated protein (MAP) kinases including extracellular signal‐related kinase (ERK) and N‐terminal‐c‐Jun‐kinase (JNK) in different cell types. To determine if the MAP kinases have a role in TNF‐α‐induced lipolysis, 3T3‐L1 adipocytes were treated with the cytokine (10 ng/ml), in the presence or absence of PD98059 or U0126 (100 µM), specific inhibitors of ERK activity. We demonstrated that U0126 or PD98059 blocked TNF‐α‐induced ERK activity and decreased TNF‐α‐induced lipolysis by 65 or 76% respectively. The peroxisome‐proliferator‐activated receptor γ (PPARγ) agonists, rosiglitazone (ros), and 15‐deoxy‐Δ‐12,14‐ prostaglandin J2 (PGJ2) have been demonstrated to block TNF‐α‐induced lipolysis. Pretreatment of adipocytes with these agents almost totally blocked TNF‐α‐induced ERK activation and reduced lipolysis by greater than 90%. TNF‐α also stimulated JNK activity, which was not affected by PD98059 or PPARγ agonist treatment. The expression of perilipin, previously proposed to contribute to the mechanism of lipolysis, is diminished in response to TNF‐α treatment. Pretreatment of adipocytes with PD98059 or ros significantly blocked the TNF‐α‐induced reduction of perilipin A protein level as determined by Western analysis. These data suggest that activation of the ERK pathway is an early event in the mechanism of TNF‐α‐induced lipolysis. © 2003 Wiley‐Liss, Inc.
Phosphorylation regulates the interaction and complex formation between wt p53 protein and PARP‐1
Authors: Węsierska‐Gądek Józefa , Wojciechowski Jacek , Schmid Gerald
Published: July 2003

We recently characterized the interaction between poly(ADP‐ribose) polymerase‐1 (PARP‐1) and the product of the tumor suppressor gene p53. We investigated which domains of human PARP‐1 and of human wild‐type (wt) p53 were involved in this protein–protein interaction. We generated baculoviral constructs encoding full length or distinct functional domains of both proteins. Full length PARP‐1 was simultaneously coexpressed in insect cells with full length wt p53 protein or its distinct truncated fragments and vice versa. Reciprocal immunoprecipitation of Sf9 cell lysates revealed that the central and carboxy‐terminal fragments of p53 were sufficient to confer binding to PARP‐1, whereas the amino‐terminal part harboring the transactivation functional domain was dispensable. On the other hand, the amino‐terminal and central fragments of PARP‐1 were necessary for complex formation with p53 protein. As the most important features of p53 protein are regulated by phosphorylation, we addressed the question of whether its phosphorylation is essential for binding between the two proteins. Baculovirally expressed wt p53 was post‐translationally modified. At least six distinct p53 isomeres were resolved by immunoblotting following two‐dimensional separation of baculovirally expressed wt p53 protein. Using specific phospho‐serine antibodies, we identified phosphorylation of baculovirally expressed p53 protein at five distinct sites. To define the role of p53 phosphorylation, pull‐down assays using untreated and dephosphorylated p53 protein were performed. Dephosphorylated p53 failed to bind PARP‐1 indicating that complex formation between both proteins is regulated by phosphorylation of p53. The marked phosphorylation of p53 at Ser392 observed in unstressed cells suggests that the phosphorylated carboxy‐terminal part of p53 undergoes complex formation with PARP‐1 resulting in masking of the NES and thereby preventing its export. The functional significance of the interaction between both proteins was investigated at two different conditions: inactivation of PARP‐1 and overexpression of PARP‐1. Our results unequivocally show that the presence of PARP‐1 regulates the basal expression of wt p53 in unstressed cells. © 2003 Wiley‐Liss, Inc.
9‐cis retinoic acid accelerates calcitriol‐induced osteocalcin production and promotes degradation of both vitamin D receptor and retinoid X receptor in human osteoblastic cells
Authors: Jääskeläinen Tiina , Ryhänen Sanna , Mäenpää Pekka H.
Published: July 2003

Abstract vitamin D receptor (VDR) and retinoid X receptor (RXR) heterodimerize to mediate the genomic actions of 1α,25‐dihydroxyvitamin D3 (1α,25(OH)2D3, calcitriol), the biologically active form of vitamin D3. In this study, we show that 9‐cis retinoic acid (9‐cisRA), the ligand for RXR, accelerates calcitriol‐induced expression of osteocalcin gene, the marker for mature osteoblasts. Calcitriol and its synthetic analog KH1060 (1 nM) induced osteocalcin secretion after a 96‐h incubation period as detected by radioimmunoassay. When these compounds were used together with 9‐cisRA, osteocalcin protein secretion was, however, detected already after 72 and 48 h, respectively. Detection of osteocalcin mRNA with quantitative PCR revealed elevated mRNA levels already after a 4‐h treatment of the cells with calcitriol, KH1060, or 9‐cisRA compared with untreated cells. In combination treatments, 9‐cisRA rapidly stimulated osteocalcin mRNA synthesis induced by the different vitamin D3 compounds. In MG‐63 cells treated with calcitriol or KH1060, the stimulation was maximal after the first 4 h and diminished thereafter. In fact, after the 48‐h incubation 9‐cisRA reduced osteocalcin mRNA levels in KH1060‐treated cells, the amount of mRNA being only 44% of the levels obtained with KH1060 alone. The reduction was accompanied by an increased degradation rate of both VDR and RXRβ in the presence of 9‐cisRA. Furthermore, 9‐cisRA increased the formation of RXRβ–VDR–VDRE complex on the osteocalcin gene VDRE. These results suggest that 9‐cisRA accelerates calcitriol‐induced osteocalcin production in human osteoblastic cells through increased formation of transcriptionally active chromatin complexes and, subsequently, promotes degradation of the heterodimeric complex of VDR and RXR. © 2003 Wiley‐Liss, Inc.
Pro‐apoptotic activity of transiently expressed BCL‐2 occurs independent of BAX and BAK
Authors: Subramanian T. , Chinnadurai G.
Published: July 2003

BCL‐2 suppresses apoptosis induced by a wide variety of stimuli in multiple cell types. Most of the in vitro studies that have examined the activity of BCL‐2 have employed stable cell lines that ectopically express BCL‐2. We have reported that BCL‐2 is expressed at high levels in the absence of the 5′‐ and 3′‐UTRs of the Bcl‐2 gene and transient high level of expression results in potent cell death (Uhlmann et al., [1998]: JBC 278:17926–17932). Expression of BCL‐2 under the transcriptional control of the cognate 5′‐ and 3′‐UTRs express lower levels of BCL‐2 and does not cause cell death. Our present results suggest that in contrast to BCL‐2, transient expression of BCL‐xL does not induce cell death and coexpression of BCL‐xL with the pro‐apoptotic BCL‐2 does not suppress cell death. The pro‐apoptotic activity of BCL‐2 appears to involve activation of the cytochrome c/caspase 9/caspase 3 pathway. Elevated levels of BCL‐2 expression results in N‐terminal cleavage of BCL‐2 at a novel site different from a previously identified caspase cleavage site at Asp 34 by a non‐caspase protease. Transient expression of a BCL‐2 mutant lacking aa 51–85 within the loop region induces efficient cell death and N‐terminal cleavage of BCL‐2 while a different deletion mutant lacking aa 30–91 induces reduced levels of cell death in the absence of BCL‐2 cleavage suggesting that N‐terminal processing of BCL‐2 may be an amplification event in BCL‐2‐mediated cell death. Overexpression of BCL‐2 in a Bax‐null human colon cancer cell line (HCT116Bax−/−) induces efficient cell death. The pro‐apoptotic activity of BCL‐2 is also observed in a Bax‐null cells in which BAK expression is inhibited by stable RNAi expression. Our results suggest that BCL‐2 contains an intrinsic pro‐apoptotic activity and can induce apoptosis independent of BAX and BAK under specific conditions. © 2003 Wiley‐Liss, Inc.
Expression and localization of extracellular matrix metalloproteinase inducer in giant cell tumor of bone
Authors: Si Andrew I.C. , Huang Lin , Xu Jiake , Kumta Shekhar‐M , Wood David , Zheng Ming H.
Published: July 2003

Matrix metalloproteinases (MMPs) are regarded as a significant regulator in tumor invasion and metastasis. Previous studies have shown that extracellular matrix metalloproteinase inducer (EMMPRIN) in tumor cells induces the synthesis of MMPs. EMMPRIN is abundantly present on the surface of tumor cells and stimulate adjacent stromal cells to synthesize MMPs to induce tumor progression. Giant cell tumor (GCT) of bone is a benign but locally aggressive primary neoplasm of bone. The spindle‐shaped mononuclear stromal cells are considered to be the tumor components of GCT, which are capable of inducing osteoclast formation by recruiting the circulating monocyte and macrophage. In this study, we proposed that EMMPRIN is associated with the biological progression and aggressiveness of GCT. We have conducted semi‐quantitative RT‐PCR to determine the correlation of EMMPRIN expression with the clinical stage of GCT. We have also examined the cellular localization of EMMPRIN in GCT using in‐situ hybridization (ISH) and Immunohistochemistry (IH). The results showed that EMMPRIN was present in GCT and its mRNA levels were associated with the clinical stage of GCT. Higher expression level of EMMPRIN was observed in GCT with advanced stage (stage III). There was a great significance (P < 0.05) of EMMPRIN expression between stage I & II and stage III GCTs. Both ISH and IH demonstrated that EMMPRIN is present at the multinuclear osteoclast‐like giant cells of GCT, with strong immunostaining on the cell membrane. The stromal‐like tumor cells were also positively stained but the intensity was weaker. Interestingly, the production of EMMPRIN in osteoclast‐like cells of GCT seems to be regulated by stromal‐like tumor cells. Receptor activator of NF‐κB ligand (RANKL), which has been previously shown to be produced by the stromal‐like tumor cells for the recruitment of osteoclast‐like giant cells in GCT, enhanced the expression of EMMPRIN mRNA during the differentiation of macrophage‐like RAW264.7 cells into osteoclasts. In short, our studies suggest that EMMPRIN may be an important regulatory factor involved in the biological behaviors of GCT. © 2003 Wiley‐Liss, Inc.
Merosin (laminin‐2/4)‐driven survival signaling: Complex modulations of Bcl‐2 homologs
Authors: Laprise Patrick , Vallée Karine , Demers Marie‐Josée , Bouchard Véronique , Poirier Ève‐Marie , Vézina Anne , Reed John C. , Rivard Nathalie , Vachon Pierre H.
Published: July 2003

We have shown previously that the promotion of myofiber survival by the basement membrane component merosin (laminin‐2 [α2β1γ1]/laminin‐4 [α2β2γ1]) is dependent on the activity of the tyrosine kinase Fyn, whereas myofiber anoikis induced by merosin deficiency is dependent on the stress‐activated protein kinase p38α. To further understand such merosin‐driven survival signaling, we analyzed the expression of five Bcl‐2 homologs (Bcl‐2, Bcl‐XL, Bax, Bak, Bad) and one non‐homologous associated molecule (Bag‐1) in normal and merosin‐deficient myotubes, with or without pharmacological inhibitors for Fyn and p38. Herein, we report that (1) merosin deficiency induces anoikis and causes decreased Bcl‐2, Bcl‐XL, and Bag‐1 levels, increased Bax and Bak levels, and decreased Bad phosphorylation; (2) Bcl‐2, Bcl‐XL, Bag‐1, and Bad phosphorylation are also decreased in anoikis‐dying, Fyn‐inhibited myotubes; (3) the inhibition of p38α in Fyn‐inhibited and/or merosin‐deficient myotubes protects against anoikis and increases Bcl‐2 levels above normal, in addition to restoring Bad phosphorylation and Bag‐1 levels to normal; (4) the overexpression of merosin in deficient myotubes also rescues from anoikis and increases Bcl‐2 levels and Bad phosphorylation above normal, in addition to restoring Bcl‐XL, Bag‐1, Bax, and Bak levels to normal; and (5) Bcl‐2 overexpression is sufficient to rescue merosin‐deficient myotubes from anoikis, even though the expression/phosphorylation levels of the other homologs analyzed are not restored to normal. These results indicate that merosin‐driven myofiber survival signaling affects complex, differential modulations of individual Bcl‐2 homologs. These further suggest that Bcl‐2 can play a major role in suppressing myofiber anoikis. © 2003 Wiley‐Liss, Inc.
TAK‐778 enhances osteoblast differentiation of human bone marrow cells
Authors: Rosa Adalberto L. , Beloti Marcio M.
Published: July 2003

TAK‐778 has been shown to induce bone growth in in vitro and in vivo models. However, there are no studies evaluating the effect of TAK‐778 on human cells. Thus, the aim of this study was to investigate osteogenesis induced by TAK‐778 on human bone marrow cells. Cells were cultured in 24‐well culture plates at a cell density of 2 × 104 cells/well in culture medium containing TAK‐778 (10−7, 10−6, and 10−5 M, each) or vehicle. During the culture period, cells were incubated at 37°C in a humidified atmosphere of 5% CO2 and 95% air. For attachment evaluation, cells were cultured for 4 and 24 h. After 7, 14, and 21 days, cell proliferation, cell viability, total protein content, alkaline phosphatase (ALP) activity, and bone‐like formation were evaluated. Data were compared by ANOVA and Duncan's multiple range test. TAK‐778 did not affect cell attachment and viability. Cell number was reduced by TAK‐778 in all time period evaluated in a dose‐dependent way. The effect of TAK‐778 on total protein content, ALP activity and bone‐like formation was a dose‐dependent increase. The present results suggest that initial cell events such as cell attachment are not affected by TAK‐778 while events that indicate osteoblast differentiation including reduced cell proliferation, and increased both ALP activity and bone‐like formation are enhanced by TAK‐778 in a time and dose‐dependent way. It means that TAK‐778 could be a useful drug to enhance new bone formation in clinical situations that require rapid restoration of physiologic function, such as orthopedic and maxillofacial surgery. © 2003 Wiley‐Liss, Inc.
Co‐culture of endothelial cells and smooth muscle cells affects gene expression of angiogenic factors
Authors: Heydarkhan‐Hagvall Sepideh , Helenius Gisela , Johansson Bengt R. , Li Julie Y. , Mattsson Erney , Risberg Bo
Published: July 2003

Endothelial cells (EC) are in contact with the underlying smooth muscle cells (SMC). The interactions between EC and SMC in the vessel wall are considered to be involved in the control of growth and function of blood vessels. A co‐culture system of EC and SMC and a method for separation of these cells was developed in order to investigate whether the presence of physical contact between EC and SMC affected the gene expression of angiogenic factors. Human EC and SMC were prepared from the great saphenous veins. Autologous EC were added on top of the confluent layer of SMC. After 72 h in co‐culture, the EC were magnetically separated from SMC with the use of superparamagnetic beads. RT‐PCR products for bFGF, bFGFR, VEGF, PDGF‐AA, PDGF‐BB, TGF‐β, and β‐actin were analyzed to study the mRNA expressions. The protein level of selected factors was studied by ELISA technique. In co‐cultured SMC there was a statistically significant higher gene expression of VEGF, PDGF‐AA, PDGF‐BB, and TGF‐β and significant lower gene expression of bFGF and its receptor than in single cultured SMC. The protein level of PDGF‐BB and TGF‐β was also significantly higher in co‐cultured SMC. In co‐cultured EC there were no significant differences in gene expression of PDGF‐AA, PDGF‐BB, and TGF‐β compared with single cultured EC. The gene expression and protein synthesis of VEGF was significantly higher in co‐cultured EC. The findings from the present study suggest that cell‐cell interactions of EC and SMC affect the gene and protein expression of angiogenic factors. © 2003 Wiley‐Liss, Inc.
Epiregulin is more potent than EGF or TGFα in promoting in vitro wound closure due to enhanced ERK/MAPK activation
Authors: Draper Bradley K. , Komurasaki Toshi , Davidson Mari K. , Nanney Lillian B.
Published: July 2003

Epiregulin (EPR) is a broad specificity EGF family member that activates ErbB1 and ErbB4 homodimers and all possible heterodimeric ErbB complexes. We have previously shown that topical EPR enhances the repair of murine excisional wounds. The purpose of this study was to determine whether EPR was more effective than EGF or TGFα in promoting in vitro wound closure and to compare the EPR induced signal transduction pathways with those activated by EGF and TGFα. Normal human epidermal keratinocytes or A431 cells were scratch wounded and treated for 24 h with varying doses of EPR, EGF or TGFα. Five‐fold lower doses of EPR were significantly better than EGF or TGFα in stimulating in vitro wound closure. Mitomycin‐c reduced EPR induced wound closure by 59%, versus a 9% and 25% decrease in EGF and TGFα induced closure. The ERK/MAPK inhibitor PD‐98059 decreased EPR induced wound closure by 88%. By contrast, the PLC inhibitor U‐73122, only reduced the EPR induced response by 21%. Immunoblot analysis revealed that 2 nM EPR stimulated a six‐fold increase in p‐ERK1/2, whereas 10 nM EGF or TGFα stimulated only a 3‐ and 2.5‐fold increase in p‐ERK1/2. When compared with EGF or TGFα, EPR is a more potent and more effective inducer of in vitro wound closure due to its ability to promote significantly greater ERK/MAPK activation. © 2003 Wiley‐Liss, Inc.
Non‐apoptogenic killing of hela cervical carcinoma cells after short exposure to the alkylating agent N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG)
Authors: Węsierska‐Gądek Józefa , Gueorguieva Marieta , Schloffer Daniela , Uhl Maria , Wojciechowski Jacek
Published: July 2003

We examined the action of N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG) on HeLa cells and compared it with that of cisplatin (CP). MNNG directly killed a substantial number of cells within 1 hour and resulted in strong DNA‐damage as evidenced by Comet measurements. Despite appearance of DNA lesions, p53 protein was not activated. Analysis of HeLa cells treated with MNNG for 1h, 3h and 6h by flow cytometry and by Hoechst staining did not reveal any sub‐G1 cell population and chromatin condensation/fragmentation characteristic for apoptosis, respectively. Also, no biochemical changes typical for apoptosis such as activation of caspase‐3 or release of cytochrome C from mitochondria were detected. Inactivation of PARP‐1 reduced the direct cytotoxicity exerted by MNNG. Our results showing that despite appearance of severe DNA lesions after short exposure of HeLa cells to MNNG neither activation of p53 response nor induction of apoptosis occurred implicate that generation of strong DNA damage is not sufficient to stabilize p53 protein in HeLa cells. Our data unequivocally show that the conscientious determination of the type of cell death induced by genotoxic agents is necessary. The assessment of the changes based on at least a few independent criteria is required to discriminate between apoptosis and necrosis. Since the alkylating agents generate DNA strand breaks, the recruitment of methods based on determination of DNA cleavage such as DNA ladder or TUNEL assay for evaluation of apoptosis is not adequate. © 2003 Wiley‐Liss, Inc.
Down‐regulation of Flt‐1 gene expression by the proteasome inhibitor MG262
Authors: Mezquita J. , Mezquita B. , Pau M. , Mezquita C.
Published: July 2003

The mechanisms involved in the anti‐angiogenic actions of the proteasome inhibitors are poorly understood. Here, we report that the gene expression of the VEGF receptor Flt‐1 (vascular endothelial growth factor receptor 1) was down‐regulated by the reversible proteasome inhibitor MG262 in explant cultures of the developing chicken pecten oculi, a vascular organ consisting of endothelial cells, pericytes, and macrophages. In addition, the inhibitor prevented the induction of Flt‐1 by lipopolysaccharide (LPS) in macrophages and down‐regulated the expression of Flt‐1 after LPS induction. Flt‐1 gene expression was also down regulated by MG262 in cultures of human microvascular endothelial cells. Interestingly, a transcript of Flt‐1, coding for a soluble form of the receptor (sFlt‐1) with anti‐angiogenic properties, was not down‐regulated in the same extent. Only a small decrease in the expression of VEGF and Ang‐2 was detected in the pecten oculi upon inhibition of the proteasome, while no major changes were observed in the expression of other angiogenic molecules, such as KDR or Ang‐1. Since recent experiments have demonstrated the importance of anti‐Flt‐1 therapy in the inhibition of tumor angiogenesis, retinal angiogenesis, arthritis, and atherosclerosis (Luttun et al. [2002]: Nat Med 8:831–840), our observation on down‐regulation of Flt‐1 in microvascular endothelial cells and macrophages by MG262 supports the postulated role of the proteasome inhibitors as potential candidates for therapeutic modulation of angiogenesis and inflammation. © 2003 Wiley‐Liss, Inc.
Establishment of a T‐Ag and NF‐κB binding elements gene co‐transfected stable HUVECs cell line
Authors: Liu Y.X. , Cao W.S. , Irani K. , Li X.J.
Published: July 2003

To set up a T‐Ag gene transfected stable human umbilical venous endothelial cells (HUVECs) cell line and a T‐Ag, nuclear transcriptional factor kappa B (NF‐κB) binding elements linked with luciferase reporter gene co‐transfected stable HUVECs cell line. Cultured HUVECs were transfected with pCI‐neo‐T‐Ag and pRSV‐luc‐3XκB by lipofectin. The G418 selected monoclones were subcultured. The expression of marker protein, vWF and the characteristic of uptake of lipids were compared by Western blotting and immunocytochemistry in non‐transfected and transfected HUVECs. The reporter gene assay was done in the presence of TNF‐α. A T‐Ag gene transfected stable HUVECs cell line and a T‐Ag and NF‐κB binding elements linked with luciferase reporter gene co‐transfected stable HUVECs cell lines were set up. The expression of vWF of these cell lines was similar with those in non‐transformed HUVECs. The function of uptaking of lipids was preserved as well in transfected cell lines. Furthermore, TNF‐α, a typical cytokine increasing the activity of NF‐κB was used to treat the transfected cells O/N. The higher luciferase reporter gene activity was seen. A pCI‐neo‐T‐Ag and pRSV‐luc‐3X κB co‐transfected stable HUVECs cell line might be used to check reporter gene activity directly. It might be a useful tool to screen drugs acting on transcription level. © 2003 Wiley‐Liss, Inc.
Effect of overexpressing fibroblast growth factor 2 protein isoforms in osteoblastic ROS 17/2.8 cells
Authors: Xiao L. , Liu P. , Sobue T. , Lichtler A. , Coffin J.D. , Hurley M.M.
Published: July 2003

Fibroblast growth factor‐2 (FGF‐2) is made by osteoblasts and modulates their function. There are high molecular weight (HMW) protein isoforms of FGF‐2 that have nuclear localization sequences and a low molecular weight (LMW) 18 kDa FGF‐2 protein that is exported from cells. Since FGF‐2 is a trophic factor and potent mitogen for osteoblasts, the goal of this study was to utilize targeted overexpression of FGF‐2 as a novel means of assessing different FGF‐2 isoforms on osteoblastic cell viability and proliferation. Either LMW or HMW human Fgf2 cDNAs were cloned downstream of 3.6 kb α1(I)—collagen 5′ regulatory elements (Col 3.6). A set of expression vectors, called Col3.6‐Fgf2 isoforms‐IRES‐GFPsaph, capable of concurrently overexpressing either LMW or HMW FGF‐2 isoforms concomitant with GFPsaph from a single bicistronic mRNA were built. Viable cell number in ROS 17/2.8 cells stably transfected with Vector (Col3.6‐IRES‐GFPsaph) versus each of the Col3.6‐Fgf2‐IRES‐GFPsaph constructs were compared. In the presence of 1 or 10% serum, DNA synthesis was increased in cells expressing any isoform of FGF‐2 compared with vector. However, cells transfected with HMW isoform had augmented DNA synthesis in 1 or 10% serum compared with cells expressing either ALL or LMW FGF‐2 isoforms. A neutralizing FGF‐2 antibody significantly reduced the mitogenic response in cells harboring ALL or the LMW FGF‐2 isoforms but did not block the mitogenic effect of cells harboring the HMW isoforms. In summary, overexpression of any isoform of FGF‐2 protein increased viable cell number and OB proliferation in the presence of low or high concentrations of serum. However, the HMW/nuclear isoforms preferentially mediate augmented OB proliferation. We conclude that differential expression of FGF‐2 proteins isoforms is important in modulating OB function. © 2003 Wiley‐Liss, Inc.
LPS induces pulmonary intravascular macrophages producing inflammatory mediators via activating NF‐κB
Authors: Chen Zheng‐Tang , Li Sheng‐Liang , Cai En‐Qi , Wu Wei‐Ling , Jin Jing‐Sheng , Zhu Bo
Published: July 2003

Pulmonary intravascular macrophages (PIMs) are often responsible for the clearance of blood‐borne pathogens, including endotoxin, lipopolysaccharide of Gram‐negative bacteria. It is well accepted that PIMs play a pivotal role in the pathogenesis of endotoxin‐induced acute lung injury. However, the mechanisms by which PIMs are involved in the lipopolysaccharide‐induced inflammatory responses remain unclear. Through the present study the following results were found: (1) When challenged with lipopolysaccharide (10 μg/ml), PIMs underwent marked cellular enlargement, intercellular adhesion plaques became longer, and some particulates were enwrapped in the pseudopods. (2) Lipopolysaccharide could up‐regulate the expression of some inflammatory mediators in PIMs, including TNF‐α, IL‐1β, IL‐6, IL‐8, and COX‐2, and these up‐regulated expression of inflammatory mediators correlated with NF‐κB activation. (3) Dexamethasone as well as acetylsalicylic acid reduced the expression of TNF‐α in lipopolysaccharide‐challenged PIMs, and the decreased expression of TNF‐α was also consistent with decreased NF‐κB activation. Our results suggest that NF‐κB activation in PIMs followed by phagocytizing lipopolysaccharide resulted in the up‐regulation of TNF‐α, IL‐1β, IL‐6, IL‐8, and COX‐2, which could be alleviated by dexamethasone. © 2003 Wiley‐Liss, Inc.
Non‐random features of loop‐size chromatin fragmentation
Authors: Szilágyi Ildikó , Varga Tamás , Székvölgyi Lóránt , Hegedüs Éva , Goda Katalin , Kaczur Viktória , Bacsó Zsolt , Nakayama Yuji , Pósafi János , Pongor Sándor , Szabó Gábor
Published: July 2003

Upon isolation of DNA from normal eukaryotic cells by standard methods involving extensive proteolytic treatment, a rather homogeneous population of loop‐size, double‐stranded DNA fragments is regularly obtained. These DNA molecules can be efficiently end‐labeled by the DNA polymerase I Klenow fragment, as well as by a 3′‐ to ‐5′‐exonuclease‐free Klenow enzyme, but not by terminal transferase (TdT) unless the ends have been filled up by Klenow, suggesting that dominantly 5′ protruding termini are generated upon fragmentation. The filled‐up termini were used for cloning the distal parts of the ∼50 kb fragments. BLAST analysis of the sequence of several clones allowed us to determine the sequence of the non‐cloned side of the breakpoints. Comparison of 25, 600 bp‐long breakpoint sequences demonstrated prevalence of repetitive elements. Consensus motives characteristic of the breakpoint sequences have been identified. Several sequences exhibit peculiar computed conformational characteristics, with sharp transition or center of symmetry located exactly at the breakpoint. Our data collectively suggest that chromatin fragmentation involves nucleolytic cleavages at fragile/hypersensitive sites delimiting loop‐size fragments in a non‐random manner. Interestingly, the sequence characteristics of the breakpoints are reminiscent of certain breakpoint cluster regions frequently subject to gene rearrangements. © 2003 Wiley‐Liss, Inc.
Characterization of mesenchymal stem cells isolated from murine bone marrow by negative selection
Authors: Baddoo Melody , Hill Katy , Wilkinson Robin , Gaupp Dina , Hughes Catherine , Kopen Gene C. , Phinney Donald G.
Published: July 2003

Mesenchymal stem cells (MSCs) are typically enriched from bone marrow via isolation of the plastic adherent, fibroblastoid cell fraction. However, plastic adherent cultures elaborated from murine bone marrow are an admixture of fibroblastoid and hematopoietic cell types. Here we report a reliable method based on immunodepletion to fractionate fibroblastoid cells from hematopoietic cells within plastic adherent murine marrow cultures. The immunodepleted cells expressed the antigens Sca‐1, CD29, CD44, CD81, CD106, and the stem cell marker nucleostemin (NST) but not CD11b, CD31, CD34, CD45, CD48, CD90, CD117, CD135, or the transcription factor Oct‐4. They were also capable of differentiating into adipocytes, chondrocytes, and osteoblasts in vitro as well as osteoblasts/osteocytes in vivo. Therefore, immunodepletion yields a cell population devoid of hematopoietic and endothelial cells that is phenotypically and functionally equivalent to MSCs. The immunodepleted cells exhibited a population doubling time of approximately 5–7 days in culture. Poor growth was due to the dramatic down regulation of many genes involved in cell proliferation and cell cycle progression as a result of immunodepletion. Exposure of immunodepleted cells to fibroblast growth factor 2 (FGF2) but not insulin‐like growth factor (IGF), murine stem cell factor, or leukemia inhibitory factor (LIF) significantly increased their growth rate. Moreover, 82% of the transcripts down regulated by immunodepletion remain unaltered in the presence of FGF2. Exposure to the later also reversibly inhibited the ability of the immunodepleted cells to differentiate into adipocytes, chondrocytes, and osteoblasts in vitro. Therefore, FGF2 appears to function as a mitogen and self‐maintenance factor for murine MSCs enriched from bone marrow by negative selection. © 2003 Wiley‐Liss, Inc.
Jun N‐terminal kinase pathway enhances signaling of monocytic differentiation of human leukemia cells induced by 1,25‐dihydroxyvitamin D3
Authors: Wang Qing , Wang Xuening , Studzinski George P.
Published: July 2003

Recent studies revealed that the MEK/ERK module of the mitogen‐activated protein kinase (MAPK) signaling cascades is up‐regulated in the early stages of 1α,25‐dihydroxyvitamin D3 (1,25D3)‐induced monocytic differentiation of human leukemia cells HL60. In the present study, we investigated whether another MAPK module, the JNK pathway, also participates in this form of differentiation. We found that the dependence on the concentration of the inducer, the vitamin‐hormone 1,25D3, in two types of human leukemia cells, HL60 and U937, and the kinetics of monocytic differentiation in HL60 cells, parallel the degree of the activation of the JNK pathway. A blockade of JNK signaling by a stable expression of dominant negative (dn) JNK1 mutant in U937 cells resulted in reduced c‐jun phosphorylation, and the differentiation of these cells was markedly decreased. Similarly, inhibition of JNK1 and JNK2 activities by the selective inhibitor SP600125 led to both dose‐dependent reduction of c‐jun and ATF‐2 phosphorylation, and of the differentiation of HL60 cells. In addition, we found that JNK activity is essential for the AP‐1 DNA binding induced by 1,25D3 in HL60 and U937 cells. The results indicate that in cultured human leukemia cells, the JNK pathway participates in the induction of monocytic differentiation by 1,25D3, probably by activating the AP‐1 transcription factor. © 2003 Wiley‐Liss, Inc.
Increased expression of Mcl‐1 is responsible for the blockage of TRAIL‐induced apoptosis mediated by EGF/ErbB1 signaling pathway
Authors: Henson Elizabeth S. , Gibson Erika M. , Villanueva Jacylyn , Bristow Nicolle A. , Haney Neil , Gibson Spencer B.
Published: July 2003

Epidermal growth factor (EGF) protects against death receptor induced apoptosis in epithelial cells. Herein, we demonstrate that EGF protection against tumor necrosis factor related apoptosis‐inducing ligand (TRAIL) induced apoptosis is mediated by increased expression of the Bcl‐2 family member myeloid cell leukemia 1 (Mcl‐1). EGF increased the mRNA and protein levels of Mcl‐1. Furthermore, expression of ErbB1 alone or in combination with ErbB2 in NIH3T3 cells up‐regulates Mcl‐1 following EGF treatment. In addition, up‐regulation of Mcl‐1 by EGF is mediated through AKT and NFκB activation since kinase inactive AKT and ΔIκB effectively blocks this up‐regulation. NFκB was also critical for the ability of EGF to prevent TRAIL induced apoptosis as a dominant negative IκB (ΔIκB) blocked NFκB activation, and relieved EGF protection against TRAIL mediated mitochondrial cytochrome‐c release and apoptosis. Finally, anti‐sense oligonucleotides directed against Mcl‐1 effectively reduced the protein levels of Mcl‐1 and blocked EGF protection against TRAIL induced mitochondrial cytochrome‐c release and apoptosis. Taken together, EGF signaling leads to increased Mcl‐1 expression that is required for blockage of TRAIL induced apoptosis. © 2003 Wiley‐Liss, Inc.
Development of a dual‐luciferase fusion gene as a sensitive marker for site‐directed DNA repair strategies
Authors: Bennett Michael , Schaack Jerome
Published: July 2003

Background

Several novel techniques have been developed recently for the site‐specific repair of DNA as an approach to gene therapy. Correction efficiencies as high as 40% have been reported, well within the range of therapeutic impact for a number of genetic diseases. Unfortunately, many of the model systems in which these methods have been employed typically target genes that are not well suited for analyzing the various techniques.

Methods

To address this, we have constructed and characterized a dual‐luciferase fusion gene as a sensitive marker for optimizing repair strategies. The genes encoding two distinct luciferase proteins were fused so that expression of one luciferase necessitated expression of the other. Engineering a stop codon in the downstream luciferase gene created an ideal tool to study the efficiency of various site‐directed DNA repair techniques as one luciferase can act as an internal control while the other is targeted for correction.

Results

Fusing two luciferase genes resulted in a single protein that produces two bioluminescent activities in a constant ratio. The utility of this system as a target for site‐directed DNA repair research was demonstrated using two of the recently developed gene repair techniques, small fragment homologous replacement and oligonucleotide‐mediated repair, to mediate correction and by the ability to detect repair efficiencies of less than 5 × 10−6 (<1 event in 200 000).

Conclusions

The ability to rapidly and accurately quantify the amount of correction using the dual‐luciferase fusion system will allow the comparison and evaluation of the many factors involved in successful gene repair and lead to the optimization of these techniques, both in cell culture and in whole animals. Copyright © 2003 John Wiley & Sons, Ltd.
Dendritic cells engineered to express the Flt3 ligand stimulate type I immune response, and induce enhanced cytoxic T and natural killer cell cytotoxicities and antitumor immunity
Authors: Liu Yongqing , Huang Hui , Chen Zhuang , Zong Li , Xiang Jim
Published: July 2003

Background

Tumor antigen presentation by dendritic cells (DCs) to T cells in lymphoid organs is crucial for induction of antitumor immune responses. Fms‐like tyrosine kinase 3 ligand (Flt3L) is a regulator of hematopoietic cell development.

Methods

To investigate the potential effect of Flt3L transgene expression on DC‐based cancer vaccines, we constructed a recombinant adenovirus AdVFlt3L expressing Flt3L, transfected DCs with AdVFlt3L, and investigated the efficacy of antitumor immunity by vaccination of DCFlt3L engineered to express Flt3L transgene.

Results

Our data demonstrated that AdVFlt3L transfection up‐regulated the expression of cytokine IL‐1β and chemokines MIP‐1α, MIP‐1β, IP‐10, MCP‐1 and MIP‐2, and stimulated DCFlt3L cell proliferation in vitro and migration toward regional lymph nodes in vivo. Our data also demonstrated that vaccination of Mut1‐pulsed DCFlt3L cells was able to stimulate (i) a type 1 immune response comprising CD4+ Th1 and CD8+ Tc1 activation and (ii) around 2‐ and 3‐fold enhanced tumor‐specific cytotoxic T lymphocyte (CTL) and non‐specific NK responses (p < 0.05) than vaccination with similarly pulsed control virus‐transfected and untransfected DCs, respectively. More importantly, vaccination of Mut1‐pulsed DCFlt3L cells induced enhanced antitumor immunity in vivo, even against poorly immunogenic 3LL tumor cells. Vaccinations of Mut1‐pulsed DCs, DCpLpA and DCFlt3L all protected mice from challenge of low dose (0.5 × 105) tumor cells. However, only vaccination of the last one was able to protect 63% (6/8) mice from challenge of high dose (3 × 105) 3LL tumor cells (p < 0.01).

Conclusions

DCs engineered to secrete Flt3L may offer a new strategy in DC‐based cancer vaccines. Copyright © 2003 John Wiley & Sons, Ltd.
Increased non‐viral gene transfer levels in mice by concentration of cationic lipid DNA complexes formed under optimized conditions
Authors: Nchinda Godwin , Zschörnig Olaf , Überla Klaus
Published: July 2003

Background

A number of non‐viral gene transfer reagents including cationic lipid DNA complexes (LDC) have been developed and were improved by changing the ratio of their components. To determine the effect of other parameters during complex formation affecting the efficacy of LDC, the conditions during complexation were varied without changing the ratios of the components.

Methods

LDC were formed at fixed ratios of an equimolar mixture of DOTAP and cholesterol to DNA to the condensing agent protamine sulfate according to different protocols at varying final concentrations. The influence of these parameters on transfection efficiency and physical properties of the complexes was determined.

Results

Changing the order of addition of compounds during complex formation affected the size distribution, the charge of the LDC, the interaction between the lipids and the accessibility of the DNA. At fixed ratios of the components, higher transfection efficiencies were observed with more condensed LDC. Complexation in higher volumes increased transduction efficiency of the complexes after intravenous inoculation. Due to restrictions on the injectable volume, the LDC were formed in the optimal volume and subsequently concentrated by ultrafiltration. The concentrated complexes maintained transduction efficiency and up to 60‐fold higher in vivo transduction levels were obtained.

Conclusions

In addition to the ratio of the components of cationic lipid DNA complexes, the final concentration and the order of addition of compounds during complex formation are critical for high transduction efficiency. Concentration of LDC formed under optimal conditions can be used to further increase in vivo gene transfer levels. Copyright © 2003 John Wiley & Sons, Ltd.
Proliferative activity and in vitro replication of HSV1716 in human metastatic brain tumours
Authors: Detta Allah , Harland June , Hanif Isma , Brown S. Moira , Cruickshank Garth
Published: July 2003

Background

The neurotropic herpes simplex virus mutant HSV1716 lacks the gene encoding the virulence factor ICP34.5 and cannot replicate in non‐dividing cells where proliferating cell nuclear antigen (PCNA) is not actively engaged in cellular DNA synthesis. In the brain, tumoral expression of PCNA therefore confers on it oncolytic specificity and may determine its efficacy. Three phase I trials in glioma patients and one in metastatic melanoma patients have established that HSV1716 is safe and replicates selectively in tumour tissue. Here we examine the in situ PCNA profiles of common human metastatic brain tumours and determine their in vitro permissivity for HSV1716 replication to ascertain their suitability for HSV1716 therapy.

Methods

Histological sections of tumour biopsies obtained from patients undergoing craniotomies were stained for PCNA expression. The replicative ability of HSV wild‐type (17+) and mutant (1716) viruses was assessed in tissue cultures of the same tumour biopsies and in cancer cell lines by plaque assay.

Results

Biopsies of all 10 metastatic tumours (3 melanoma, 4 carcinoma and 3 adenocarcinoma) as well as 4 glioblastoma multiforme were positive for PCNA immunoexpression and supported the replication of HSV1716. The PCNA‐positive cells in the metastatic tumours were distributed comparatively in larger and more contiguous areas than in glioblastoma (1.69 ± 1.61 mm2 vs. 0.73 ± 0.77 mm2) and numbered 29.0 ± 12.4 and 12.6 ± 4.7%, respectively.

Conclusions

The results show that human cerebral metastatic tumours have generally larger and more contiguous proliferative areas, support efficient HSV1716 replication, and are thus potential candidates for such oncolytic viral therapy. Copyright © 2003 John Wiley & Sons, Ltd.
Phase I immunotherapy with a modified vaccinia virus (MVA) expressing human MUC1 as antigen‐specific immunotherapy in patients with MUC1‐positive advanced cancer
Authors: Rochlitz Christoph , Figlin Robert , Squiban Patrick , Salzberg Marc , Pless Miklos , Herrmann Richard , Tartour Eric , Zhao Yongxiang , Bizouarne Nadine , Baudin Martine , Acres Bruce
Published: July 2003

Background

The MUC1 protein is a highly glycosylated mucin normally found at the apical surface of mucin‐secreting epithelial cells in many types of tissues. MUC1 is expressed, but heavily underglycosylated, in different human tumors. TG4010 is a viral suspension of a recombinant vaccinia vector (MVA) containing DNA sequences coding for the human MUC1 antigen and interleukin‐2 (IL‐2). This product was developed for use as a vaccine in cancer patients whose tumors express the MUC1 antigen. The objective of the present study was to determine the safety of the product and to define the dose of TG4010 to be used in further clinical trials.

Materials and methods

Thirteen patients with different solid tumors were treated by repeated intramuscular injection with increasing doses of TG4010 in two separate phase I studies, one in Europe (Basel—CR) and one in the United States (UCLA—RF): a total of 6 patients were treated at a dose of 5 × 106 pfu, 3 patients at 5 × 107 pfu, and 4 patients at 108 pfu. Safety, efficacy, and different immunological tests were the endpoints of the study.

Results

Tolerance of TG4010 was excellent, and side effects mainly consisted of injection site pain and influenza‐like symptoms. There was no apparent detrimental effect of repeated injections of the vaccinia virus. Four of thirteen evaluable patients showed stabilization of their disease for 6 to 9 months. One lung cancer patient who was initially progressing after the first injections later showed a marked decrease in the size of his metastases that lasted for 14 months. Some T cell proliferative immune responses were seen in five patients.

Conclusions

The administration of TG4010 was generally well tolerated in patients with metastatic tumors, and transient disease stabilization was observed in several patients, warranting further clinical studies with the product. Copyright © 2003 John Wiley & Sons, Ltd.
Lentivirally transduced dendritic cells as a tool for cancer immunotherapy
Authors: Breckpot Karine , Dullaers Melissa , Bonehill Aude , Van Meirvenne Sonja , Heirman Carlo , De Greef Catherine , van der Bruggen Pierre , Thielemans Kris
Published: July 2003

Background

Dendritic cells (DC) are the professional antigen‐presenting cells of the immune system, fully equipped to prime naive T cells and thus essential components for cancer immunotherapy.

Methods

We tested the influence of several elements (cPPT, trip, WPRE, SIN) on the transduction efficiency of human DC. Human and murine DC were transduced with tNGFR‐encoding lentiviruses to assess the effect of transduction on phenotype and function. Human DC were transduced with lentiviruses encoding huIi80MAGE‐A3 and murine DC with huIi80tOVA to test antigen presentation.

Results

A self‐inactivating (SIN) lentiviral vector containing the trip element was most efficient in transducing human DC. The transduction of DC with trip/SIN tNGFR encoding lentiviral vectors at MOI 15 resulted in stable gene expression in up to 94.6% (murine) and 88.2% (human) of the mature DC, without perturbing viability, phenotype and function. Human huIi80MAGE‐A3‐transduced DC were able to stimulate MAGE‐A3‐specific CD4+ and CD8+ T cell clones and could prime both MAGE‐A3‐specific CD4+ and CD8+ T cells in vitro. Murine huIi80tOVA‐transduced DC were able to present OVA peptides in the context of MHC class I and class II in vitro and induced a strong OVA‐specific cytotoxic T lymphocyte response in vivo, that was protective against subsequent challenge with OVA‐expressing tumor cells.

Conclusions

We show that, using lentiviral vectors, efficient gene transfer in human and murine DC can be obtained and that these DC can elicit antigen‐specific immune responses in vitro and in vivo. The composition of the transfer vector has a major impact on the transduction efficiency. Copyright © 2003 John Wiley & Sons, Ltd.