Low-serum Medium Research Articles

Neoplastic Reversal of Human Ovarian Carcinoma Cells Transfected with Connexin43

Gap junctional intercellular communication and expression of gap junction proteins (connexins) are decreased frequently in neoplastic cells including human ovarian carcinoma cells. In order to test the hypothesis that these changes contribute to the neoplastic phenotype of ovarian carcinoma cells, we transfected human ovarian carcinoma SKOV-3 cells with connexin43. Stable, connexin43-expressing transfectants were characterized for cell proliferation in vitro in normal, low-serum, and serum-free culture medium, for tumorigenicity in nude mice, and for sensitivity to adriamycin in vitro. Transfected clones expressed higher levels of connexin43 and gap junctional intercellular communication, reduced proliferation and greater dependence upon serum for growth in vitro, decreased tumor formation, increased sensitivity to adriamycin, and reduced expression of p-glycoprotein. These data suggest that gap junctional intercellular communication and/or connexin43 expression suppresses the neoplastic phenotype of ovarian carcinoma cells and their downregulation is involved in neoplastic transformation of ovarian epithelial cells. The increased sensitivity to adriamycin and elevated expression of p-glycoprotein by the transfected cells also suggest that gap junctional intercellular communication and connexin43 expression are involved in drug sensitivity and might be manipulated to enhance the clinical response.

Wnt/beta-catenin signaling suppresses apoptosis in low serum medium and induces morphologic change in rodent fibroblasts.

Wnt/beta-catenin signaling plays important roles in tumorigenesis in certain tumors as well as during development. However, the mechanism of tumorigenesis mediated by this signaling remains to be elucidated. We investigated the response of rodent fibroblasts to activation of Wnt/beta-catenin signaling by treatment with conditioned medium containing soluble Wnt-3a protein (W3a-CM) and by expression of a constitutive active beta-catenin gene harbored by an adenovirus vector. W3a-CM induced transcriptional activation of a beta-catenin/T-cell factor (Tcf)-responsive promoter in rodent fibroblasts such as NIH3T3, Rat-1, Swiss3T3 and Balb3T3 cells. In these cells, an increase in saturation density and an inhibition of apoptosis and/or promotion of growth in low-serum medium were induced by treatment with W3a-CM. In Rat-1 cells, morphologic changes were also induced. All these alterations were reversible. Moreover, the inhibition of apoptosis of NIH3T3 cells in low-serum medium and the morphologic changes in Rat-1 cells, but not the increase in saturation density, were also induced by ectopic expression of a constitutive active beta-catenin gene. These results suggested that activation of Wnt/beta-catenin signaling induces inhibition of apoptosis and morphologic changes in these cells.

Insulin-like growth factors (IGF-I and IGF-II) inhibit C2 skeletal myoblast differentiation and enhance TNF alpha-induced apoptosis.

IGF-I and IGF-II are thought to be unique in their ability to promote muscle cell differentiation. Murine C2 myoblasts differentiate when placed into low serum media (LSM), accompanied by increased IGF-II and IGF binding protein-5 (IGFBP-5) production. Addition of 20 ng/ml TNF alpha on transfer into LSM blocked differentiation, IGF-II and IGFBP-5 secretion and induced apoptosis. We, therefore, wished to assess whether IGFs could protect against the effects of TNF alpha. Neither inhibition of differentiation or induction of apoptosis was rescued by co-incubation with IGF-I or IGF-II. A lower dose of TNF alpha (1 ng/ml) while not inducing apoptosis still inhibited myoblast differentiation by 56% +/- 12, (P < 0.001), indicating that induction of apoptosis is not the sole mechanism by which TNF alpha inhibits myoblast differentiation. Addition of IGF-I or IGF-II alone reduced differentiation by 49% +/- 15 and 33% +/- 20, respectively, (P < 0.001), although neither induced apoptosis. For muscle cells to differentiate, they must arrest in G0. We established that addition of IGF-I, IGF-II or TNF alpha to the myoblasts promoted proliferation. The myoblasts could not exit the cell cycle as efficiently as controls and differentiation was thus reduced. Unexpectedly, co-incubation of IGF-I or IGF-II with 1 ng/ml TNF alpha enhanced the inhibition of differentiation and induced apoptosis. In the absence of apoptosis we show an association between IGF-induced inhibition of differentiation and increased IGFBP-5 secretion. These results indicate that the effects of the IGFs on muscle may depend on the cytokine environment. In the absence of TNF alpha, the IGFs delay differentiation and promote myoblast proliferation whereas in the presence of TNF alpha the IGFs induce apoptosis.

Reduction of spontaneous mutagenesis in mismatch repair-deficient and proficient cells by dietary antioxidants

Cells lacking mismatch repair (MMR) exhibit elevated levels of spontaneous mutagenesis. Evidence exists that MMR is involved in repair of some DNA lesions besides mismatches. If some oxidative DNA lesions are substrates for MMR, then the excess mutagenesis in MMR − cells might be blocked by dietary antioxidants. Effects of the dietary antioxidants ascorbate, α-tocopherol, (−)-epigallocatechin gallate (EGCG) and lycopene on spontaneous mutagenesis were studied using mismatch repair-deficient (hMLH1 −) human colon carcinoma HCT116 cells and HCT116/ch3 cells, in which normal human chromosome 3 has been added to restore mismatch repair. HCT116 cells have a 22-fold higher spontaneous mutation rate compared with HCT116/ch3 cells. HCT116 cells cultured in 1% fetal bovine serum (FBS) have twice the spontaneous mutation rate of those cultured in 10% FBS, most likely due to reduction in serum antioxidants in the low serum medium. As expected, α-tocopherol (50 μM) and ascorbate (284 μM) reduced spontaneous mutagenesis in HCT116 cells growing in 1% serum more dramatically than in cells cultured in 10% serum. The strongest antimutagenic compound was lycopene (5 μM), which reduced spontaneous mutagenesis equally (about 70%) in HCT116 cells growing in 10 and 1% FBS and in HCT116/ch3 cells. Since lycopene was equally antimutagenic in cells growing in low and high serum, it may have another antimutagenic mechanism in addition to its antioxidant effect. Surprisingly, EGCG (10 μM) was toxic to cells growing in low serum. It also reduced spontaneous mutagenesis equally (nearly 40%) in HCT116 and HCT116/ch3 cells. The large proportion of spontaneous mutagenesis that can be blocked by antioxidants in mismatch repair-deficient cells support the hypothesis that a major cause of their excess mutagenesis is endogenous oxidants. Blocking spontaneous mutagenesis, perhaps with a cocktail of antioxidants, should reduce the risk of cancer in people with a genetic defect in mismatch repair as well as other individuals.

IL-12/IL-18-Dependent IFN-γ Release by Murine Dendritic Cells

Dendritic cells (DC) develop in GM-CSF-stimulated cultures from murine bone marrow progenitors in serum-free (or low serum) medium. CD11c(+) myeloid DC from 7-day cultures stimulated with TNF-alpha, IFN-alpha, IFN-gamma, or LPS up-regulated surface expression of CD40 and CD86 costimulator and MHC class II molecules, did not up-regulate the low "spontaneous" release of IL-18, and did not release IFN-gamma. Stimulation of in vitro-generated DC with exogenous IL-12 and IL-18 (but not with IL-4 or LPS plus IL-18) induced IFN-gamma expression and release in 15-20% of the DC (detectable by FACS analyses or ELISA). Endogenous IL-12 p70 produced by DC in response to ligation of CD40 stimulated IFN-gamma release when exogenous IL-18 was supplied. In vivo-generated, splenic CD8alpha(+) and CD8alpha(-) DC (from immunocompetent and immunodeficient H-2(d) and H-2(b) mice) cultured with IL-12 and IL-18 released IFN-gamma. The presence of LPS during the stimulation of DC with IL-18 plus endogenous (CD40 ligation) or exogenous IL-12 did not affect their IFN-gamma release. In contrast, splenic DC pretreated in vitro or in vivo by LPS strikingly down-regulated IFN-gamma release in response to stimulation by IL-18 and (endogenous or exogenous) IL-12. Hence, DC are a source of early IFN-gamma generated in response to a cascade of cytokine- and/or cell-derived signals that can be positively and negatively regulated.

AN2/NG2 protein-expressing glial progenitor cells in the murine CNS: isolation, differentiation, and association with radial glia.

During early neural development, the lineage specification of initially pluripotent progenitor cells is associated with proliferation, differentiation, and migration. Oligodendroglial progenitor cells migrate from their sites of origin to reach the axons that they will myelinate. We have described a cell-surface protein, AN2, expressed by oligodendroglial progenitor cells in vitro and showed that antibodies against AN2 inhibited the migration of cultured primary oligodendroglial progenitor cells, suggesting that the AN2 antigen plays a role in their migration. Recently, results from MALDI mass spectroscopy showed that AN2 is the mouse homologue of the rat NG2 protein. In this study, we have analyzed cells staining with AN2 antibodies during development and in the adult murine central nervous system (CNS), carried out double stainings with antibodies against NG2, and investigated the differentiation potential of cells in vitro after isolation from early postnatal brain using AN2 antibodies. AN2 and NG2 antibodies stained totally overlapping populations of cells in the CNS. AN2/NG2 expressing cells in embryonic and postnatal brain expressed the PDGF-alpha-receptor and in postnatal brain exhibited electrophysiological properties typical of glial progenitor cells. Cells isolated from early postnatal brain using AN2 monoclonal antibody developed into oligodendrocytes in low serum medium or into astrocytes in the presence of fetal calf serum. In the embryonic spinal cord, cells staining with AN2 antibodies were found closely apposed to radial glial cells, suggesting that glial precursors, like neurons, may use radial glia as scaffolds for migration.

Positive and Negative Regulation of Myogenic Differentiation of C2C12 Cells by Isoforms of the Multiple Homeodomain Zinc Finger Transcription Factor ATBF1

The ATBF1 gene encodes two protein isoforms, the 404-kDa ATBF1-A, possessing four homeodomains and 23 zinc fingers, and the 306-kDa ATBF1-B, lacking a 920-amino acid N-terminal region of ATBF1-A which contains 5 zinc fingers. In vitro, ATBF1-A was expressed in proliferating C2C12 myoblasts, but its expression levels decreased upon induction of myogenic differentiation in low serum medium. Forced expression of ATBF1-A in C2C12 cells resulted in repression of MyoD and myogenin expression and elevation of Id3 and cyclin D1 expression, leading to inhibition of myogenic differentiation in low serum. In contrast, transfection of C2C12 cells with the ATBF1-B isoform led to an acceleration of myogenic differentiation, as indicated by an earlier onset of myosin heavy chain expression and formation of a higher percentage of multinucleated myotubes. The fourth homeodomain of ATBF1-A bound to an AT-rich element adjacent to the E1 E-box of the muscle regulatory factor 4 promoter mediating transcriptional repression. The ATBF1-A-specific N-terminal region possesses general transcription repressor activity. These results suggest that ATBF1-A plays a role in the maintenance of the undifferentiated myoblast state, and its down-regulation is a prerequisite to initiate terminal differentiation of C2C12 cells.

Tissue engineering of functional cardiac muscle: molecular, structural, and electrophysiological studies.

The primary aim of this study was to relate molecular and structural properties of in vitro reconstructed cardiac muscle with its electrophysiological function using an in vitro model system based on neonatal rat cardiac myocytes, three-dimensional polymeric scaffolds, and bioreactors. After 1 wk of cultivation, we found that engineered cardiac muscle contained a 120- to 160-microm-thick peripheral region with cardiac myocytes that were electrically connected through gap junctions and sustained macroscopically continuous impulse propagation over a distance of 5 mm. Molecular, structural, and electrophysiological properties were found to be interrelated and depended on specific model system parameters such as the tissue culture substrate, bioreactor, and culture medium. Native tissue and the best experimental group (engineered cardiac muscle cultivated using laminin-coated scaffolds, rotating bioreactors, and low-serum medium) were comparable with respect to the conduction velocity of propagated electrical impulses and spatial distribution of connexin43. Furthermore, the structural and electrophysiological properties of the engineered cardiac muscle, such as cellularity, conduction velocity, maximum signal amplitude, capture rate, and excitation threshold, were significantly improved compared with our previous studies.

Tissue transglutaminase is essential for neurite outgrowth in human neuroblastoma SH-SY5Y cells

Tissue transglutaminase is a normal constituent of the central and peripheral nervous systems and in rats transglutaminase activity in brain and spinal cord is highest during fetal stages when axonal outgrowth is occurring. Further, treatment of human neuroblastoma SH-SY5Y cells with retinoic acid results in the cells withdrawing from the cell cycle and extending neurites, in the same time frame that tissue transglutaminase expression significantly increases. Considering these and other previous findings, this study was carried out to determine whether tissue transglutaminase is involved in neuronal differentiation of SH-SY5Y cells. For these studies SH-SY5Y cells stably overexpressing wild-type tissue transglutaminase, an inactive tissue transglutaminase mutant (C277S) or an antisense tissue transglutaminase construct (which decreased endogenous tissue transglutaminase below detectable levels) were used. SH-SY5Y cells overexpressing wild-type tissue transglutaminase spontaneously differentiated into a neuronal phenotype when grown in low-serum media. In contrast, cells overexpressing inactive tissue transglutaminase or the antisense tissue transglutaminase continued to proliferate and exhibit a flat polygenic morphology even when maintained in low-serum conditions. In addition, increased tissue transglutaminase expression in response to retinoic acid was abolished in the antisense tissue transglutaminase cells, and antisense and mutant tissue transglutaminase expressing cells did not extend neurites in response to retinoic acid. Moreover, wild-type and inactive tissue transglutaminase exhibited differential intracellular localization. These data indicate that tissue transglutaminase is necessary and sufficient for neuronal differentiation of human neuroblastoma SH-SY5Y cells.

Effect of antioxidants on the apoptosis of CHO cells and production of tissue plasminogen activator in suspension culture

The effects of antioxidants on the apoptosis of Chinese hamster ovary (CHO) 1-15,500 cells and production of tissue plasminogen activator (tPA) in suspension culture were investigated. After cell growth to 2 × 10 5 cells/ml in Ham's F12 medium containing 10% newborn bovine serum (NBS) in a spinner bottle, CHO cells were maintained for 6 d in Ham's F12 medium containing 0 or 0.4% NBS and 10 mM antioxidants, namely, l-ascorbic acid 2-phosphate (VCP) or the reduced form of glutathione (GSH). The viable cell concentrations at day 6 in the serum-free culture with GSH and in the low-serum culture wiht VCP or GSH were 0.57, 1.04 and 1.69 × 10 5 cells/ml, respectively, while those in the serum-free and low-serum cultures without the antioxidants were only 2.33 and 1.17 × 10 3 cells/ml, respectively. The percentages of apoptotic cells in the serum-free and low-serum cultures with VCP (73.2, 44.6%) and GSH (76.9, 38.6%) measured using a flowcytometer after annexin V-FITC/propidium iodide staining were markedly lower than those in the cultures without antioxidants (96.3, 92.5%). The percentage of cells having a high mitochondrial membrane potential among the viable cells in the cultures with antioxidants determined using a flowcytometer after 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide staining was clearly higher than those in the cultures without the antioxidants. The production of tPA in the serum-free and low-serum media with VCP (0.282, 2.92 mg/ l) or GSH (1.01, 1.61 mg/ l) was markedly higher than that in the cultures without the antioxidants (0.275, 0.689 mg/ l). Consequently, the suppression of apoptosis through the maintenance of the membrane potential of mitochondria by VCP or GSH resulted in a marked increase in tPA production by CHO cells in the serum-free and low-serum cultures.

Effect of Antioxidants on the Apoptosis of CHO Cells and Production of Tissue Plasminogen Activator in Suspension Culture.

The effects of antioxidants on the apoptosis of Chinese hamster ovary (CHO) 1-15,500 cells and production of tissue plasminogen activator (tPA) in suspension culture were investigated. After cell growth to 2 x 10(5) cells/ml in Ham's F12 medium containing 10% newborn bovine serum (NBS) in a spinner bottle, CHO cells were maintained for 6 d in Ham's F12 medium containing 0 or 0.4% NBS and 10 mM antioxidants, namely, l-ascorbic acid 2-phosphate (VCP) or the reduced form of glutathione (GSH). The viable cell concentrations at day 6 in the serum-free culture with GSH and in the low-serum culture wiht VCP or GSH were 0.57, 1.04 and 1.69 x 10(5) cells/ml, respectively, while those in the serum-free and low-serum cultures without the antioxidants were only 2.33 and 1.17 x 10(3) cells/ml, respectively. The percentages of apoptotic cells in the serum-free and low-serum cultures with VCP (73.2, 44.6%) and GSH (76.9, 38.6%) measured using a flowcytometer after annexin V-FITC/propidium iodide staining were markedly lower than those in the cultures without antioxidants (96.3, 92.5%). The percentage of cells having a high mitochondrial membrane potential among the viable cells in the cultures with antioxidants determined using a flowcytometer after 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide staining was clearly higher than those in the cultures without the antioxidants. The production of tPA in the serum-free and low-serum media with VCP (0.282, 2.92 mg/l) or GSH (1.01, 1.61 mg/l) was markedly higher than that in the cultures without the antioxidants (0.275, 0.689 mg/l). Consequently, the suppression of apoptosis through the maintenance of the membrane potential of mitochondria by VCP or GSH resulted in a marked increase in tPA production by CHO cells in the serum-free and low-serum cultures.

Effects of prolonged exposure to hypoxia on morphological changes of endothelial cells plated on fibrin gel

Since tissue oxygenation has a profound effect on capillary growth, the effect of pO2 on endothelial cell functions was studied. Under normoxic conditions, EA.hy926 endothelial cells and HUVEC plated onto fibrin gels in low-serum culture medium underwent rapid and profound morphological changes within 12 to 48 hours depending on the cell line used. Their characteristic cobblestone organisation was transformed into a network of cord-like or tube-like structures. We showed that when exposed to low oxygen concentrations for 3 days, HUVEC and EA.hy926 have their ability to rearrange reduced to around 50 %. With EA.hy926 this effect was amplified by 79% after 9 days of hypoxia. The altered behaviour of hypoxia-adapted cells was not caused by a loss in their fibrinolytic activity. In fact, the fibrin degradation rate and the generated fibrin fragments appeared identical in normoxia and hypoxia. Confocal microscopy and gel densitometry showed that in normoxia the remaining undegraded fibrin gel underwent a dynamic remodeling whereas in hypoxia it remained undisturbed. It is likely that hypoxia induces modification in the factors that integrate matrix information and cytoskeletal organisation in order to contract fibrin.

Insulin-like growth factor-mediated muscle cell survival: central roles for Akt and cyclin-dependent kinase inhibitor p21.

Polypeptide growth factors activate specific transmembrane receptors, leading to the induction of multiple intracellular signal transduction pathways which control cell function and fate. Recent studies have shown that growth factors promote cell survival by stimulating the serine-threonine protein kinase Akt, which appears to function primarily as an antiapoptotic agent by inactivating death-promoting molecules. We previously established C2 muscle cell lines lacking endogenous expression of insulin-like growth factor II (IGF-II). These cells underwent apoptotic death in low-serum differentiation medium but could be maintained as viable myoblasts by IGF analogues that activated the IGF-I receptor or by unrelated growth factors such as platelet-derived growth factor BB (PDGF-BB). Here we show that IGF-I promotes muscle cell survival through Akt-mediated induction of the cyclin-dependent kinase inhibitor p21. Treatment of myoblasts with IGF-I or transfection with an inducible Akt maintained muscle cell survival and enhanced production of p21, and ectopic expression of p21 was able to sustain viability in the absence of growth factors. Blocking of p21 protein accumulation through a specific p21 antisense cDNA prevented survival regulated by IGF-I or Akt but did not block muscle cell viability mediated by PDGF-BB. Our results define Akt as an intermediate and p21 as a critical effector of an IGF-controlled myoblast survival pathway that is active during early myogenic differentiation and show that growth factors are able to maintain cell viability by inducing expression of pro-survival molecules.

A Polymorphic Protease-activated Receptor 2 (PAR2) Displaying Reduced Sensitivity to Trypsin and Differential Responses to PAR Agonists

Protease-activated receptor 2 (PAR2) is a trypsin-activated member of a family of G-protein-coupled PARs. We have identified a polymorphic form of human PAR2 (PAR(2)F240S) characterized by a phenylalanine to serine mutation at residue 240 within extracellular loop 2, with allelic frequencies of 0.916 (Phe(240)) and 0.084 (Ser(240)) for the wild-type and mutant alleles, respectively. Elevations in intracellular calcium were measured in permanently transfected cell lines expressing the receptors. PAR(2)F240S displayed a significant reduction in sensitivity toward trypsin ( approximately 3.7-fold) and the PAR2-activating peptides, SLIGKV-NH(2) ( approximately 2.5-fold) and SLIGRL-NH(2) ( approximately 2.8-fold), but an increased sensitivity toward the selective PAR2 agonist, trans-cinnamoyl-LIGRLO-NH(2) ( approximately 4-fold). Increased sensitivity was also observed toward the selective PAR-1 agonist, TFLLR-NH(2) ( approximately 7-fold), but not to other PAR-1 agonists tested. Furthermore, we found that TLIGRL-NH(2) and a PAR4-derived peptide, trans-cinnamoyl-YPGKF-NH(2), were selective PAR(2)F240S agonists. By introducing the F240S mutation into rat PAR2, we observed shifts in agonist potencies that mirrored the human PAR(2)F240S, suggesting that Phe(240) is involved in determining agonist specificity of PAR2. Finally, differences in receptor signaling were paralleled in a cell growth assay. We suggest that the distinct pharmacological profile induced by this polymorphism will have important implications for the design of PAR-targeted agonists/antagonists and may contribute to, or be predictive of, an inflammatory disease.

The effect of Huwentoxin-I on Ca 2+ channels in differentiated NG108-15 cells, a patch-clamp study

Huwentoxin-I (HWTX-I), a 3.75 kDa peptide toxin isolated from the venom of the spider Selenocosmia huwena, was found to be a reversible presynaptic inhibitor by our previous work. Using whole-cell patch clamp methods, we found that HWTX-I had no significant effect on the TTX-sensitive Na + current or the delayed rectifier K + current ( K r) in low-serum medium cultured NG108-15 cells, but High-Voltage-Activated Ca 2+ channel expressed in prostaglandin E 1 differentiated NG108-15 cells could be potently inhibited by HWTX-I (EC 50 ≈ 100 nM), while it hardly affected low-voltage-activated Ca 2+ channel. Among types of high-voltage-activated Ca 2+ channel, HWTX-I selectively inhibited N-type Ca 2+ channel and had only very weak effect on L-type Ca 2+ channel in prostaglandin E 1 differentiated NG108-15 cells.

Dual control of muscle cell survival by distinct growth factor-regulated signaling pathways.

In addition to their ability to stimulate cell proliferation, polypeptide growth factors are able to maintain cell survival under conditions that otherwise lead to apoptotic death. Growth factors control cell viability through regulation of critical intracellular signal transduction pathways. We previously characterized C2 muscle cell lines that lacked endogenous expression of insulin-like growth factor II (IGF-II). These cells did not differentiate but underwent apoptotic death in low-serum differentiation medium. Death could be prevented by IGF analogues that activated the IGF-I receptor or by unrelated growth factors such as platelet-derived growth factor BB (PDGF-BB). Here we analyze the signaling pathways involved in growth factor-mediated myoblast survival. PDGF treatment caused sustained activation of extracellular-regulated kinases 1 and 2 (ERK1 and -2), while IGF-I only transiently induced these enzymes. Transient transfection of a constitutively active Mek1, a specific upstream activator of ERKs, maintained myoblast viability in the absence of growth factors, while inhibition of Mek1 by the drug UO126 blocked PDGF-mediated but not IGF-stimulated survival. Although both growth factors activated phosphatidylinositol 3-kinase (PI3-kinase) to similar extents, only IGF-I treatment led to sustained stimulation of its downstream kinase, Akt. Transient transfection of a constitutively active PI3-kinase or an inducible Akt promoted myoblast viability in the absence of growth factors, while inhibition of PI3-kinase activity by the drug LY294002 selectively blocked IGF- but not PDGF-mediated muscle cell survival. In aggregate, these observations demonstrate that distinct growth factor-regulated signaling pathways independently control myoblast survival. Since IGF action also stimulates muscle differentiation, these results suggest a means to regulate myogenesis through selective manipulation of different signal transduction pathways.

Zinc protects against apoptosis of endothelial cells induced by linoleic acid and tumor necrosis factor α

Zinc requirements of the vascular endothelium may be increased in inflammatory conditions, ie, atherosclerosis, in which apoptotic cell death is prevalent. We hypothesized that zinc deficiency may potentiate disruption of endothelial cell integrity mediated by fatty acids and inflammatory cytokines by enhancing pathways that lead to apoptosis and up-regulation of caspase genes. Endothelial cells were maintained in low-serum medium or grown in culture media containing selected chelators, ie, diethylenetriaminepentaacetate or N,N,N', N'-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN), with or without zinc supplementation. Subsequently, cells were treated with linoleic acid, tumor necrosis factor alpha (TNF-alpha), or both. We studied the effect of zinc deficiency and supplementation on the induction of apoptosis by measuring caspase-3 activity, cell binding of annexin V, and DNA fragmentation. Our results indicated that linoleic acid and TNF-alpha independently, but more markedly in concert, up-regulated caspase-3 activity and induced annexin V binding and DNA fragmentation. Zinc deficiency, especially when induced by TPEN, dramatically increased apoptotic cell death induced by cytokines and lipids compared with control cultures. Supplementation of low-serum- or chelator-treated endothelial cells with physiologic amounts of zinc caused a marked attenuation of apoptosis induced by linoleic acid and TNF-alpha. Morphologic changes of cells observed during zinc deficiency were prevented by zinc supplementation. Media supplementation with other divalent cations (eg, calcium and magnesium) did not mimic the protective role of zinc against apoptosis. Our data indicate that zinc is vital to vascular endothelial cell integrity, possibly by regulating signaling events to inhibit apoptotic cell death.

Modulation of cell growth and transformation by doxycycline-regulated FGF-2 expression in NIH-3T3 cells

The single-copy fibroblast growth factor 2 (FGF-2) gene encodes four coexpressed isoforms of different molecular masses. The 18-kDa FGF-2 is primarily localized in the cytoplasm, whereas the higher molecular mass isoforms (HMW FGF-2) localize to the nucleus and nucleolus. The overexpression of either 18-kDa FGF-2 or HMW FGF-2 promotes cell transformation in a dose-dependent manner. In NIH 3T3 cells, the selective overexpression of HMW FGF-2 but not of 18-kDa FGF-2 confers upon the cells the unique phenotype of growth in low serum-containing medium. Thus, the distinct intracellular localization and the level of expression of FGF-2 are pivotal requirements for the differential effects of FGF-2 isoforms on the cellular phenotype. On this basis, we established a doxycycline-regulatable FGF-2 expression system that permitted us to regulate the expression of each isoform in a time- and dose-dependent manner. We analyzed the growth properties of cells in the presence and absence of doxycycline in both normal and low serum-containing medium and in soft agar. The doxycycline-activated expression of 18-kDa FGF-2 did not allow growth in low serum medium. The growth of cells expressing HMW FGF-2 was increased by doxycycline under all three conditions, and a relationship between the level of HMW FGF-2 expression and cell growth was observed for all three conditions. This doxycycline-regulatable FGF-2 expression system provides a mechanism to analyze changes in FGF-2 targeted pathways and genes and to characterize pathways specifically activated by either the 18-kDa FGF-2 or the HMW FGF-2 isoforms.

Modulation of cell growth and transformation by doxycycline-regulated FGF-2 expression in NIH-3T3 cells.

The single-copy fibroblast growth factor 2 (FGF-2) gene encodes four coexpressed isoforms of different molecular masses. The 18-kDa FGF-2 is primarily localized in the cytoplasm, whereas the higher molecular mass isoforms (HMW FGF-2) localize to the nucleus and nucleolus. The overexpression of either 18-kDa FGF-2 or HMW FGF-2 promotes cell transformation in a dose-dependent manner. In NIH 3T3 cells, the selective overexpression of HMW FGF-2 but not of 18-kDa FGF-2 confers upon the cells the unique phenotype of growth in low serum-containing medium. Thus, the distinct intracellular localization and the level of expression of FGF-2 are pivotal requirements for the differential effects of FGF-2 isoforms on the cellular phenotype. On this basis, we established a doxycycline-regulatable FGF-2 expression system that permitted us to regulate the expression of each isoform in a time- and dose-dependent manner. We analyzed the growth properties of cells in the presence and absence of doxycycline in both normal and low serum-containing medium and in soft agar. The doxycycline-activated expression of 18-kDa FGF-2 did not allow growth in low serum medium. The growth of cells expressing HMW FGF-2 was increased by doxycycline under all three conditions, and a relationship between the level of HMW FGF-2 expression and cell growth was observed for all three conditions. This doxycycline-regulatable FGF-2 expression system provides a mechanism to analyze changes in FGF-2 targeted pathways and genes and to characterize pathways specifically activated by either the 18-kDa FGF-2 or the HMW FGF-2 isoforms.

Stimulation of coronary vasculogenesis/angiogenesis by hypoxia in cultured embryonic hearts.

Hypoxia is known to stimulate vascular growth by up-regulating vascular endothelial growth factor (VEGF), but little is known about the function of hypoxia in the development of the coronary vasculature, and the relationship between hypoxia and VEGF in this event. To test the effects of hypoxia and VEGF on coronary vasculogenesis/angiogenesis in the developing heart, ventricles from 6-day-old quail embryos were cultured on three-dimensional collagen gels. After 2 days of growth in normal medium and 1 day of starvation in low serum medium (0.5% fetal bovine serum), the heart explants were further cultured under various oxygen levels for another 24, 48, and 72 hr. Angioblasts and endothelial cells, which migrated out from the heart explants, were identified by QH1 antibody using immunofluorescence and confocal microscopy. In the normoxic culture environment, the endothelial cells began to proliferate and migrate out from the heart explants after 3 days of growth; they formed tubes mainly after another 72 hr. In contrast, this vascular growth was accelerated under hypoxic conditions, as evidenced by increased tube formation with significant differences observed at 48 hr. On the other hand, hyperoxia delayed this process. Reverse transcription-polymerase chain reaction results indicated that VEGF (including VEGF(122), VEGF(166), and VEGF(190)) was up-regulated in the heart explants under hypoxia and down-regulated under hyperoxia. VEGF neutralizing antibody added to the culture medium partially blocked this vascular growth. We conclude from this study that hypoxia can stimulate or up-regulate coronary vasculogenesis/angiogenesis and that VEGF signaling plays a major role in this event. Dev Dyn 1999;216:28-36.