Murine Fibroblast Cell Line Research Articles

Analysis of Retroviral Vector Insertion Sites after T-Cell Directed Gene Therapy.

Gene transfer into peripheral blood lymphocytes has several potential applications including the correction of genetic diseases and therapeutic approaches for HIV-1 infection and cancer. Integrating gene transfer system based on murine oncoretroviruses are a convenient tool for such strategies. However, the recent occurrence of uncontrolled clonal T cell expansions in two patients treated with retroviral gene transfer for X-linked severe combined immune deficiency has raised the concern of the risk of insertional oncogenesis associated with the clinical use of integrating viral systems. In vitro studies have indicated that murine viral vectors tend to integrate in the vicinity of transcription start regions of the genome, thus providing a possible mechanism for oncogene activation, however, data from clinical gene transfer trials is lacking.We are following patients affected with adenosine deaminase (ADA) deficiency who have received T-lymphocyte-directed, retroviral-mediated gene transfer starting in 1990. The first treated patient received the last infusion of gene-corrected cells 12 years ago, has never shown any sign of lymphoproliferation and still carries ~20% of gene-corrected peripheral blood lymphocytes. We set out to study the integration sites in the cells of this patient with the aim of mapping the regions involved by retroviral integrations, determining their localization with respect to known genes, and assessing whether a preferred pattern could be defined. Genomic DNA was prepared from stored lymphocyte samples dating 1991, 1992, 1995, 1998, 2000, and 2003. By inverse PCR and ligation-mediated PCR, we have identified ~860 bona fide insertion sites. Search for homology within the human genome using BLAT returned ~330 unique hits that involved a variety of genes, including transcription factors and oncogenes (e.g. RUNX1, STAT5, FYN). To evaluate the distribution pattern of these integration sites, 2000 randomly generated data sets of genomic coordinates were assembled and their distribution relative to annotations of the human genome was analyzed. A preliminary comparison of the random distribution to our experimental samples showed that retroviral integrations in cells obtained from the patient were significantly skewed toward regions within 2 kb of genes (p<0.002) and CpG islands (p<0.001).These results suggest that, similar to what observed in murine fibroblast and human cancer cell lines, transcriptionally active regions of the genome may be preferred targets of retroviral vectors in human primary T lymphocytes. At the same time, our observations show that the resulting integration events are compatible with long-term, event-free in vivo survival of gene-modified cells in clinical settings.

VEGF induces Mcl-1 up-regulation and protects multiple myeloma cells against apoptosis

AbstractInterleukin-6 (IL-6) triggers multiple myeloma (MM) cell proliferation and protects against apoptosis by up-regulating myeloid cell leukemia 1 (Mcl-1). Vascular endothelial growth factor (VEGF) induces modest proliferation of MM cells and induces IL-6 secretion in a paracrine loop involving MM cells and bone marrow stromal cells. Using murine embryonic fibroblast cell lines as a model (Mcl-1wt/wt and Mcl-1Δ/null MEFs), we here demonstrate that deletion of Mcl-1 reduces fetal bovine serum (FBS)-, VEGF-, and IL-6-induced proliferation. We also show that VEGF up-regulates Mcl-1 expression in a time- and dose-dependent manner in 3 human MM cell lines and MM patient cells. Importantly, we demonstrate that the pan-VEGF inhibitor, GW654652, inhibits VEGF-induced up-regulation of Mcl-1 and, as with Mcl-1 siRNA, is associated with decreased proliferation and induction of apoptosis. Finally, we show that VEGF protects MM patient cells against FBS starvation-induced apoptosis. Our studies therefore demonstrate that VEGF-induced MM cell proliferation and survival are mediated via Mcl-1, providing the preclinical framework for novel therapeutics targeting Mcl-1 and/or VEGF to improve patient outcome in MM.

Triptolide, an active component of the Chinese herbal remedy Tripterygium wilfordii Hook F, inhibits production of nitric oxide by decreasing inducible nitric oxide synthase gene transcription.

The ethyl acetate (EA) extract of Tripterygium wilfordii Hook F (TWHF) and its major active component, triptolide, have been reported to be effective in the treatment of rheumatoid arthritis and other autoimmune inflammatory diseases. Nitric oxide (NO) has been recognized as an important mediator of inflammation. This study was therefore undertaken to examine the effects of the EA extract and triptolide on the production of NO and inducible NO synthase (iNOS) gene expression and transcription in vivo and in vitro. Peritoneal macrophages from C57BL/6J mice treated orally with the EA extract of TWHF were assayed for NO production and iNOS messenger RNA (mRNA) expression by reverse transcriptase-polymerase chain reaction. The murine fibroblast cell line NIH3T3 was also assessed for NO production and iNOS mRNA expression, as well as for iNOS promoter activation, Oct-1 nuclear binding capacity, and Oct-1 protein content by transient transfection, electrophoretic mobility shift assay, and immunoblotting, respectively. NO production and iNOS mRNA expression by macrophages from C57BL/6J mice immunized with trinitrophenyl-bovine serum albumin in Freund's complete adjuvant were significantly inhibited by oral administration of the EA extract (52.3% and 59.8% of control, respectively, at one-eighth of the dose that is lethal for 50% of the animals [LD(50)] and 21.0% and 38.1% of control, respectively, at one-fourth the LD(50)). Moreover, the EA extract and triptolide significantly inhibited NO production in vitro in activated peritoneal macrophages, which reflected a decreased level of iNOS mRNA. Finally, triptolide inhibited promoter activity of the iNOS gene and induction of the activity of the regulator of iNOS transcription, Oct-1. The EA extract of TWHF and triptolide inhibit transcription of the iNOS gene. This may contribute to the antiinflammatory effects of this traditional herbal remedy.

Expression of a functional human tumor necrosis factor-α (hTNF-α) in yeastSaccharomyces cerevisiae

The recombinant soluble human tumor necrosis factor-alpha (hTNF-α) was expressed in a yeastSaccharomyces cerevisiae and its cytotoxicity was evaluated. A cDNA encoding hTNF-α was placed under the control of two different promoters: a glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter and a yeast hybridADH2-GPD promoter, consisting of alcohol dehydrogenase II (ADH2) and theGPD promoter. A Northern blot analysis revealed that, although variation in the expression level of hTNF-α existed among transformants, the higher expression was obtained with theGPD promoter. Expressed hTNF-α protein (rhTNF-α) was successfully secreted into the culture medium, producing 2.5 mg per liter of culture filtrate, with no changes in cell growth. The bioassay for observing the cytotoxicity to the murine L929 fibroblast cell line, with serial dilution of rhTNF-α, indicated that the secreted rhTNF-α was bioactive and its doseresponse was improved eight to ten times over that of theE. coli-derived rhTNF-α.

Porcine adenoviral vectors evade preexisting humoral immunity to adenoviruses and efficiently infect both human and murine cells in culture

Preexisting immunity against human adenoviruses (HAd) limits the efficiency of transduction of HAd vectors in humans. In addition, development of a vector-specific immune response after the first inoculation with a HAd vector further lowers vector uptake following readministration. We investigated the usefulness of porcine adenovirus serotype 3 (PAd3)-based vectors as a supplement to HAd vectors. Here we demonstrate that preexisting HAd-specific neutralizing antibodies in humans do not cross-neutralize PAd3. In order to generate E1A-deleted PAd3 vectors, an E1-complementing cell line of porcine origin was produced. E1A-deleted PAd3 vector expressing green fluorescent protein; GFP (PAd-GFP) and E1-deleted HAd5 vector expressing GFP (HAd-GFP) transduced human cell lines with comparable efficiencies. Both of these vectors efficiently transduced murine MT1A2 breast cancer cell line, while PAd-GFP transduced murine NIH 3T3 fibroblast cell line significantly better ( P < 0.05) than HAd-GFP. These results suggest that PAd3 vectors would be promising supplement to HAd vectors as a delivery vehicle for recombinant vaccines and gene therapy applications.

A sustained activation of PI3K/NF-kappaB pathway is critical for the survival of chronic lymphocytic leukemia B cells.

The progressive rise of mature CD5+ B lymphocytes, despite the low proportion of proliferating cells, has led to the notion that B cell chronic lymphocytic leukemia (B-CLL) is primarily related to defective apoptosis. The microenvironment likely plays a prominent role because the malignant cells progressively accumulate in vivo, whereas they rapidly undergo spontaneous apoptosis when cultured in vitro. To assess microenvironment-mediated survival signals, B-CLL cells were cultured with a murine fibroblast cell line, Ltk-, with and without an agonistic antibody to CD40. Spontaneous apoptosis was associated with the loss of Akt and NF-kappaB activities. Interactions with fibroblasts sustained a basal level of Akt and NF-kappaB activities, which was dependent on phosphatidylinositol-3 kinase (PI3K). Constitutive activity of the PI3K pathway in B-CLL cells when cultured with fibroblasts prevented the downregulation of the prosurvival Bcl-2 family protein Bcl-xL and the caspase inhibitor proteins FLIPL and XIAP, and consequently caspase-3 activation and apoptosis. CD40 crosslinking in B-CLL cells did not further prevent murine fibroblasts-mediated apoptosis but induced cell proliferation, which was associated with an increase of Akt and NF-kappaB activation compared with cells cultured with fibroblasts alone. The PI3K pathway seems to play a pivotal role in B-CLL cell survival and growth.

Assessment of the potential irritation and photoirritation of novel amino acid-based surfactants by in vitro methods as alternative to the animal tests.

The ultraviolet-A radiation damage effects on skin and eyes will be increased by phototoxic compounds which could be present in pharmaceutical or cosmetic formulations. Great efforts have been made in the last years to find surfactants to replace those with phototoxic potential in commercial use. Series of different in vitro models for phototoxicity, included to validated neutral red uptake (NRU) 3T3 phototoxicity assay are useful screening tools. The phototoxic effects of a novel family of glycerol amino acid-based surfactant compounds were examined via these assays. Human red blood cells and two immortalised cell lines, murine fibroblast cell line 3T3, and one human keratinocyte cell line, HaCaT, were the in vitro models employed to predict potential photoirritation. The phototoxic end-points assessed were hemolysis (human red blood cell test) and resazurin transformation to resorufin and NRU in cell culture methods. The results suggest that no phototoxic effects by any new amino acid derived-surfactants, could be identified.

303. Impaired Nuclear Transport and Virus Uncoating Limit Recombinant Adeno-Associated Virus 2 Vector-Mediated Transduction of Primary Murine Hematopoietic Cells

The transduction efficiency of adeno-associated virus 2 (AAV) vectors varies greatly in different cells and tissues. Whereas muscle and brain cells are transduced efficiently, controversies abound regarding hematopoietic cell transduction by AAV vectors. For human hematopoietic cells, we documented this problem to be related to the extent of AAV receptor expression (J. Virol., 71: 8262–8267, 1997). Murine hematopoietic cells express the receptor, yet are transduced poorly. Using a murine fibroblast cell line, we reported that the lack of transduction was due to inefficient intracellular trafficking of AAV from cytosol into the nucleus (J. Virol., 74: 992–996, 2000), and that treatment with hydroxyurea (HU) facilitated nuclear transport of AAV in these cells (J. Virol., 75: 4080–4090, 2001). In the present studies, we extended these observations to primitive murine hematopoietic cells. Murine c-kit+, lin− hematopoietic cells were transduced with recombinant AAV-lacZ vectors. Forty-eight hrs. post-transduction, nuclear and cytoplasmic fractions were isolated and low Mr DNA samples extracted from these fractions were analyzed on Southern blots. Approximately 85% of AAV genomes were present in the cytoplasmic fraction. However, when donor mice were injected intra-peritoneally with HU at 1 mg/g body weight, 24 hrs. prior to isolation of cells, the extent of AAV genomes detected in the cytoplasmic fraction was reduced to ~40%, with a corresponding increase in the nuclear fraction, which increased to ~60%, indicating that in vivo administration of HU facilitated nuclear transport of AAV. However, when DNA samples were electrophoresed on denaturing gels and analyzed on Southern blots, it was apparent that a significant fraction of the AAV genome present in the nuclear fraction from cells obtained from HU-treated mice was single-stranded. We tested the hypothesis whether single-stranded genomes were derived from virions that failed to undergo uncoating in the nucleus. Primitive hematopoietic cells, with and without HU-administration in vivo, were transduced with recombinant AAV-lacZ vectors, and 48-hrs. post-transduction, equivalent fractions were either mock-treated, or digested exhaustively with DNase I, and analyzed on DNA slot-blots using a lacZ probe. Whereas majority of the signal, which was resistant to DNase I, was detected in the cytoplasmic fraction in cells obtained from untreated mice, as expected, a substantial fraction of the signal in the nuclear fraction in cells obtained from HU-treated mice was also resistant to DNase I, suggesting that although HU facilitated nuclear transport of AAV, most of the virions failed to undergo uncoating, thereby leading to only a partial improvement in viral second-strand DNA synthesis and transgene expression. Thus, the identification of a yet another obstacle — virus uncoating — has implications in the optimal use of recombinant AAV vectors in hematopoietic stem cell gene therapy.

Retrovirus-Induced Lung Cancer: Mechanisms of Transformation of Alveolar Type II Epithelial Cells

The jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma, which is the only known example of a retrovirus-induced lung cancer. The finding that the JSRV env gene is sufficient to induce the transformation of murine and avian fibroblast cell lines has led to an investigation of the mechanism by which this gene induces transformation. Reports from this and other laboratories have presented conflicting data regarding the requirement for a known SH2-binding motif, which is found within the transmembrane domain of the JSRV env gene, in cellular transformation.

457. Fibronectin Peptide Reduces the Immune Response to Adenovirus and Enhances Transgene Expression

Preexisting immunity against human adenoviruses (HAd) limits the efficiency of transduction of HAd vectors in humans. In addition, development of a vector-specific immune response following the first inoculation with a HAd vector further lowers vector uptake following readministration. We investigated the usefulness of porcine adenovirus serotype 3 (PAd3)-based vectors as a supplement to HAd vectors. Here we demonstrate that preexisting HAd-specific neutralizing antibodies in humans do not cross-neutralize PAd3. In order to generate E1A-deleted PAd3 vectors, an E1-complementing cell line of porcine origin was produced. To compare transduction efficiencies of PAd3 and human adenovirus type 5 (HAd5) vectors, PAd-GFP (E1A-deleted PAd3 vector expressing green fluorescent protein; GFP) and HAd-GFP (E1-deleted HAd5 vector expressing GFP) were generated. PAd-GFP and HAd-GFP transduced human cell lines with comparable efficiencies. Both vectors efficiently transduced murine MT1A2 breast cancer cell line, while PAd-GFP transduced murine NIH-3T3 fibroblast cell line significantly better (P<0.05) than HAd-GFP. These results suggest that PAd3 vector system would be a promising supplement to HAd vectors as a delivery vehicle for recombinant vaccines and gene therapy applications.

Assessment of the potential irritation and photoirritation of novel amino acid-based surfactants by in vitro methods as alternative to the animal tests

The ultraviolet-A radiation damage effects on skin and eyes will be increased by phototoxic compounds which could be present in pharmaceutical or cosmetic formulations. Great efforts have been made in the last years to find surfactants to replace those with phototoxic potential in commercial use. Series of different in vitro models for phototoxicity, included to validated neutral red uptake (NRU) 3T3 phototoxicity assay are useful screening tools. The phototoxic effects of a novel family of glycerol amino acid-based surfactant compounds were examined via these assays. Human red blood cells and two immortalised cell lines, murine fibroblast cell line 3T3, and one human keratinocyte cell line, HaCaT, were the in vitro models employed to predict potential photoirritation. The phototoxic end-points assessed were hemolysis (human red blood cell test) and resazurin transformation to resorufin and NRU in cell culture methods. The results suggest that no phototoxic effects by any new amino acid derived-surfactants, could be identified.

129. Infectivity Enhancement of an Adenovirus Vector Via Genetic Incorporation of the Reovirus Spike Protein Sigma 1

During the last decade, human adenoviruses (Ad) have been exploited as vectors for gene transfer and gene therapy of various monogenic and polygenic diseases, with limited success. Early gene therapy efforts revealed that the clinical benefit of these gene delivery modalities was irrevocably linked to specific localization of the therapeutic agent. Many clinically relevant tissues are refractory to Ad5 infection, including several cancer tissue types, due to negligible CAR levels. Thus, development of novel Ad vectors demonstrating CAR-independent tropism may lead directly to therapeutic gain. Based on a clear understanding of native Ad cell recognition, development of genetically targeted vectors has rationally focused on the fiber knob domain, the primary determinant of Ad tropism. Reovirus serotype type 3 (T3) utilizes a fiber-like attachment protein, called sigma 1, to infect human cells. The sigma 1 molecule is comprised of an N-terminal tail region of alpha helix and eight beta-repeats, and a C-terminal globular head domain. Sigma 1 contains two receptor-binding domains (RBDs): one within the fiber tail that binds sialic acid (SA), and the other in the globular head that binds to junction adhesion molecule (JAM). The head RBD plays a key role in determining the tropism of T3 reoviruses, which have CAR-independent tropism. Based on these observations, we hypothesized that replacement of the Ad5 fiber with reovirus attachment protein sigma 1, a fiber-like molecule, would result in an Ad5 vector with a novel, CAR-independent tropism. We constructed a dual-fiber Ad5 vector, designated Ad5-sigma 1, encoding a luciferase reporter gene and two fibers: the first fiber is a chimeric fiber comprised of the tail domain of Ad5 fiber fused to the reovirus sigma 1 and the second fiber is the wild-type Ad5 fiber. Virus particles were rescued and purified and characterization of the dual-fiber Ad5-sigma 1 virions confirmed the presence of both fibers. Functionally, Ad5-sigma 1 was found to be able to utilize both receptors, CAR and JAM, for virus attachment to cells. In CAR positive cells, Ad5-sigma 1 gene transfer was reduced 80% by competition with recombinant Ad5 knob. Gene transfer was also inhibited by antibodies directed against JAM and sigma 1 in the L929 murine fibroblast cell line, known to express JAM and SA but not CAR. Ad5-sigma 1 provided 223-fold greater gene transfer versus the isogenic control vector in the L929 cells. In addition, Ad5-sigma 1 gene transfer was increased 6-fold in SA-positive, low-CAR chinese hamster ovary cells versus the isogenic control Ad5 vector. Furthermore, Ad5-sigma 1 provided 3.1- to 10.7- fold greater gene transfer versus control in a panel of ovarian cancer cell lines, known for relatively low CAR expression. We developed an infectivity enhanced Ad5 vector to address the key issue of limited Ad5 vector efficiency. The expanded tropism of the Ad5-sigma1 vector represents a crucial attribute for an Ad-based gene therapy vector specifically for ovarian cancer, and may represent an agent with future clinical utility.

Phosphorylation of BCL-2 regulates ER Ca2+ homeostasis and apoptosis.

Phosphorylation of BCL-2 within an unstructured loop inhibits its antiapoptotic effect. We found that phosphorylated BCL-2 predominantly localized to the endoplasmic reticulum (ER) and tested whether phosphorylation would control its activity at this organelle, where Ca(2+) dynamics serve as a critical control point for apoptosis. Phosphorylation greatly inhibits the ability of BCL-2 to lower [Ca(2+)](er) and protect against Ca(2+)-dependent death stimuli. Cells expressing nonphosphorylatable BCL-2(AAA) exhibited increased leak of Ca(2+) from the ER and further diminished steady-state [Ca(2+)](er) stores when compared to cells expressing BCL-2(wt). Consequently, when BCL-2 is phosphorylated, Ca(2+) discharge from the ER is increased, with a secondary increase in mitochondrial Ca(2+) uptake. We also demonstrate that phosphorylation of BCL-2 inhibits its binding to proapoptotic family members. This inhibitory mechanism manifested at the ER, where phosphorylated BCL-2 was unable to bind proapoptotic members. [Ca(2+)](er) proved coordinate with the capacity of BCL-2 to bind proapoptotic BH3-only members, further integrating the apoptotic pathway and Ca(2+) modulation. Unexpectedly, the regulation of ER Ca(2+) dynamics is a principal avenue whereby BCL-2 phosphorylation alters susceptibility to apoptosis.

Assessment of primary eye and skin irritants by in vitro cytotoxicity and phototoxicity models: an in vitro approach of new arginine-based surfactant-induced irritation.

Extensive efforts have been made, recently, to find surfactants with lower irritation potential than those presently commercially available, for use in pharmaceutical and cosmetic preparations. Cytotoxic and phototoxic effects of a novel family of dicationic arginine-diglyceride surfactant compounds, 1,2-diacyl,3-O-(l-arginyl)-rac-glycerol with alkyl chain lengths in the range from 8 to 14 carbon atoms, were compared to three commercial surfactants. The end-points used to assess toxicity were the red blood cell lysis assay and uptake of the vital dye neutral red 24h after dosing (NRU), respectively. Two immortalized cell lines, murine fibroblast cell line, 3T3, and one human keratinocyte cell line, HaCaT, were used as in vitro models to predict the potential phototoxicity which could result in irritation, determined by resazurin reduction to resorufin and neutral red uptake (NRU). All tested surfactants had cytotoxicity effects as demonstrated by and decrease of NR uptake, which showed a clear concentration-response relationship. Concentrations resulting in 50% inhibition of NR uptake (IC(50)) range from 1 microM(-1) (hexadecyl trimethyl ammonium bromide) to 565 microM(-1) (12,12-l-arginine). Erythrocyte haemolysis also showed a clear concentration-response relationship, the 50% of haemolysis ranged from 37 microM(-1) (10,10-l-arginine) to 151 microM(-1) (sodium lauryl sulphate). Phototoxicity was performed with 12,12-l-acetyl-arginine, the most stable chemical structure. The validated 3T3 NRU photoxicity assay was used and revealed a phototoxic potential.

Calcium and its role in the nuclear translocation and activation of cytosolic phospholipase A2 in cells rendered sensitive to TNF-induced apoptosis by cycloheximide.

In these experiments, we investigated the role of calcium as a second messenger in the apoptotic activation of cytosolic phospholipase A(2) (cPLA(2)). As our model, we used a murine fibroblast cell line (C3HA) that was induced to undergo apoptosis by a combination of TNF and cycloheximide. Using fura 2 Ca(2+) imaging, we found strong evidence for an intracellular calcium response after 1 h of treatment, which correlated with the onset of phosphatidylserine externalization, but preceded effector procaspase processing by several hours. The response was strongest in the perinuclear region, where mean levels rose 83% (144 +/- 14 nM in untreated cells vs 264 +/- 39 nM in treated), while cells displaying morphological evidence of apoptosis had the highest levels of calcium (250-1000 nM). Verapamil blocked this response, indicating an extracellular source for the calcium. Fluorescence microscopy revealed a pattern of nuclear translocation of cPLA(2) during apoptosis, which was also blocked by verapamil, indicating an important role for calcium in this process. In addition, we found that verapamil prevented the release of [(3)H]arachidonic acid from C3HA cells induced to undergo apoptosis by the chemotherapeutic agents vinblastine, melphalan, and cis-platinum. Together, these data suggest that calcium is important for cPLA(2) activation by diverse apoptotic stimuli.

Expression of Cytosolic Phospholipase A2α in Murine C12 Cells, a Variant of L929 Cells, Induces Arachidonic Acid Release in Response to Phorbol Myristate Acetate and Ca2+ Ionophores, but Not to Tumor Necrosis Factor-α

Tumor necrosis factor-α (TNFα)-induced cell death is regulated through the release of arachidonic acid (AA) by group IVA cytosolic phospholipase A2 (cPLA2α) in the murine fibroblast cell line L929. However, the signaling pathway by which TNFα activates cPLA2α remained to be solved. We examined AA release in L929 cells, in a variant of L929 (C12 cells) lacking cPLA2α, and in C12 cells transfected with cPLA2α expression vectors. In transient and stable clones of C12 cells expressing cPLA2α, Ca2+ ionophore A23187 and phorbol myristate acetate (PMA) stimulated AA release within 90 min, although no response to TNFα was observed within 6 h. These results suggest that C12 cells may lack the components necessary for TNFα-induced AA release, in addition to cPLA2α. PMA is known to stimulate AA release via phosphorylation of Ser505 in cPLA2α by activating extracellular signal-regulated kinases (ERK1/2). However, PMA-induced AA release from C12 cells expressing mutant cPLA2αS505A (mutation of Ser505 to Ala), which is not phosphorylated by ERK1/2, was similar to that from L929 cells and C12 cells expressing wild-type cPLA2α. The role of Ser505 phosphorylation in AA release induced by PMA is also discussed.

Effects of endostatin-vascular endothelial growth inhibitor chimeric recombinant adenoviruses on antiangiogenesis.

To investigate the inhibitory effects of endostatin-vascular endothelial growth inhibitor (VEGI151) recombinant adenoviruses on neovascularization. We used recombinant adenoviruses to treat human vascular endothelial cell line ECV304, human hepatocellular carcinoma cell line HepG2, and murine fibroblast cell line L929, in order to study the chimeric gene expression in these cell lines. Chick choriallantic membrane (CAM) model, rabbit inflammatory corneal neovascularization (CNV) model, and liver cancer-bearing nude mice model were employed to investigate the negative biological effect of fusion molecules on neovascularization in vivo. Western blot showed that the molecular weight of fusion protein was about 41 kD after infection of ECV304, HepG2 and L929 cells with supernatant of AdhENDO-VEGI151. The fusion protein showed a specific inhibitory effect on the proliferation of ECV304 cells, but no inhibitory effect on the growth of HepG2 and L929 cells (F=13112.13, P=0.0001). In the chick choriallantic membrane (CAM) assay, the expressed fusion protein significantly inhibited neovascularization. Rabbit inflammatory corneal neovascularization (CNV) induced by intrastromal sutures resulted in a uniform neovascular response. In this model, direct subconjunctival injection of AdhENDO-VEGI151 expressed the fusion protein in vivo and suppressed the development of CNV. Topical application of AdhENDO-VEGI151 led to a significant suppression of CNV (F=1413.11, P=0.0001), as compared with the control group of AdLacZ. Immunohistochemical staining showed the fusion protein dominantly expressed in corneal epithelium. Compared with the control group of AdLacZ (4075.9+/-1849.9 mm(3)), the average tumor size of group AdhENDO-VEGI151 reduced in size (487.7+/-241.2 mm(3)) (F=14.80, P=0.0085), with an inhibition rate of 88.03%. Immunohistochemical staining showed the adenoviruses carried the fusion gene expressed on liver cancer cell membrane. MVD decreased more significantly in treated mice (30.75+/-3.31%) than in AdLacZ control (50.25+/-8.65%) (F=17.72, P=0.0056) with an inhibition rate of 39%. Fusion protein expressed by recombinant adenoviruses has a significant inhibitory effect on neovascularization.

Analysis of Mycobacterium species for the presence of a macrolide toxin, mycolactone.

Mycobacterium ulcerans is an environmental organism which is responsible for the disease Buruli ulcer, a necrotizing skin disease emerging in west Africa. M. ulcerans produces the polyketide-derived macrolide mycolactone, which is required for the immunosuppression and tissue damage which characterizes Buruli ulcer. We have extracted lipids from the cell envelope and culture filtrate from 52 isolates of Mycobacterium species, analyzed them with thin-layer chromatography, and tested them in a murine fibroblast cell line (L929) cytotoxicity assay to investigate whether these mycobacterial species produce mycolactone. For these studies chloroform-methanol (2:1, vol/vol) extracts were prepared from representative fast- and slow-growing mycobacterial species. Isolates tested included 16 uncharacterized, slow-growing, environmental mycobacterial species isolated from areas in which M. ulcerans infection is endemic. Although several strains of mycobacteria studied produced cytopathic lipids, none of these produced a phenotype on cultured cells consistent with that produced by mycolactone. Two mycobacterial species, M. scrofulaceum and M. kansasii, and eight of the environmental mycobacterial isolates contained cell-associated lipids cytopathic to fibroblasts at concentrations of 33 to 1,000 microg/ml. In contrast, mycolactone produces cytotoxicity at less than 2 ng/ml. Analysis of 16S rRNA sequences from the eight environmental isolates suggests that these are novel mycobacterial species. Results from these studies suggest that, although production of cytopathic lipids is relatively common among mycobacterial species, the production of mycolactone as a cell-associated or secreted molecule appears so far to be restricted to M. ulcerans.

Inhibition of p38 mitogen-activated protein kinase reduces TNF-induced activation of NF-κB, elicits caspase activity, and enhances cytotoxicity

Among other cellular responses, tumor necrosis factor (TNF) induces different forms of cell death and the activation of the p38 mitogen-activated protein kinase (MAPK). The influence of p38 MAPK activation on TNF-induced apoptosis or necrosis is controversially discussed. Here, we demonstrate that pharmacological inhibition of p38 MAPK enhances TNF-induced cell death in murine fibroblast cell lines L929 and NIH3T3. Furthermore, overexpression of dominant-negative versions of p38 MAPK or its upstream kinase MKK6 led to increased cell death in L929 cells. While overexpression of the p38 isoforms alpha and beta did not protect L929 cells from TNF-induced toxicity, overexpression of constitutively active MKK6 decreased TNF-induced cell death. Although the used inhibitors of p38 MAPK decreased the phosphorylation of the survival kinase PKB/Akt, this effect could be ruled out as cause of the observed sensitization to TNF-induced cytotoxicity. Finally, we demonstrate that the nuclear factor κB (NF-κB)-dependent gene expression, shown as an example for the anti-apoptotic gene cellular inhibitor of apoptosis (c-IAP2), was reduced by p38 MAPK inhibition. In consequence, we found that inhibition of p38 MAPK led to the activation of the executioner caspase-3.

ErbB2 and EGFR are downmodulated during the differentiation of 3T3-L1 preadipocytes.

The expression of receptors belonging to the epidermal growth factor receptor subfamily has been largely studied these last years in epithelial cells mainly as involved in cell proliferation and malignant progression. Although much work has focused on the role of these growth factor receptors in the differentiation of a variety of tissues, there is little information in regards to normal stromal cells. We investigated erbB2 expression in the murine fibroblast cell line Swiss 3T3L1, which naturally or hormonally induced undergoes adipocyte differentiation. We found that the Swiss 3T3-L1 fibroblasts express erbB2, in addition to EGFR, and in a quantity comparable to or even greater than the breast cancer cell line T47D. Proliferating cells increased erbB2 and EGFR levels when reaching confluence up to 4- and 10-fold, respectively. This expression showed a significant decrease when growth-arrested cells were stimulated to differentiate with dexamethasone and isobutyl-methylxanthine. Differentiated cells presented a decreased expression of both erbB2 and EGFR regardless of whether the cells were hormonally or spontaneously differentiated. EGF stimulation of serum-starved cells increased erbB2 tyrosine phosphorylation and retarded erbB2 migration in SDS-PAGE, suggesting receptor association and activation. Heregulin-alpha1 and -beta1, two EGF related factors, had no effect on erbB2 or EGFR phosphorylation. Although 3T3-L1 cells expressed heregulin, its specific receptors, erbB3 and erbB4, were not found. This is the first time in which erbB2 is reported to be expressed in an adipocytic cell line which does not depend on non EGF family growth factors (thyroid hormone, growth hormone, etc.) to accomplish adipose differentiation. Since erbB2 and EGFR expression were downmodulated as differentiation progressed it is conceivable that a mechanism of switching from a mitogenic to a differentiating signaling pathway may be involved, through regulation of the expression of these growth factor receptors.