Showing papers on "Cell culture published in 2000"
TL;DR: Using cDNA microarrays to explore the variation in expression of approximately 8,000 unique genes among the 60 cell lines used in the National Cancer Institute's screen for anti-cancer drugs provided a novel molecular characterization of this important group of human cell lines and their relationships to tumours in vivo.
Abstract: We used cDNA microarrays to explore the variation in expression of approximately 8,000 unique genes among the 60 cell lines used in the National Cancer Institute's screen for anti-cancer drugs. Classification of the cell lines based solely on the observed patterns of gene expression revealed a correspondence to the ostensible origins of the tumours from which the cell lines were derived. The consistent relationship between the gene expression patterns and the tissue of origin allowed us to recognize outliers whose previous classification appeared incorrect. Specific features of the gene expression patterns appeared to be related to physiological properties of the cell lines, such as their doubling time in culture, drug metabolism or the interferon response. Comparison of gene expression patterns in the cell lines to those observed in normal breast tissue or in breast tumour specimens revealed features of the expression patterns in the tumours that had recognizable counterparts in specific cell lines, reflecting the tumour, stromal and inflammatory components of the tumour tissue. These results provided a novel molecular characterization of this important group of human cell lines and their relationships to tumours in vivo.
2,192 citations
TL;DR: The paired box transcription factor Pax7 was isolated by representational difference analysis as a gene specifically expressed in cultured satellite cell-derived myoblasts and it was demonstrated that satellite cells and muscle-derived stem cells represent distinct cell populations.
2,148 citations
TL;DR: The results suggest that preterm, as compared with term, cord blood is richer in mesenchymal progenitors, similar to haematopoietic progenitor cells.
Abstract: Haemopoiesis is sustained by two main cellular components, the haematopoietic cells (HSCs) and the mesenchymal progenitor cells (MPCs). MPCs are multipotent and are the precursors for marrow stroma, bone, cartilage, muscle and connective tissues. Although the presence of HSCs in umbilical cord blood (UCB) is well known, that of MPCs has been not fully evaluated. In this study, we examined the ability of UCB harvests to generate in culture cells with characteristics of MPCs. Results showed that UCB-derived mononuclear cells, when set in culture, gave rise to adherent cells, which exhibited either an osteoclast- or a mesenchymal-like phenotype. Cells with the osteoclast phenotype were multinucleated, expressed TRAP activity and antigens CD45 and CD51/CD61. In turn, cells with the mesenchymal phenotype displayed a fibroblast-like morphology and expressed several MPC-related antigens (SH2, SH3, SH4, ASMA, MAB 1470, CD13, CD29 and CD49e). Our results suggest that preterm, as compared with term, cord blood is richer in mesenchymal progenitors, similar to haematopoietic progenitors.
1,667 citations
TL;DR: A method for obtaining dopaminergic (DA) and serotonergic neurons in high yield from mouse ES cells in vitro is presented and it is demonstrated that the ES cells can be obtained in unlimited numbers and that these neuron types are generated efficiently.
Abstract: Embryonic stem (ES) cells are clonal cell lines derived from the inner cell mass of the developing blastocyst that can proliferate extensively in vitro and are capable of adopting all the cell fates in a developing embryo. Clinical interest in the use of ES cells has been stimulated by studies showing that isolated human cells with ES properties from the inner cell mass or developing germ cells can provide a source of somatic precursors. Previous studies have defined in vitro conditions for promoting the development of specific somatic fates, specifically, hematopoietic, mesodermal, and neurectodermal. In this study, we present a method for obtaining dopaminergic (DA) and serotonergic neurons in high yield from mouse ES cells in vitro. Furthermore, we demonstrate that the ES cells can be obtained in unlimited numbers and that these neuron types are generated efficiently. We generated CNS progenitor populations from ES cells, expanded these cells and promoted their differentiation into dopaminergic and serotonergic neurons in the presence of mitogen and specific signaling molecules. The differentiation and maturation of neuronal cells was completed after mitogen withdrawal from the growth medium. This experimental system provides a powerful tool for analyzing the molecular mechanisms controlling the functions of these neurons in vitro and in vivo, and potentially for understanding and treating neurodegenerative and psychiatric diseases.
1,346 citations
TL;DR: Keratinocyte replicative potential is limited by a p16INK4a-dependent mechanism, the activation of which can occur independent of telomere length, and abrogation of this mechanism together with telomerase expression immortalizes keratinocytes without affecting other major growth control or differentiation systems.
Abstract: Normal human cells exhibit a limited replicative life span in culture, eventually arresting growth by a process termed senescence. Progressive telomere shortening appears to trigger senescence in normal human fibroblasts and retinal pigment epithelial cells, as ectopic expression of the telomerase catalytic subunit, hTERT, immortalizes these cell types directly. Telomerase expression alone is insufficient to enable certain other cell types to evade senescence, however. Such cells, including keratinocytes and mammary epithelial cells, appear to require loss of the pRB/p16(INK4a) cell cycle control mechanism in addition to hTERT expression to achieve immortality. To investigate the relationships among telomerase activity, cell cycle control, senescence, and differentiation, we expressed hTERT in two epithelial cell types, keratinocytes and mesothelial cells, and determined the effect on proliferation potential and on the function of cell-type-specific growth control and differentiation systems. Ectopic hTERT expression immortalized normal mesothelial cells and a premalignant, p16(INK4a)-negative keratinocyte line. In contrast, when four keratinocyte strains cultured from normal tissue were transduced to express hTERT, they were incompletely rescued from senescence. After reaching the population doubling limit of their parent cell strains, hTERT(+) keratinocytes entered a slow growth phase of indefinite length, from which rare, rapidly dividing immortal cells emerged. These immortal cell lines frequently had sustained deletions of the CDK2NA/INK4A locus or otherwise were deficient in p16(INK4a) expression. They nevertheless typically retained other keratinocyte growth controls and differentiated normally in culture and in xenografts. Thus, keratinocyte replicative potential is limited by a p16(INK4a)-dependent mechanism, the activation of which can occur independent of telomere length. Abrogation of this mechanism together with telomerase expression immortalizes keratinocytes without affecting other major growth control or differentiation systems.
997 citations
TL;DR: It is reported that certain oestrogen derivatives selectively kill human leukaemia cells but not normal lymphocytes, and that mechanism-based combinations of SOD inhibitors with free-radical-producing agents may have clinical applications.
Abstract: Superoxide dismutases (SOD) are essential enzymes that eliminate superoxide radical (O2-) and thus protect cells from damage induced by free radicals. The active O2- production and low SOD activity in cancer cells may render the malignant cells highly dependent on SOD for survival and sensitive to inhibition of SOD. Here we report that certain oestrogen derivatives selectively kill human leukaemia cells but not normal lymphocytes. Using complementary DNA microarray and biochemical approaches, we identify SOD as a target of this drug action and show that chemical modifications at the 2-carbon (2-OH, 2-OCH3) of the derivatives are essential for SOD inhibition and for apoptosis induction. Inhibition of SOD causes accumulation of cellular O2- and leads to free-radical-mediated damage to mitochondrial membranes, the release of cytochrome c from mitochondria and apoptosis of the cancer cells. Our results indicate that targeting SOD may be a promising approach to the selective killing of cancer cells, and that mechanism-based combinations of SOD inhibitors with free-radical-producing agents may have clinical applications.
809 citations
TL;DR: In this paper, the effects of in vitro expansion on BMSC pluripotentiality, proliferative ability, and bone-forming efficiency in vivo were investigated, and the lifespan and differentiation kinetics of five of these clones were determined.
747 citations
TL;DR: This article discusses the various models used for studying the preadipocyte differentiation process and focuses on those genetic events that link effectors to induction of adipocyte gene expression, with the mouse 3T3-L1 cell culture line described in detail.
Abstract: The major function of adipocytes is to store triacylglycerol in periods of energy excess and to mobilize this energy during times of deprivation. The short-term control of these lipogenic and lipolytic processes is carefully modulated by hormonal signals from the bloodstream, which provide an inventory of the body's metabolic state. Long-term changes in fat storage needs are accomplished by altering both the size and number of fat cells within the body because terminally differentiated adipocytes cannot divide. Alterations in the number of fat cells within the body must be accomplished by the differentiation of preadipocytes, which act as the renewable source of adipocytes. Our understanding of the events that occur during preadipocyte differentiation has advanced considerably in the last few years and has relied mainly on the use of tissue culture models of adipogenesis. This article will discuss the various models used for studying the preadipocyte differentiation process, with the mouse 3T3-L1 cell culture line described in detail. We focus on those genetic events that link effectors to induction of adipocyte gene expression.
734 citations
TL;DR: Efficient and reproducible gene targeting in fetal fibroblasts to place a therapeutic transgene at the ovine α1(I) procollagen (COL1A1) locus is described and the production of live sheep by nuclear transfer is described.
Abstract: It is over a decade since the first demonstration that mouse embryonic stem cells could be used to transfer a predetermined genetic modification to a whole animal. The extension of this technique to other mammalian species, particularly livestock, might bring numerous biomedical benefits, for example, ablation of xenoreactive transplantation antigens, inactivation of genes responsible for neuropathogenic disease and precise placement of transgenes designed to produce proteins for human therapy. Gene targeting has not yet been achieved in mammals other than mice, however, because functional embryonic stem cells have not been derived. Nuclear transfer from cultured somatic cells provides an alternative means of cell-mediated transgenesis. Here we describe efficient and reproducible gene targeting in fetal fibroblasts to place a therapeutic transgene at the ovine alpha1(I) procollagen (COL1A1) locus and the production of live sheep by nuclear transfer.
682 citations
Journal Article•
TL;DR: The results suggest that hydroxamic acid-based hybrid polar compounds inhibit prostate cancer cell growth and may be useful, relatively nontoxic agents for the treatment of prostate carcinoma.
Abstract: Suberoylanilide hydroxamic acid (SAHA) is the prototype of a family of hybrid polar compounds that induce growth arrest in transformed cells and show promise for the treatment of cancer. SAHA induces differentiation and/or apoptosis in certain transformed cells in culture and is a potent inhibitor of histone deacetylases. In this study, we examined the effects of SAHA on the growth of human prostate cancer cells in culture and on the growth of the CWR22 human prostate xenograft in nude mice. SAHA suppressed the growth of the LNCaP, PC-3, and TSU-Pr1 cell lines at micromolar concentrations (2.5-7.5 microM). SAHA induced dose-dependent cell death in the LNCaP cells. In mice with transplanted CWR222 human prostate tumors, SAHA (25, 50, and 100 mg/kg/day) caused significant suppression of tumor growth compared with mice receiving vehicle alone; treatment with 50 mg/kg/day resulted in a 97% reduction in the mean final tumor volume compared with controls. At this dose, there was no detectable toxicity as evaluated by weight gain and necropsy examination. Increased accumulation of acetylated core histones was detected in the CWR22 tumors within 6 h of SAHA administration. SAHA induced prostate-specific antigen mRNA expression in CWR22 prostate cancer cells, resulting in higher levels of serum prostate-specific antigen than predicted from tumor volume alone. The results suggest that hydroxamic acid-based hybrid polar compounds inhibit prostate cancer cell growth and may be useful, relatively nontoxic agents for the treatment of prostate carcinoma.
648 citations
TL;DR: It is demonstrated that responses to substrate rigidity play a major role in distinguishing the growth behavior of normal cells from that of transformed cells, and that proper mechanical feedback is required for regulating cell shape, cell growth, and survival.
Abstract: One of the hallmarks of oncogenic transformation is anchorage-independent growth (27). Here we demonstrate that responses to substrate rigidity play a major role in distinguishing the growth behavior of normal cells from that of transformed cells. We cultured normal or H-ras-transformed NIH 3T3 cells on flexible collagen-coated polyacrylamide substrates with similar chemical properties but different rigidity. Compared with cells cultured on stiff substrates, nontransformed cells on flexible substrates showed a decrease in the rate of DNA synthesis and an increase in the rate of apoptosis. These responses on flexible substrates are coupled to decreases in cell spreading area and traction forces. In contrast, transformed cells maintained their growth and apoptotic characteristics regardless of substrate flexibility. The responses in cell spreading area and traction forces to substrate flexibility were similarly diminished. Our results suggest that normal cells are capable of probing substrate rigidity and that proper mechanical feedback is required for regulating cell shape, cell growth, and survival. The loss of this response can explain the unregulated growth of transformed cells.
TL;DR: It is shown that exogenous addition of activated matrix metalloprotease (MMP) 2 stimulates migration onto Ln-5 in breast epithelial cells via cleavage of the γ2 subunit, and this model suggests a model whereby expression of MT1-MMP is the primary trigger for migration over LN-5, whereas MMP2, which is activated by MT1,MMP, may play an ancillary role, perhaps by amplifying the MT1
Abstract: Laminin-5 (Ln-5) is an extracellular matrix substrate for cell adhesion and migration, which is found in many epithelial basement membranes. Mechanisms eliciting migration on Ln-5 need to be elucidated because of their relevance to tissue remodeling and cancer metastasis. We showed that exogenous addition of activated matrix metalloprotease (MMP) 2 stimulates migration onto Ln-5 in breast epithelial cells via cleavage of the γ2 subunit. To investigate the biological scope of this proteolytic mechanism, we tested a panel of cells, including colon and breast carcinomas, hepatomas, and immortalized hepatocytes, selected because they migrated or scattered constitutively in the presence of Ln-5. We found that constitutive migration was inhibited by BB94 or TIMPs, known inhibitors of MMPs. Limited profiling by gelatin zymography and Western blotting indicated that the ability to constitutively migrate on Ln-5 correlated with expression of plasma membrane bound MT1-MMP metalloprotease, rather than secretion of MMP2, since MMP2 was not produced by three cell lines (one breast and two colon carcinomas) that constitutively migrated on Ln-5. Moreover, migration on Ln-5 was reduced by MT1-MMP antisense oligonucleotides both in MMP2+ and MMP2− cell lines. MT1-MMP directly cleaved Ln-5, with a pattern similar to that of MMP2. The hemopexin-like domain of MMP2, which interferes with MMP2 activation, reduced Ln-5 migration in MT1-MMP+, MMP2+ cells, but not in MT1-MMP+, MMP2− cells. These results suggest a model whereby expression of MT1-MMP is the primary trigger for migration over Ln-5, whereas MMP2, which is activated by MT1-MMP, may play an ancillary role, perhaps by amplifying the MT1-MMP effects. Codistribution of MT1-MMP with Ln-5 in colon and breast cancer tissue specimens suggested a role for this mechanism in invasion. Thus, Ln-5 cleavage by MMPs may be a widespread mechanism that triggers migration in cells contacting epithelial basement membranes.
TL;DR: These findings suggest that immature osteoblasts and endothelial cells control stem cell homing, retention, and repopulation by secreting SDF-1, which also participates in host defense responses to DNA damage.
Abstract: The chemokine stromal-derived factor-1 (SDF-1) controls many aspects of stem cell function. Details of its regulation and sites of production are currently unknown. We report that in the bone marrow, SDF-1 is produced mainly by immature osteoblasts and endothelial cells. Conditioning with DNA-damaging agents (ionizing irradiation, cyclophosphamide, and 5-fluorouracil) caused an increase in SDF-1 expression and in CXCR4-dependent homing and repopulation by human stem cells transplanted into NOD/SCID mice. Our findings suggest that immature osteoblasts and endothelial cells control stem cell homing, retention, and repopulation by secreting SDF-1, which also participates in host defense responses to DNA damage.
TL;DR: It is reported that cholesterol is essential for uptake of mycobacteria by macrophages, and entering host cells at cholesterol-rich domains of the plasma membrane may ensure their subsequent intracellular survival in TACO-coated phagosomes.
Abstract: Mycobacteria are intracellular pathogens that can invade and survive within host macrophages, thereby creating a major health problem worldwide. The molecular mechanisms involved in mycobacterial entry are still poorly characterized. Here we report that cholesterol is essential for uptake of mycobacteria by macrophages. Cholesterol accumulated at the site of mycobacterial entry, and depleting plasma membrane cholesterol specifically inhibited mycobacterial uptake. Cholesterol also mediated the phagosomal association of TACO, a coat protein that prevents degradation of mycobacteria in lysosomes. Thus, by entering host cells at cholesterol-rich domains of the plasma membrane, mycobacteria may ensure their subsequent intracellular survival in TACO-coated phagosomes.
Journal Article•
TL;DR: A2E is implicate as an initiator of blue light-induced apoptosis of RPE cells, a finding that indicates an apoptotic form of cell death.
Abstract: Purpose To determine whether the lipofuscin fluorophore A2E participates in blue light-induced damage to retinal pigmented epithelial (RPE) cells. Methods Human RPE cells (ARPE-19) accumulated A2E from 10, 50, and 100 microM concentrations in media, the levels of internalized A2E ranging from less than 5 to 64 ng/10(5) cells, as assayed by quantitative high-performance liquid chromatography (HPLC). Restricted zones (0.5-mm diameter spots) of confluent cultures were subsequently exposed to 480 +/- 20-nm (blue) or 545 +/- 1-nm (green) light for 15 to 60 seconds. Phototoxicity was quantified at various periods after exposure by fluorescence staining of the nuclei of membrane-compromised cells, by TdT-dUTP terminal nick-end labeling (TUNEL) of apoptotic cells and by Annexin V labeling for phosphatidylserine exposure. Results Nonviable cells were located in blue light- exposed zones of A2E-containing RPE cells, whereas cells situated outside the illuminated areas remained viable. As shown by fluorescence labeling of the nuclei of membrane-damaged cells and by the presence of TUNEL-positive cells, the numbers of nonviable cells increased with exposure duration and as a function of the concentration of A2E used to load the cells before illumination. The numbers of blue light-induced TUNEL-positive cells also increased in advance of the increase in labeling of membrane-compromised cells, a finding that, together with Annexin V labeling, indicates an apoptotic form of cell death. Conversely, blue light- exposed RPE cells that did not contain A2E remained viable. In addition, illumination with green light resulted in the appearance of substantially fewer nonviable cells. Conclusions These studies implicate A2E as an initiator of blue light-induced apoptosis of RPE cells.
TL;DR: Evidence is presented that CREB-binding protein (CBP), a transcriptional co-activator that orchestrates nuclear response to a variety of cell signaling cascades, is incorporated into nuclear inclusions formed by polyglutamine-containing proteins in cultured cells, transgenic mice and tissue from patients with SBMA.
Abstract: Spinal and bulbar muscular atrophy (SBMA) is one of eight inherited neurodegenerative diseases known to be caused by CAG repeat expansion The expansion results in an expanded polyglutamine tract, which likely confers a novel, toxic function to the affected protein Cell culture and transgenic mouse studies have implicated the nucleus as a site for pathogenesis, suggesting that a critical nuclear factor or process is disrupted by the polyglutamine expansion In this report we present evidence that CREB-binding protein (CBP), a transcriptional co-activator that orchestrates nuclear response to a variety of cell signaling cascades, is incorporated into nuclear inclusions formed by polyglutamine-containing proteins in cultured cells, transgenic mice and tissue from patients with SBMA We also show CBP incorporation into nuclear inclusions formed in a cell culture model of another polyglutamine disease, spinocerebellar ataxia type 3 We present evidence that soluble levels of CBP are reduced in cells expressing expanded polyglutamine despite increased levels of CBP mRNA Finally, we demonstrate that over-expression of CBP rescues cells from polyglutamine-mediated toxicity in neuronal cell culture These data support a CBP-sequestration model of polyglutamine expansion disease
TL;DR: It is shown that the use of recombinant lentiviruses enables efficient transfer and faithful integration of the human β-globin gene together with large segments of its locus control region, which should be of therapeutic benefit in patients with severe defects in haemoglobin production.
Abstract: The stable introduction of a functional beta-globin gene in haematopoietic stem cells could be a powerful approach to treat beta-thalassaemia and sickle-cell disease. Genetic approaches aiming to increase normal beta-globin expression in the progeny of autologous haematopoietic stem cells might circumvent the limitations and risks of allogeneic cell transplants. However, low-level expression, position effects and transcriptional silencing hampered the effectiveness of viral transduction of the human beta-globin gene when it was linked to minimal regulatory sequences. Here we show that the use of recombinant lentiviruses enables efficient transfer and faithful integration of the human beta-globin gene together with large segments of its locus control region. In long-term recipients of unselected transduced bone marrow cells, tetramers of two murine alpha-globin and two human betaA-globin molecules account for up to 13% of total haemoglobin in mature red cells of normal mice. In beta-thalassaemic heterozygous mice higher percentages are obtained (17% to 24%), which are sufficient to ameliorate anaemia and red cell morphology. Such levels should be of therapeutic benefit in patients with severe defects in haemoglobin production.
TL;DR: In this paper, the internal tandem duplication of the human Flt3 gene in approximately 20% of acute myeloid leukemia (AML) cases was identified, and the wild-type and the mutant FLt3 genes were transfected into two IL-3-dependent cell lines, 32D and BA/F3 cells.
Abstract: We have recently identified an internal tandem duplication of the human Flt3 gene in approximately 20% of acute myeloid leukemia (AML) cases. In the present study, the wild-type and the mutant Flt3 genes were transfected into two IL-3-dependent cell lines, 32D and BA/F3 cells. Mutant Flt3-transfected cells exhibited autonomous growth while wild-type Flt3-transfected cells with the continuous stimulation of Flt3 ligand exhibited a minimal proliferation. Cells expressing mutant Flt3 showed constitutive activation of STAT5 and MAP kinase. In contrast, Flt3 ligand stimulation caused rapid activation of MAP kinase but not STAT5 in cells expressing wild-type Flt3. Finally, we found constitutive activation of MAP kinase and STAT5 in all clinical samples of AML patients with mutant Flt3. Our study shows the significance of internal tandem duplication of Flt3 receptors for leukemia cell expansion.
TL;DR: The results indicate that mitotic activity enhances transfections not only by lipoplexes but also by polyplexes, but not a viral system which has an efficient nuclear entry machinery, suggesting that transfection close to M phase is facilitated perhaps by nuclear membrane breakdown.
Abstract: The aim of this study was to investigate the influence of cell cycle on transfection efficiency. Counterflow centrifugal elutriation was used which avoids possible side-effects from chemical treatment of cells. With this method, cell populations were fractionated by means of size and density, and fractions corresponding to discrete cell cycle phase-specific populations were transfected with various nonviral methods (Lipofectamine, TfpLys and TfPEI), adenovirus-enhanced transferrinfection (AVET system) and recombinant adenovirus. Transfection efficiency was found to be strongly dependent on the cell cycle stage at the time of transfection. Luciferase activity from cells transfected with polycation- or lipid-based transfection systems was 30- to more than 500-fold higher when transfection was performed during S or G2 phase compared with cells in G1 phase which have the lowest expression levels. In contrast, this effect was not observed with recombinant adenovirus which varied only four-fold. Our results indicate that mitotic activity enhances transfection not only by lipoplexes but also by polyplexes, but not a viral system which has an efficient nuclear entry machinery, suggesting that transfection close to M phase is facilitated perhaps by nuclear membrane breakdown. Furthermore, low transfection success into G1 cells indicates that DNA complexes deposited in G1 cells are probably not retained long enough to take advantage of mitosis effects or that passage of transfected cells through S phase is inhibitory.
TL;DR: It is found that EGF accelerated repair of scrape‐wounded monolayers and that the EGFR‐selective inhibitor, tyrphostin AG1478, inhibited both EGF‐stimulated and basal wound closure whereas dexamethasone was without effect.
Abstract: Epithelial damage and airway remodeling are consistent features of bronchial asthma and are correlated with disease chronicity, severity, and bronchial hyperreactivity. To examine the mechanisms that control bronchial epithelial repair, we investigated expression of the epidermal growth factor receptor (c-erbB1, EGFR) in asthmatic bronchial mucosa and studied repair responses in vitro. In biopsies from asthmatic subjects, areas of epithelial damage were frequently observed and exhibited strong EGFR immunostaining. EGFR expression was also high in morphologically intact asthmatic epithelium. Using image analysis, EGFR immunoreactivity (% of total epithelial area, median (range) was found to increase from 9.4 (4.1-20.4) in normal subjects (n=10) to 18.4 (9.3-28.9) in mild asthmatics (P<0.01, n=13) and 25.4 (15.4-31.8) in severe asthmatics (P<0.00, n=5). Epithelial EGFR immunoreactivity remained elevated in patients treated with corticosteroids and was positively correlated with subepithelial reticular membrane thickening. Using 16HBE 14o- bronchial epithelial cells, we found that EGF accelerated repair of scrape-wounded monolayers and that the EGFR-selective inhibitor, tyrphostin AG1478, inhibited both EGF-stimulated and basal wound closure whereas dexamethasone was without effect. Intrinsic activation of the EGFR was confirmed by analysis of tyrosine phosphorylated proteins, which revealed a rapid, damage-induced phosphorylation of the EGFR, irrespective of the presence of exogenous EGF. To assess the relationship between EGFR-mediated repair and tissue remodeling, release of the profibrogenic mediator TGF-beta2 was also measured. Scrape wounding increased release of TGF-beta2 from epithelial monolayers and EGF had no additional stimulatory effect. However, when repair was retarded with AG1478, the amount of TGF-beta2 increased significantly. These data indicate that the EGFR may play an important role in bronchial epithelial repair in asthma and that impairment of this function may augment airway remodeling.
TL;DR: It is shown that delayed and persistent activation of ERKs is associated with glutamate-induced oxidative toxicity in HT22 cells and immature primary cortical neuron cultures, and that U0126, a specific inhibitor of the ERK-activating kinase, MEK-1/2, protects both HT22cells and immaturePrimary cortex neuron cultures from glutamate toxicity.
TL;DR: Overexpression of the Bcr/Abl protein mediated through gene amplification is associated with and probably determines resistance of human leukemic cells to STI571 in vitro.
TL;DR: It is concluded that the pathogenically critical process of Abeta oligomers, principally dimers, in primary human neurons and in neuronal and nonneural cell lines begins intraneuronally.
Abstract: The progressive aggregation and deposition of amyloid beta-protein (Abeta) in brain regions subserving memory and cognition is an early and invariant feature of Alzheimer's disease, the most common cause of cognitive failure in aged humans. Inhibiting Abeta aggregation is therapeutically attractive because this process is believed to be an exclusively pathological event. Whereas many studies have examined the aggregation of synthetic Abeta peptides under nonphysiological conditions and concentrations, we have detected and characterized the oligomerization of naturally secreted Abeta at nanomolar levels in cultures of APP-expressing CHO cells [Podlisny, M. B., Ostaszewski, B. L., Squazzo, S. L., Koo, E. H., Rydell, R. E., Teplow, D. B., and Selkoe, D. J. (1995) J. Biol. Chem. 270, 9564-9570 (1); Podlisny, M. B., Walsh, D. M., Amarante, P., Ostaszewski, B. L., Stimson, E. R., Maggio, J. E., Teplow, D. B., and Selkoe, D. J. (1998) Biochemistry 37, 3602-3611 (2)]. To determine whether similar species occur in vivo, we probed samples of human cerebrospinal fluid (CSF) and detected SDS-stable dimers of Abeta in some subjects. Incubation of CSF or of CHO conditioned medium at 37 degrees C did not lead to new oligomer formation. This inability to induce oligomers extracellularly as well as the detection of oligomers in cell medium very early during the course of pulse-chase experiments suggested that natural Abeta oligomers might first form intracellularly. We therefore searched for and detected intracellular Abeta oligomers, principally dimers, in primary human neurons and in neuronal and nonneural cell lines. These dimers arose intracellularly rather than being derived from the medium by reuptake. The dimers were particularly detectable in neural cells: the ratio of intracellular to extracellular oligomers was much higher in brain-derived than nonbrain cells. We conclude that the pathogenically critical process of Abeta oligomerization begins intraneuronally.
TL;DR: It is suggested that TNF acts distal to IGF-I, BMPs, and LMP-1 in the progression toward the osteoblast phenotype as shown by continued attachment and metabolism in culture, trypan blue exclusion, and Alamar Blue cytotoxicity assay.
Abstract: Tumor necrosis factor-a (TNF-a) has a key role in skeletal disease in which it promotes reduced bone formation by mature osteoblasts and increased osteoclastic resorption. Here we show that TNF inhibits differentiation of osteoblasts from precursor cells. TNF-a treatment of fetal calvaria precursor cells, which spontaneously differentiate to the osteoblast phenotype over 21 days, inhibited differentiation as shown by reduced formation of multilayered, mineralizing nodules and decreased secretion of the skeletal-specific matrix protein osteocalcin. The effect of TNF was dose dependent with an IC50 of 0.6 ng/ml, indicating a high sensitivity of these precursor cells. Addition of TNF-a from days 2‐21, 2‐14, 7‐14, and 7‐10 inhibited nodule formation but addition of TNF after day 14 had no effect. Partial inhibition of differentiation was observed with addition of TNF on only days 7‐ 8, suggesting that TNF could act during a critical period of phenotype selection. Growth of cells on collagen-coated plates did not prevent TNF inhibition of differentiation, suggesting that inhibition of collagen deposition into matrix by proliferating cells could not, alone, explain the effect of TNF. Northern analysis revealed that TNF inhibited the expression of insulin-like growth factor I (IGF-I). TNF had no effect on expression of the osteogenic bone morphogenic proteins (BMPs-2, -4, and -6), or skeletal LIM protein (LMP-1), as determined by semiquantitative RT-PCR. Addition of IGF-I or BMP-6 to fetal calvaria precursor cell cultures enhanced differentiation but could not overcome TNF inhibition, suggesting that TNF acted downstream of these proteins in the differentiation pathway. The clonal osteoblastic cell line, MC3T3-E1‐14, which acquires the osteoblast phenotype spontaneously in postconfluent culture, was also studied. TNF inhibited differentiation of MC3T3-E1‐14 cells as shown by failure of mineralized matrix formation in the presence of calcium and phosphate. TNF was not cytotoxic to either cell type as shown by continued attachment and metabolism in culture, trypan blue exclusion, and Alamar Blue cytotoxicity assay. These results demonstrate that TNF-a is a potent inhibitor of osteoblast differentiation and suggest that TNF acts distal to IGF-I, BMPs, and LMP-1 in the progression toward the osteoblast phenotype. (Endocrinology 141: 3956 ‐3964, 2000)
TL;DR: The cell culture and nuclear transfer techniques described here should allow the use of genetic modification procedures to produce tissues and organs from cloned pigs with reduced immunogenicity for use in xenotransplantation.
Abstract: Here we describe a procedure for cloning pigs by the use of in vitro culture systems. Four healthy male piglets from two litters were born following nuclear transfer of cultured somatic cells and subsequent embryo transfer. The initiation of five additional pregnancies demonstrates the reproducibility of this procedure. Its important features include extended in vitro culture of fetal cells preceding nuclear transfer, as well as in vitro maturation and activation of oocytes and in vitro embryo culture. The cell culture and nuclear transfer techniques described here should allow the use of genetic modification procedures to produce tissues and organs from cloned pigs with reduced immunogenicity for use in xenotransplantation.
TL;DR: It is demonstrated that differentiated bone tissue was formed throughout 3D scaffolds after 2 weeks in culture using an optimized initial cell density, whereas mineralization of the tissue only occurred after 3 weeks.
Abstract: New three-dimensional (3D) scaffolds for bone tissue engineering have been developed throughout which bone cells grow, differentiate, and produce mineralized matrix. In this study, the percentage of cells anchoring to our polymer scaffolds as a function of initial cell seeding density was established; we then investigated bone tissue formation throughout our scaffolds as a function of initial cell seeding density and time in culture. Initial cell seeding densities ranging from 0.5 to 10 x 10(6) cells/cm(3) were seeded onto 3D scaffolds. After 1 h in culture, we determined that 25% of initial seeded cells had adhered to the scaffolds in static culture conditions. The cell-seeded scaffolds remained in culture for 3 and 6 weeks, to investigate the effect of initial cell seeding density on bone tissue formation in vitro. Further cultures using 1 x 10(6) cells/cm(3) were maintained for 1 h and 1, 2, 4, and 6 weeks to study bone tissue formation as a function of culture period. After 3 and 6 weeks in culture, scaffolds seeded with 1 x 10(6) cells/cm(3) showed similar tissue formation as those seeded with higher initial cell seeding densities. When initial cell seeding densities of 1 x 10(6) cells/cm(3) were used, osteocalcin immunolabeling indicative of osteoblast differentiation was seen throughout the scaffolds after only 2 weeks of culture. Von Kossa and tetracycline labeling, indicative of mineralization, occurred after 3 weeks. These results demonstrated that differentiated bone tissue was formed throughout 3D scaffolds after 2 weeks in culture using an optimized initial cell density, whereas mineralization of the tissue only occurred after 3 weeks. Furthermore, after 6 weeks in culture, newly formed bone tissue had replaced degrading polymer.
TL;DR: These studies provide clear evidence that irradiated cells can induce a bystander mutagenic response in neighboring cells not directly traversed by alpha particles and that cell-cell communication process play a critical role in mediating the bystander phenomenon.
Abstract: Ever since the discovery of X-rays was made by Rontgen more than a hundred years ago, it has always been accepted that the deleterious effects of ionizing radiation such as mutation and carcinogenesis are attributable mainly to direct damage to DNA. Although evidence based on microdosimetric estimation in support of a bystander effect appears to be consistent, direct proof of such extranuclear/extracellular effects are limited. Using a precision charged particle microbeam, we show here that irradiation of 20% of randomly selected AL cells with 20 alpha particles each results in a mutant fraction that is 3-fold higher than expected, assuming no bystander modulation effect. Furthermore, analysis by multiplex PCR shows that the types of mutants induced are significantly different from those of spontaneous origin. Pretreatment of cells with the radical scavenger DMSO had no effect on the mutagenic incidence. In contrast, cells pretreated with a 40 μM dose of lindane, which inhibits cell–cell communication, significantly decreased the mutant yield. The doses of DMSO and lindane used in these experiments are nontoxic and nonmutagenic. We further examined the mutagenic yield when 5–10% of randomly selected cells were irradiated with 20 alpha particles each. Results showed, likewise, a higher mutant yield than expected assuming no bystander effects. Our studies provide clear evidence that irradiated cells can induce a bystander mutagenic response in neighboring cells not directly traversed by alpha particles and that cell–cell communication process play a critical role in mediating the bystander phenomenon.
TL;DR: The results suggest that the effects of p21 induction on gene expression in senescent cells may contribute to the pathogenesis of cancer and age-related diseases.
Abstract: Induction of cyclin-dependent kinase inhibitor p21(Waf1/Cip1/Sdi1) triggers cell growth arrest associated with senescence and damage response. Overexpression of p21 from an inducible promoter in a human cell line induces growth arrest and phenotypic features of senescence. cDNA array hybridization showed that p21 expression selectively inhibits a set of genes involved in mitosis, DNA replication, segregation, and repair. The kinetics of inhibition of these genes on p21 induction parallels the onset of growth arrest, and their reexpression on release from p21 precedes the reentry of cells into cell cycle, indicating that inhibition of cell-cycle progression genes is a mechanism of p21-induced growth arrest. p21 also up-regulates multiple genes that have been associated with senescence or implicated in age-related diseases, including atherosclerosis, Alzheimer's disease, amyloidosis, and arthritis. Most of the tested p21-induced genes were not activated in cells that had been growth arrested by serum starvation, but some genes were induced in both forms of growth arrest. Several p21-induced genes encode secreted proteins with paracrine effects on cell growth and apoptosis. In agreement with the overexpression of such proteins, conditioned media from p21-induced cells were found to have antiapoptotic and mitogenic activity. These results suggest that the effects of p21 induction on gene expression in senescent cells may contribute to the pathogenesis of cancer and age-related diseases.
TL;DR: In vitro results suggest that dectin-1 on DC may bind to as yet undefined ligand(s) on T cells, thereby delivering T cell co-stimulatory signals.
TL;DR: The A(1)R/D( 1)R heteromerization may be one molecular basis for the demonstrated antagonistic modulation of A(2)R of D(1]R receptor signaling in the brain and seems to be essential for the blockade of A-1-R agonist-induced A(3)R-induced coclustering and for the desensitization of the D(0)R agonists cAMP accumulation.
Abstract: The possible molecular basis for the previously described antagonistic interactions between adenosine A(1) receptors (A(1)R) and dopamine D(1) receptors (D(1)R) in the brain have been studied in mouse fibroblast Ltk(-) cells cotransfected with human A(1)R and D(1)R cDNAs or with human A(1)R and dopamine D(2) receptor (long-form) (D(2)R) cDNAs and in cortical neurons in culture. A(1)R and D(1)R, but not A(1)R and D(2)R, were found to coimmunoprecipitate in cotransfected fibroblasts. This selective A(1)R/D(1)R heteromerization disappeared after pretreatment with the D(1)R agonist, but not after combined pretreatment with D(1)R and A(1)R agonists. A high degree of A(1)R and D(1)R colocalization, demonstrated in double immunofluorescence experiments with confocal laser microscopy, was found in both cotransfected fibroblast cells and cortical neurons in culture. On the other hand, a low degree of A(1)R and D(2)R colocalization was observed in cotransfected fibroblasts. Pretreatment with the A(1)R agonist caused coclustering (coaggregation) of A(1)R and D(1)R, which was blocked by combined pretreatment with the D(1)R and A(1)R agonists in both fibroblast cells and in cortical neurons in culture. Combined pretreatment with D(1)R and A(1)R agonists, but not with either one alone, substantially reduced the D(1)R agonist-induced accumulation of cAMP. The A(1)R/D(1)R heteromerization may be one molecular basis for the demonstrated antagonistic modulation of A(1)R of D(1)R receptor signaling in the brain. The persistence of A(1)R/D(1)R heteromerization seems to be essential for the blockade of A(1)R agonist-induced A(1)R/D(1)R coclustering and for the desensitization of the D(1)R agonist-induced cAMP accumulation seen on combined pretreatment with D(1)R and A(1)R agonists, which indicates a potential role of A(1)R/D(1)R heteromers also in desensitization mechanisms and receptor trafficking.