Low doses of 3-aminobenzamide, a poly(ADP-ribose) polymerase inhibitor, stimulate angiogenesis by regulating expression of urokinase type plasminogen activator and matrix metalloprotease 2.

Background: Poly(ADP-Ribose) polymerase (PARP) activity has been demonstrated fundamental in many cellular processes, including DNA repair, cell proliferation and differentiation. In particular, PARP activity has been recently found to affect proliferation, migration, and tube formation of human umbilical vein endothelial cells. In recent times, PARP inhibitors have entered in clinical trials to potentiate cancer treatments by preventing DNA repair, but little is known about the effects performed by different drug concentrations on neoangiogenesis, an essential step in tumor growth.

Differentiating and apoptotic dose-dependent effects in (-)-alpha-bisabolol-treated human endothelial cells.

The effect on angiogenesis of (-)-alpha-bisabolol [(-)-6-methyl-2-(4-methyl-3-cyclohexen-1-yl)-5-hepten-2-ol] (1), a widely distributed plant sesquiterpene alcohol, was investigated for the first time. Human endothelial cells treated with 1 were analyzed for their ability to differentiate and organize in microvessels and for their sensitivity to this compound in terms of cytotoxicity and cell growth inhibition. Within 24 h of the treatment with 5 microM 1, cells underwent massive death.

Inhibition of PARP activity by PJ-34 leads to growth impairment and cell death associated with aberrant mitotic pattern and nucleolar actin accumulation in M14 melanoma cell line.

The capability of PARP activity inhibitors to prevent DNA damage recovery suggested the use of these drugs as chemo- and radio-sensitisers for cancer therapy. Our research, carried out on cultured human M14 melanoma cells, was aimed to examine if PJ-34, a potent PARP activity inhibitor of second generation, was per se able to affect the viability of these cancer cells without any DNA damaging agents. Using time-lapse videomicroscopy, we evidenced that 10 microM PJ-34 treatment induced severe mitotic defects leading to dramatic reduction of cell proliferation and to cell death.

Oxidative DNA damage repair and parp 1 and parp 2 expression in Epstein-Barr virus-immortalized B lymphocyte cells from young subjects, old subjects, and centenarians.

Oxidative DNA damage has been implicated in the aging process and in some of its features such as telomere shortening and replicative senescence. Poly(ADP-ribosyl)ation is involved in many molecular and cellular processes, including DNA damage detection and repair, chromatin modification, transcription, and cell death pathways. We decided to examine the behavior of poly(ADP-ribosyl)ation in centenarians, i.

Poly(ADP-ribosyl)ation, a molecular switch of transcription, shows an attractive relationship with urokinase expression.

Poly(ADP-ribosyl)ation is a posttranslational modification of proteins that consists in the transfer of ADP-ribose units from NAD+ onto protein acceptors to form long and branched polymers. PARP activity is stimulated either by genotoxic stimuli or by environmental factors. The negative charged polymers alter functional activity of several proteins involved in genome stability, gene expression, cell proliferation and differentiation.

FGF2-mediated upregulation of urokinase-type plasminogen activator expression requires a MAP-kinase dependent activation of poly(ADP-ribose) polymerase.

Poly(ADP-ribosyl)ation is a post-translational modification of protein occurring in the nucleus by poly(ADP-ribose) polymerase enzyme activity. The main role of poly(ADP-ribose) polymerase system as "nick sensor" and DNA breaks repair is based on its activation via DNA strand breaks. Furthermore, poly(ADP-ribose) polymerase modifies the binding to DNA of several transcriptional factors by poly(ADP-ribosyl)ation, thereby regulating also transcriptional gene expression.

Comparative levels of DNA breaks and sensitivity to oxidative stress in aged and senescent human fibroblasts: a distinctive pattern for centenarians.

Basal and H(2)O(2)-induced DNA breaks as well as DNA repair activity and efficacy of the antioxygenic system were determined in human dermal fibroblasts explanted from either (i) young donors and passaged serially to reach replicative senescence or (ii) young, old and centenarian donors and shortly propagated in culture. These fibroblasts have been employed as an in vitro and ex vivo model, respectively, to evaluate comparatively DNA integrity during senescence (increasing population doubling levels) and aging (increasing donor age). Constitutive levels of DNA total strand breaks, as determined by the alkaline extraction procedure, changed moderately among the different cell lines, which exhibited, however, significant differences in the amount of either single or double strand breaks.

A method for separation of heparin species from biological samples by ethanol precipitation of compounds solubilized in guanidine hydrochloride.

In this paper we describe a procedure to determine glycosaminoglycan and oligosaccharide composition of biological samples such as cell cultures or tissue explants. We demonstrate that heparin species of different molecular mass can be easily fractionated by sequential ethanol precipitation in 4.0 M guanidine hydrochloride.

Heparin binding peptides co-purify with glycosaminoglycans from human plasma.

Glycosaminoglycans (GAGs) are complexed with plasma proteins and proteolysis of plasma reduced the protein-GAG ratio about 140-fold. After dialysis, analysis by gradient PAGE revealed heparinase-1-sensitive GAGs, thus suggesting that heparin could be among the plasma GAGs. However, after dialysis most of the plasma GAGs were still not 'free'.

Premature induction of aging in sublethally H2O2-treated young MRC5 fibroblasts correlates with increased glutathione peroxidase levels and resistance to DNA breakage.

Human MRC5 fibroblasts, at different passages in cultures, were used as an in vitro model to assess variations and/or induction of aging parameters under basal conditions or following sublethal oxidative stress by H2O2. DNA sensitivities to oxidatively-induced breakage, rather than basal levels of damaged DNA, were significantly different between cultures at low and high population doubling level (PDL): old cells maintained most of their DNA integrity even at high concentrations of H2O2, while young cells showed more extensive DNA damage which developed in a dose-dependent fashion. However, young cells pretreated with low doses of H2O2 exhibited increased resistance against further oxidative damage to DNA thus reproducing a senescent-like profile of sensitivity.

Urokinase-dependent angiogenesis in vitro and diacylglycerol production are blocked by antisense oligonucleotides against the urokinase receptor.

The plasminogen activator system is known to play a crucial role in the angiogenesis process by modulating the adhesive properties of endothelial cells to the extracellular matrix and cell-cell interaction. In the present study, we demonstrated that the urokinase-type plasminogen activator (u-PA) induced neovascular growth in the avascular rabbit cornea and dose-dependently promoted growth, chemotaxis, and matrix invasion of cultured endothelial cells. Interaction between u-PA and its receptor appears to be mandatory for the angiogenic effect of u-PA because monoclonal antibodies anti-u-PA and anti-u-PA receptor (u-PAR) blocked the proangiogenic effects of u-PA at the endothelial cell level.

Interaction of urokinase-type plasminogen activator with its receptor rapidly induces activation of glucose transporters.

The interaction of urokinase-type plasminogen activator (u-PA) or of u-PA amino-terminal fragment (u-PA-ATF) with the cell surface receptor (u-PAR) was found to stimulate an increase of glucose uptake in many cell lines, ranging from normal and transformed human fibroblasts, mouse fibroblasts transfected with human u-PAR, and cells of epidermal origin. Such increase of glucose uptake reached a peak within 5-10 min, depending on the cell line, and occurred through the facilitative glucose transporters (GLUTs), since it was inhibited by cytochalasin B. Each cell line showed a specific mosaic of glucose transporter isoforms, GLUT2 being the most widespread and GLUT1 the most abundant, when present.

Induction, effects, and quantification of sublethal oxidative stress by hydrogen peroxide on cultured human fibroblasts.

Conditions to induce and parameters to evaluate sublethal oxidative stress of cultured human fibroblasts have been investigated in the attempt to identify markers for a more accurate quantification of cell injury. Sublethal oxidative stress was obtained by treating fibroblasts with 0.5 mM H2O2 in DMEM plus 5% FCS for times not exceeding 60 min.

Production of second messengers following chemotactic and mitogenic urokinase-receptor interaction in human fibroblasts and mouse fibroblasts transfected with human urokinase receptor.

We studied urokinase-type plasminogen activator (u-PA)-dependent chemotaxis and DNA synthesis in both human fibroblasts and LB6 mouse fibroblasts transfected with human u-PA receptor (u-PAR) gene (LB6 clone 19). Both cell lines have receptors for the amino-terminal fragment of u-PA (u-PA-ATF). We observed that u-PA and u-PA-ATF stimulated chemotactic migration of both LB6 clone 19 cells and human fibroblasts, which could be impaired by down-regulation of protein kinase C (PKC) with phorbol myristate acetate (PMA).

Changes in pyridine and adenine nucleotide levels in Friend erythroleukaemia cells during growth and differentiation.

Pyridine and adenine nucleotide levels were measured in Friend erythroleukaemia cells (FELC) stimulated to growth and induced to differentiate by hexamethylene bisacetamide (HMBA) and N'-methylnicotinamide (N'-MNAM). A three- to fourfold increase in the NADP(H) was found to parallel cell growth stimulation in both the presence and absence of differentiation inducers. NAD(H) increased about twofold in control and to a minor extent in HMBA-treated FELC but did not vary significantly in N'-MNAM-treated cells.

Role of specific membrane receptors in urokinase-dependent migration of human keratinocytes.

On the basis of both 125I-labeled plasminogen activator binding analysis and transmission electron microscopy studies of the interaction of a plasminogen activator/gold complex with cell membranes, we have found that human keratinocytes have specific receptors for human urokinase-type plasminogen activator distributed on the cell surface as singlets, or as small or large clusters. The use in binding experiments of the purified A chain of urokinase-plasminogen activator and of anti-A chain monoclonal antibodies has indicated that cell receptors are specific for a sequence present on the A chain, as previously reported for other cells. The interaction of both the native molecule and the purified A chain with such receptors stimulates mobilization of keratinocytes in an in vitro cell model system (Boyden chamber), when present in the lower compartment of the migration apparatus in nanomolar concentrations.

Modulation of urokinase receptors on human synovial cells and osteoarthritic chondrocytes by diacetylrhein.

On the basis of both 125I-labelled urokinase-like plasminogen activator binding analysis and transmission electron microscopy of an urokinase-gold complex, we have shown the presence of specific receptors for human urokinase on the cell membrane of human synovial cells. By radio-ligand binding experiments we have shown the existence of similar receptors on the surface of human chondrocytes. In both cases the specific binding is attributable to interaction between the receptor and the A chain of the ligand, as previously shown in other cell model systems.

Inhibition of spontaneous growth and induced differentiation of murine erythroleukaemia cells by paraquat and atrazine.

The effect of the herbicides paraquat and atrazine on erythroid differentiation has been studied in mouse erythroleukaemic cells. The addition of atrazine or paraquat was shown to inhibit both spontaneous growth and hexamethylene-bis-acetamide (HMBA)-induced differentiation of undifferentiated erythroleukaemic cells. This effects was dose-dependent and occurred at concentrations of less than 10 ppm for both herbicides.

Plasminogen activators and tiaprofenic acid in inflammation. A preliminary study.

Treatment with tiaprofenic acid appreciably reduced the level of plasminogen activators in the medium of 3T3-Balb mouse fibroblasts, as revealed by both a fibrin plate assay and amidolytic determination with chromogenic substrates. At the same time, tiaprofenic acid was able to inhibit the production of plasminogen activators induced by phorbol myristate acetate, a powerful inflammation and tumour promoter, added to the cell monolayers. By isolating the inhibitors of plasminogen activators it was possible to show that the decrease of fibrinolytic activity produced by tiaprofenic acid is not related to an increase of inhibitors.

Relationship between pyridine nucleotide levels and ribonucleotide reductase activity in Yoshida ascites hepatoma AH130.

We measured both pyridine nucleotide levels and ribonucleotide reductase-specific activity in Yoshida ascites hepatoma cells as a function of growth in vivo and during recruitment from non-cycling to cycling state in vitro. Oxidized nicotinamide adenine dinucleotide (NAD+) and reduced nicotinamide adenine dinucleotide (NADP) levels remained unchanged during tumour growth, while NADP+ and reduced nicotinamide adenine dinucleotide phosphate (NADPH) levels were very high in exponentially growing cells and markedly decreased in the resting phase. Ribonucleotide reductase activity paralleled NADP(H) (NADP+ plus NADPH) intracellular content.

Effects of liver regeneration on tRNA contents and aminoacyl-tRNA synthetase activities and sedimentation patterns.

The tRNA content and aminoacyl-tRNA synthetases of regenerating liver in the phase of rapid growth were compared with those of livers from both intact and sham-operated rats. At 48 h after hepatectomy, the amount of active tRNA (called 'total acceptor capacity') is significantly higher in regenerating liver than in control livers, owing to a general, possibly not uniform, increase in the various tRNA families, which suggests that it may contribute to the increased protein synthesis and to decreased protein degradation as well. The activities of most, but not of all, aminoacyl-tRNA synthetases in cell sap of regenerating liver tend to be greater than normal.

Metabolic aspects of cell cycle regulation in normal and cancer cells.

Several studies are reviewed dealing with the mechanisms which regulate the cell cycle progression in normal and cancer cells. Using Yoshida AH 130 ascites tumor cells, it has been found that the G1-S transition of these cells is impaired by specific inhibitors of the electron flow through the respiratory chain (antimycin A), although respiratory ATP can be replaced by glycolytic ATP. The above transition can be also inhibited by the addition of physiologic substrates, mainly pyruvate, by a mechanism which appears linked to a modification of the cellular redox state and can be totally reversed by adding adenine to the culture medium.

The respiration-linked limiting step of tumor cell transition from the non-cycling to the cycling state: its inhibition by oxidizable substrates and its relationships to purine metabolism.

The recruitment into the cycling state of resting Yoshida AH 130 hepatoma cells was studied with respect to its dependence on respiration in an experimental system wherein the overall energy requirement for this recruitment can be supplied by the glycolytic ATP. The G1-S transition of these cells, unaffected by 2,4-dinitrophenol (DNP) at concentrations which uncouple the respiratory phosphorylation, is impaired either by blocking the electron flow to oxygen by antimycin A or by adding an excess of some oxidizable substrates, chiefly pyruvate and oxalacetate. An experimental analysis, focused on pyruvate activity, showed that the inhibition of cell recruitment into S is not related to the depressing effects of this substrate on aerobic glycolysis of tumor cells, nor is it modified by forcing, in the presence of DNP, pyruvate oxidation through the tricarboxylic acid cycle as well as the overall oxygen consumption.