FGF-2 suppresses cellular senescence of human mesenchymal stem cells by down-regulation of TGF-beta2

Human mesenchymal stem cells (hMSCs) are able to both self-replicate and differentiate into a variety of cell types. Fibroblast growth factor-2 (FGF-2) stimulates the growth of hMSCs in vitro, but its mechanisms have not been clarified yet. In this study, we investigated whether cellular senescence was involved in the stimulation of hMSCs growth by FGF-2 and the expression levels of transforming growth factor-beta1 and -beta2 (TGF-betas). Because hMSCs were induced cellular senescence due to long-term culture, FGF-2 decreased the percentage of senescent cells and suppressed G1 cell growth arrest through the suppression of p21(Cip1), p53, and p16(INK4a) mRNA expression levels. Furthermore, the levels of TGF-betas mRNA expression in hMSCs were increased by long-term culture, but FGF-2 suppressed the increase of TGF-beta2 mRNA expression due to long-term culture. These results suggest that FGF-2 suppresses the hMSCs cellular senescence dependent on the length of culture through down-regulation of TGF-beta2 expression.     

Homologous recombination of exogenous DNA fragments with genomic DNA in somatic cells of mice.

We compared liposomes and empty viral capsids for their use as vehicles for DNA transfer into cells and animals. DNA binding capacity was high for liposomes, but DNase I protection of DNA bound to liposomes was only moderate in comparison to DNA incorporated into viral capsids. Cellular uptake of radiolabeled and physiologically active DNA was also compared. For animal studies we chose an endogenous retroposon as target gene. To identify recombinational events we replaced a part of this gene with an artificial sequence not present in the mouse genome. The recombination rate for DNA fragments transfected in Polyoma capsids in live mice was higher than for liposome mediated transfection. Homologous recombination could be observed for both DNA transfer methods, mediated by positively charged liposomes (DOTMA) and by empty Polyoma viral capsids.     

Failure of accumulation of cellular RNA in hamster cells stimulated to synthesize DNA by infection with adenovirus 2

AF8 cells are temperature-sensitive mutants of the cell cycle derived from baby hamster kidney (BHK) cells which arrest in the G1 phase when incubated at the nonpermissive temperature. RNA accumulation was studied in these cells by flow cytofluorimetry following serum stimulation or adenovirus 2 infection. Serum stimulation caused an increase in the amount of RNA per cell, which reached a maximum in S and G2 cells, as repeatedly reported in the literature. However, adenovirus 2 infection caused a fraction of cells to enter S phase without any concomitant increase in the amount of RNA per cell.     

Nuclear accumulation and activation of p53 in embryonic stem cells after DNA damage

Background  

P53 is a key tumor suppressor protein. In response to DNA damage, p53 accumulates to high levels in differentiated cells and activates target genes that initiate cell cycle arrest and apoptosis. Since stem cells provide the proliferative cell pool within organisms, an efficient DNA damage response is crucial.     

E1A stimulates FGF-2 release promoting differentiation of primary endothelial cells

Basic Fibroblast Growth Factor (FGF-2) is a growth and survival factor and represents one of the most potent differentiation agents of vascular system. In the present study we describe that adenoviral oncoprotein E1A regulates FGF-2 production and determines the acquisition of a pro-angiogenic phenotype in primary bovine aortic endothelial cells (BAEC). Following their transfection, wild type E1A proteins 12S and 13S (wtE1A) stimulated BAEC to differentiate on reconstituted basement membrane matrix (Matrigel). This outcome was paralleled by invasion and migration enhancement in wtE1A-transfected cells. This stimulating effect was absent with the E1A mutant dl646N. Accordingly, zymography and RT - PCR analyses showed that matrix metalloproteinase-9 protein- and mRNA-levels increased following wtE1A transfection. Interestingly, wtE1A-transfected BAEC showed FGF-2 mRNA- and protein-levels higher than controls. Further, FGF-2 neutralization reduced the amount of MMP-9 released in the supernatant of E1A-transfected cells and strongly inhibited BAEC differentiation, thus suggesting that wtE1A activates BAEC by a mechanism, at least partially, dependent on a FGF-2 autocrine/paracrine loop.     

Methylglyoxal induces cellular damage by increasing argpyrimidine accumulation and oxidative DNA damage in human lens epithelial cells

Methylglyoxal (MGO) is a cytotoxic metabolite and modifies tissue proteins through the Maillard reaction, resulting in advanced glycation end products (AGEs), which can alter protein structure and functions. Several MGO-derived AGEs have been described, including argpyrimidine, a fluorescent product of the MGO reaction with arginine residues. Herein, we evaluated the cytotoxic role of MGO in human lens epithelial cell line (HLE-B3). HLE-B3 cells were exposed to 400 μM MGO in the present or absence of pyridoxamine for 24 h. We then examined the formation of argpyrimidine, apoptosis and oxidative stress in HLE-B3 cells. In MGO-treated HLE-B3 cells, the accumulation of argpyrimidine was markedly increased, and caspase-3 and 8-hydroxydeoxyguanosine (8-OHdG) were highly expressed, which paralleled apoptotic cell death. However, pyridoxamine (AGEs inhibitor) prevented the argpyrimidine formation and apoptosis of MGO-treated HLE-B3 cells. These results suggested that the accumulation of argpyrimidine and oxidative DNA damage caused by MGO are involved in apoptosis of HLE-B3 cells.     

Angiotensin II-induced MMP-2 release from endothelial cells is mediated by TNF-alpha

Angiotensin II (ANG II) has been etiologically linked to vascular disease; however, its role in the alterations of endothelial function that occur in vascular disorders is not completely understood. Matrix metalloproteinases (MMPs) and proinflammatory cytokines are involved in the pathological remodeling of blood vessels that occurs in vascular disease. In this study we evaluated the effects of ANG II on tumor necrosis factor (TNF)- and MMP-2 production in endothelial cells. Human umbilical vein endothelial cells (HUVECs) were stimulated with ANG II (0.1–10 µM) for 24 h, in the presence or absence of antagonists of ANG II type 1 (AT₁R) and type 2 (AT₂R) receptors, and the production and release of TNF- and MMP-2 were assessed. ANG II increased TNF- mRNA and protein expression and the release of bioactive TNF-. Moreover, ANG II induced MMP-2 release and reduced the secretion of tissue inhibitor of MMP (TIMP)-2 from endothelial cells. To elucidate whether endogenous TNF- could mediate the effects of ANG II on MMP-2 release, cells were pretreated with anti-TNF- neutralizing antibodies or pentoxifylline (an inhibitor of TNF- synthesis). TNF- inhibition prevented the secretion of MMP-2 induced by ANG II. Furthermore, AT₁R antagonism with candesartan prevented the formation of MMP-2 and TNF- and the reduction of TIMP-2 induced by ANG II. These results indicate that ANG II, via AT₁R, modulates the secretion of TNF- and MMP-2 from endothelial cells and that TNF- mediates the effects of ANG II on MMP-2 release. remodeling; vasoactive mediators; inflammation     

Effect of exogenous ATP upon inositol phosphate production, cationic fluxes and insulin release in pancreatic islet cells

Endogenous ATP is thought to play a key regulatory role in nutrient-stimulated insulin release. The present study deals with the effect of exogenous ATP and its stable analog alpha, beta-methylene ATP upon pancreatic islet function. Both alpha, beta-methylene ATP (5.0 microM to 0.2 mM) and ATP (0.3-3.0 mM) caused a rapid and concentration-related increase in insulin output by rat islets incubated or perfused at an intermediate concentration of D-glucose (8.3 mM). The effect of the ATP analog faded out at both lower and higher D-glucose concentrations. In the presence of 8.3 mM D-glucose, ATP also increased both 86Rb and 45Ca outflow from prelabelled islets. The cationic response to ATP persisted in the absence of extracellular Ca2+ and, hence, was reminiscent of that evoked by cholinergic agents. Like carbamylcholine, ATP caused a dose-related increase in the production of [3H]inositol phosphates from prelabelled islets or tumoral islet cells (RINm5F line). The latter effect was duplicated by alpha, beta-methylene ATP and unaffected by atropine. It is speculated that ATP, liberated together with insulin at the exocytotic site, might participate in a positive feedback control of insulin release.     

Rapid degradation of Cdt1 upon UV-induced DNA damage is mediated by SCFSkp2 complex

Cdt1 is a licensing factor for DNA replication, the function of which is tightly controlled to maintain genome integrity. Previous studies have indicated that the cell cycle-dependent degradation of Cdt1 is triggered at S phase to prevent re-replication. In this study, we found that Cdt1 is degraded upon DNA damage induced by either UV treatment or gamma-irradiation (IR). Although the IR-triggered degradation of Cdt1 was caffeine-insensitive, the UV-triggered degradation of Cdt1 was caffeine-sensitive. This indicates that the cells treated with UV utilize the checkpoint pathway, which differs from that triggered by IR. A recent study has suggested that Cdt1 is phosphorylated, ubiquitylated, and degraded at the G(1)/S boundary in the normal cell cycle. Treatment with MG132, a proteasome inhibitor, inhibited the degradation of Cdt1 and resulted in the accumulation of the phosphorylated form of Cdt1 after UV treatment. In the case of UV treatment, phosphorylation of Cdt1 induced the recruitment of Cdt1 to a SCF(Skp2) complex. Moreover, ectopic overexpression of Cdt1 after UV treatment interfered the inhibition of DNA synthesis. These results indicate that Cdt1 is a target molecule of the cell cycle checkpoint in UV-induced DNA damage.     

Uptake of exogenous DNA by pea seedlings and tobacco cells

1.

(1) The uptake of Pseudomonas aeruginosa DNA by pea seedlings, and uptake of tobacco DNA or P. aeruginosa DNA by tobacco cells in shake cultures has been investigated. The fate of the DNA has been followed by CsCl density gradient equilibrium centrifugation, using radiolabeled donor DNA of high density.     

Ca2+ oscillations mediated by exogenous GTP in Paramecium cells: assessment of possible Ca2+ sources

We applied exogenous guanosine trisphosphate, GTP, to Paramecium tetraurelia cells injected with Fura Red for analysing changes of free intracellular Ca(2+) concentrations, [Ca(2+)](i), during periodic back-/forward swimming thus induced. Strain ginA (non-responsive to GTP) shows no Ca(2+) signal upon GTP application. In strain nd6 (normal Ca(2+) signalling) an oscillating [Ca(2+)](i) response with a prominent first peak occurs upon GTP stimulation, but none after mock-stimulation or after 15 min adaptation to GTP. While this is in agreement with previous electrophysiological analyses, we now try to identify more clearly the source(s) of Ca(2+). Stimulation of nd6 cells, after depletion of Ca(2+) from their cortical stores (alveolar sacs), shows the same Ca(2+) oscillation pattern but with reduced amplitudes, and a normal behavioural response is observed. Stimulation with GTP, supplemented with the Ca(2+) chelator BAPTA, results in loss of the first prominent Ca(2+) peak, in reduction of the following Ca(2+) amplitudes, and in the absence of any behavioural response. Both these observations strongly suggest that for the initiation of GTP-mediated back-/forward swimming Ca(2+) from the extracellular medium is needed. For the maintenance of the Ca(2+) oscillations a considerable fraction must come from internal stores, probably other than alveolar sacs, rather likely from the endoplasmic reticulum.     

Nuclear control of tumorigenicity in cells reconstructed by PEG-induced fusion of cell fragments

The techniques of somatic cell hybridization have provided a valuable means of studying mechanisms of regulation of mammalian cell differentiation and transformation. Most previous studies have indicated that fusions between tumorigenic and nontumorigenic cells result in hybrid cells that are usually tumorigenic. In recent years it has been demonstrated that the phenotypic expression of tumorigenicity is at least partially due to the extensive chromosome loss that occurs in most interspecific and some intraspecific hybrid cells. In the present study we have utilized enucleation techniques that permit cells to be divided into nuclear (karyoplast) and cytoplasmic (cytoplast) cell fragments. Even though these nuclear and cytoplasmic fragments are metabolically stable for short periods of time, in our hands they ultimately degenerate. Viable cells can be reconstructed by PEG-induced fusion of karyoplasts to cytoplasts. Since reconstructed cells apparently do not segregate chromosomes, they may provide a clearer understanding of the interactions between the nucleus and the cytoplasm in the control of the expression of tumorigenicity. We have reconstructed cells using karyoplasts from the tumorigenic Y-1 cell line and cytoplasts from a nontumorigenic cell line, A-MT-BU-A1. In addition we have reconstructed cells containing Y-1 cytoplasts and A-MT-BU-A1 karyoplasts. The reconstructed cells porduced were assayed for tumorigenicity by their ability to grow in soft agar and in nude mice. The results of these experiments indicate that the reconstructed cells containing a tumorigenic nucleus and a nontumorigenic cytoplasm ultimately are tumorigenic and conversely the reconstructed cells containing a nontumorigenic nucleus and a tumorigenic cytoplasm are nontumorigenic. These experiments support the concept that with these cell lines the nucleus (karyoplast) is sufficient to control the phenotypic expression of tumorigenicity.     

Porphyrin accumulation in mitochondria is mediated by 2-oxoglutarate carrier

Heme (Fe-protoporphyrin IX), an endogenous porphyrin derivative, is an essential molecule in living aerobic organisms and plays a role in a variety of physiological processes such as oxygen transport, respiration, and signal transduction. For the biosynthesis of heme or the mitochondrial heme proteins, heme or its biosynthetic precursor porphyrin must be transported into mitochondria from cytosol. The mechanism of porphyrin accumulation in the mitochondrial inner membrane is unclear. In the present study, we analyzed the mechanism of mitochondrial translocation of porphyrin derivatives. We showed that palladium meso-tetra(4-carboxyphenyl)porphyrin (PdTCPP), a phosphorescent porphyrin derivative, accumulated in the mitochondria of several cell lines. Using affinity latex beads, we showed that 2-oxoglutarate carrier (OGC), the mitochondrial transporter of 2-oxoglutarate, bound to PdTCPP, and in vitro PdTCPP inhibited 2-oxoglutarate uptake into mitochondria in a competitive manner (Ki = 15 microM). Interestingly, all types of porphyrin derivatives examined in this study competitively inhibited 2-oxoglutarate uptake into mitochondria, including protoporphyrin IX, coproporphyrin III, and hemin. Furthermore, mitochondrial accumulation of porphyrins was inhibited by 2-oxoglutarate or OGC inhibitor. These results suggested that porphyrin accumulation in mitochondria is mediated by OGC and that porphyrins are able to competitively inhibit 2-oxoglutarate uptake into mitochondria. This is the first report of a putative mechanism for accumulation of porphyrins in the mitochondrial inner membrane.     

Filipin prevents pathological prion protein accumulation by reducing endocytosis and inducing cellular PrP release

Conversion of the normal membrane-bound prion protein (PrP-sen) to its pathological isoform (PrP-res) is a key event in the pathogenesis of transmissible spongiform encephalopathies. Although the subcellular sites of conversion are poorly characterized, several lines of evidence have suggested the involvement of membrane lipid rafts in the conversion process. Here we report that copper stimulates the endocytosis of PrP-sen via a caveolin-dependent pathway in both microglia and neuroblastoma cells. We show that the polyene antibiotic filipin both limits endocytosis of PrP-sen and dramatically reduces the amount of membrane-bound PrP-sen. This reduction results from a rapid and massive release of full matured PrP-sen into the culture medium. Finally, we demonstrate that filipin is a potent inhibitor of PrP-res formation into chronically infected neuroblastoma cells. Our results reinforce the role of rafts in PrP trafficking and raise the possibility that the release of PrP-sen from the plasma membrane decreases the amount of available substrate PrP-sen at the conversion sites.     

Trehalose accumulation during cellular stress protects cells and cellular proteins from damage by oxygen radicals

The disaccharide trehalose, which accumulates dramatically during heat shock and stationary phase in many organisms, enhances thermotolerance and reduces aggregation of denatured proteins. Here we report a new role for trehalose in protecting cells against oxygen radicals. Exposure of Saccharomyces cerevisiae to a mild heat shock (38 degrees C) or to a proteasome inhibitor (MG132) induced trehalose accumulation and markedly increased the viability of the cells upon exposure to a free radical-generating system (H(2)O(2)/iron). When cells were returned to normal growth temperature (28 degrees C) or MG132 was removed from the medium, the trehalose content and resistance to oxygen radicals decreased rapidly. Furthermore, a mutant unable to synthesize trehalose was much more sensitive to killing by oxygen radicals than wild-type cells. Providing trehalose exogenously enhanced the resistance of mutant cells to H(2)O(2). Exposure of cells to H(2)O(2) caused oxidative damage to amino acids in cellular proteins, and trehalose accumulation was found to reduce such damage. After even brief exposure to H(2)O(2), the trehalose-deficient mutant exhibited a much higher content of oxidatively damaged proteins than wild-type cells. Trehalose accumulation decreased the initial appearance of damaged proteins, presumably by acting as a free radical scavenger. Therefore, trehalose accumulation in stressed cells plays a major role in protecting cellular constituents from oxidative damage.     

Nuclear accumulation of truncated atrophin-1 fragments in a transgenic mouse model of DRPLA

Dentatorubral and pallidoluysian atrophy (DRPLA) is a member of a family of progressive neurodegenerative diseases caused by polyglutamine repeat expansion. Transgenic mice expressing full-length human atrophin-1 with 65 consecutive glutamines exhibit ataxia, tremors, abnormal movements, seizures, and premature death. These mice accumulate atrophin-1 immunoreactivity and inclusion bodies in the nuclei of multiple populations of neurons. Subcellular fractionation revealed 120 kDa nuclear fragments of mutant atrophin-1, whose abundance increased with age and phenotypic severity. Brains of DRPLA patients contained apparently identical 120 kDa nuclear fragments. By contrast, mice overexpressing atrophin-1 with 26 glutamines were phenotypically normal and did not accumulate the 120 kDa fragments. We conclude that the evolution of neuropathology in DRPLA involves proteolytic processing of mutant atrophin-1 and nuclear accumulation of truncated fragments.     

Angiotensin II-induced ET and PGI2 release in rat aortic endothelial cells is mediated by PKC.

Angiotensin II (Ang II) has been shown to stimulate the release of immunoreactive endothelin (ET) from cultured bovine ECs. Also, Ang II activates phospholipase A2 (PLA2) in various tissues, resulting in the release of arachidonic acid and formation of prostaglandins. We used rat aortic endothelial cells to investigate the role of protein kinase C (PKC) in Ang II-induced release of both ET and prostacyclin (PGI2). The amount of ET and PGI2 produced were determined by radioimmunoassay. Ang II-induced the release of both ET and PGI2. Pretreatment with 10(-6) M of any one of the PKC inhibitors: 1-(5-isoquinolinesulfonyl) piperazine(CL), staurosporine, 1-(5-isoquinolinesulfonylmethyl)piperazine(H7), and calphostin C blocked AII-induced release of both ET and PGI2. In rat aortic endothelial cells that were treated with either AII or PDBu, PKC enzyme assay showed PKC was translocated from the cytosol to the membrane which indicates activation. This suggests that PKC mediates AII-induced ET and PGI2 release. In summary, AII activates PKC which inhibits rat aortic endothelial cells ET and PGI2 formation, and this inhibition can be overcome by pretreatment with PKC inhibitors.