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V Tomasi

Publications and source records attributed to V Tomasi.

At least 19 recordsLinked to original sources
PubMed2006

RNA interference as a key to knockdown overexpressed cyclooxygenase-2 gene in tumour cells.

Silencing those genes that are overexpressed in cancer and contribute to the survival and progression of tumour cells is the aim of several researches. Cyclooxygenase-2 (COX-2) is one of the most intensively studied genes since it is overexpressed in most tumours, mainly in colon cancer. The use of specific COX-2 inhibitors to treat colon cancer has generated great enthusiasm. Yet, the side effects of some inhibitors emerging during long-term treatment have caused much concern. Genes silencing by RNA interference (RNAi) has led to new directions in the field of experimental oncology. In this study, we detected sequences directed against COX-2 mRNA, that potently downregulate COX-2 gene expression and inhibit phorbol 12-myristate 13-acetate-induced angiogenesis in vitro in a specific, nontoxic manner. Moreover, we found that the insertion of a specific cassette carrying anti-COX-2 short hairpin RNA sequence into a viral vector (pSUPER.retro) greatly increased silencing potency in a colon cancer cell line (HT29) without activating any interferon response. Phenotypically, COX-2 deficient HT29 cells showed a significant impairment of their in vitro malignant behaviour. Thus, the retroviral approach enhancing COX-2 knockdown, mediated by RNAi, proved to be an useful tool to better understand the role of COX-2 in colon cancer. Furthermore, the higher infection efficiency we observed in tumour cells, if compared to normal endothelial cells, may disclose the possibility to specifically treat tumour cells without impairing endothelial COX-2 activity.

Carcinogens↗
PubMed2001

Colocalization prostacyclin (PGI2) synthase--caveolin-1 in endothelial cells and new roles for PGI2 in angiogenesis.

In vascular cells, prostacyclin (PGI2) synthase (PGI2s) has been localized in the endoplasmic reticulum of endothelial cells and in the nuclear and plasma membrane of smooth muscle cells. In human umbilical vein endothelial (HUVE) cells, we detected the enzyme in abundant cytoplasmic vesicles apparently originating from the plasma membrane and similar to those stained by gold-albumin, which interacts with a caveolar receptor. This prompted us to try a direct confocal microscopy approach aimed at colocalizing gold-albumin, caveolin-1, and PGI2 synthase. Moreover, the staining of HUVE cells with an anti-BiP7Grp78 antibody (a marker of endoplasmic reticulum) shows a perinuclear localization, sharply separated from PGI2 synthase localization. The results indicate that more than 80% of the enzyme resides in cellular sites costaining with caveolin-1 antibody and gold-albumin. This evidence was confirmed by the demonstration that PGI2 synthase and caveolin-1 coimmunoprecipitate in HUVE cell lysates and that they are associated to detergent-insoluble membrane domains in the same low-density fractions of a sucrose gradient. In addition, depletion of cellular cholesterol by mevalonate and methyl-beta-cyclodextrin leads to the shift of PGI2 synthase and caveolin-1 to higher density fractions of the gradient. Biochemical evidence about colocalization was supported by the use of a fusion protein glutathione S-transferase (GST)/caveolin-1, which retained either PGI2s purified from ram seminal vesicles or PGI2s present in HUVE cell lysates. Binding of PGI2s to caveolin "scaffolding domain" and to C-terminal region was deduced by using full-length GST--Cav-1, GST--Cav 61--101, and GST C- and N-terminal fusion proteins. A double approach based on the usage of filipin as a specific caveolae-disrupting agent and antisense oligonucleotides targeting PGI2 synthase mRNA suggests that the production of PGI2 in caveolae is likely to be connected to the regulation of angiogenesis, at least in vitro.

6-Ketoprostaglandin F1 alpha↗
PubMed2001

The Rossmann fold of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a nuclear docking site for antisense oligonucleotides containing a TAAAT motif.

The subcellular localisation of oligodeoxynucleotides (ODN) is a major limitation for their use against nuclear targets. In this study we demonstrate that an antisense ODN directed against cytosolic phospholipase A(2) (cPLA2) mRNA is efficiently taken up and accumulates in the nuclei of endothelial cells (HUVEC), human monocytes and HeLa cells. Gel shift experiments and incubation of cells with oligonucleotide derivatives show that the anti-cPLA2 oligo binds a 37 kDa protein in nuclear extracts. The TAAAT sequence was identified as the major binding motif for the nuclear protein in competition experiments with mutated ODNs. Modification of the AAA triplet resulted in an ODN which failed to localise in the nucleus. Moreover, inserting a TAAAT motif into an ODN localising in the cytosol did not modify its localisation. The 37 kDa protein was purified and identified after peptide sequencing as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). It was shown by confocal microscopy that GAPDH co-localises with anti-cPLA2 ODN in the nucleus and commercial GAPDH effectively binds the oligo. Competition experiments with increasing concentration of NAD(+) co-factor indicate that the GAPDH Rossmann fold is a docking site for antisense oligonucleotides containing a TAAAT motif.

Amino Acid Sequence↗
PubMed2000

Knockdown of caveolin-1 by antisense oligonucleotides impairs angiogenesis in vitro and in vivo.

Knock-out of the gene coding for caveolin-1, the main organizer of caveolae, has not yet been performed. We devised a strategy to knock-down caveolin-1 gene expression using antisense oligodeoxynucleotides (ODNs). Seven ODNs, covering different regions of caveolin-1 mRNA, were screened by Western blot analysis of caveolin-1 levels. The most active and specific was found to reduce caveolin-1 protein levels by 70% at 1 microM concentration and its action, as demonstrated by a marked reduction (about 50%) in caveolin-1 mRNA levels, was due to a true antisense mechanism. In HUVEC treated with the active ODN, caveolae were undetectable by confocal and electron microscopy, while their number was not affected when cells were treated with a scrambled ODN. Using the fibrin gel 3 D angiogenesis test we established that the active (but not the scrambled) ODN strongly suppressed capillary-like tube formation. Moreover, an antisense tailored against chicken caveolin-1 mRNA, when tested using the chorio-allantoic membrane technique, dramatically reduced vessel formation at doses (10-20 microg) under which control ODNs were ineffective and devoid of toxicity. Thus, it is likely that caveolin-1 down regulation, followed by caveolae disruption, impairs angiogenesis in vitro and in vivo.

Angiogenesis Inhibitors↗
PubMed1998

Evidence that photodynamic stress kills Zellweger fibroblasts by a nonapoptotic mechanism.

Zellweger fibroblasts, which are devoid of peroxisomes and fail to synthesize plasmalogens, are very sensitive to the killing effect triggered by UV-activated 12-(1-pyrene) dodecanoic acid (P12). Although in some studied performed, it is assumed that reactive oxygen species (ROS) may damage plasma membrane causing necrosis, other studies suggest that ROS are involved in apoptotic cell death induced by a wide variety of stimuli. Analysing the P12 dose-response in Zellweger fibroblasts, we observed that at high doses (1-2 microM), more than 75% of the cells died after 24 h. This behaviour suggested that, at high doses, P12 kills the cells by unspecific lytic mechanisms or by necrosis, while at low doses (0.1-0.5 microM), an apoptotic mechanism could be involved. Cytofluorimetric analysis of Zellweger fibroblasts-treated with activated P12 (0.5 microM) did not show morphological modifications typical of apoptotic cell death. This was supported by comparative staining of fibroblast nuclei, DNA gel electrophoresis and identification of poly(ADP-ribose) polymerase (PARP) cleavage and Bcl-2 expression, assayed by Western blots. Thus, our results, while confirming the importance of plasmalogens in the protection against ROS, establish that apoptosis is not involved in photodynamic death induced by activated P12. Therefore, we can expect that in gene transfer experiments, the rescue of Zellweger cells will be dependent only on the correction of peroxisomal biogenesis.

Apoptosis↗
PubMed1995

Post-transcriptional regulation of thromboxane A2 synthase in U937 cells.

Translation of mRNAs is a process usually tightly coupled to transcription of genes. However, there are examples of mRNA species which accumulate without being translated. Some mRNAs present in oocytes and ferritin mRNA are the most studied models. Studying the biogenesis of thromboxane A2 (TXA2) in the promonocytic line U937, we have noted that in proliferating cells high levels of TXA2 synthase mRNA are detectable by Northern blot, whereas no TXA2 could be recovered in the medium. This has been explained on the basis of Western blot experiments: TXA2 synthase was not detectable in proliferating cells, while a band of about 55 kd appears after treatment with the differentiating agent TPA. Immunofluorescence detection by confocal microscopy was in agreement with Immunoblot results. Thus, in U937 cells, TPA behaves as a regulator of translation of TXA2 synthase mRNA. We have further observed that the induced enzyme in U937 cells has many characteristics in common with the human monocytic enzyme: a long half life (> 24 hrs), a marked stability during catalysis and similar Km and Vmax values. Thus, U937 cells are a good model to study the mechanism by which a mRNA is efficiently translated only after differentiation has been triggered.

Animals↗
PubMed1995

Prostacyclin (PGI2) synthase is a constitutively expressed enzyme in human endothelial cells.

Biogenesis of prostanoids is under the control of some polypeptide growth factors. Cytosolic phospholipase A2, a form specific for arachidonic acid containing phospholipids, is activated by a translocation mechanism regulated by growth factors, while prostaglandin H synthase isoforms are induced de novo in several cell types. No information is available as far as PGI2 synthase is concerned. Human umbilical vein endothelial cells were cultured under conditions favoring proliferation or differentiation or capillary-like network formation in the presence of collagen gels. Basic fibroblast growth factor (bFGF 0.5-4 ng/ml) was used as a mitogen, interleukin-1 alpha (IL-1 alpha 10-60 UI/ml) as a differentiating agent, and prostacyclin (PGI2) biosynthesis was evaluated. Under the first condition, basal PGI2 production was unaffected while, in the presence of IL-1 alpha, a marked stimulation of PGI2 synthesis was observed. It is known that IL-1 alpha is a potent inducer of PGH synthase, while it is not known whether PGI2 synthase is also induced. Two lines of evidence indicate that PGI2 synthase is a constitutively expressed not inducible enzyme: (a) proliferating nonproducing cells when added with PGH2 produce an amount of PGI2 not different from the amount produced by cells stimulated with IL-1 alpha; (b) under this condition PGI2 synthase was immunodetectable either by immunofluorescence detected by confocal microscopy or by ELISA and, on microsomes isolated from endothelial cells, by Western blotting. It is concluded that the limiting step in the conversion arachidonate-PGI2 is represented solely by the level of PGH synthase. These results strongly suggest, but do not prove, the constitutive nature of the enzyme. The final demonstration requires the availability of a probe to detect mRNA level, a trial we are carrying out at the moment.

Cell Division↗
PubMed1995

Possible involvement of prostaglandin H synthase-1 induction in the differentiation of U-937 cells.

The promyelocytic human cell line U937, cultured in the presence of TPA and/or vit. D3, differentiates to monocytes and to macrophage-like cells. A potent stimulus for differentiation is represented also by colony stimulating factor-1 (CSF-1). Since this factor is a strong inducer of PGH synthase in human monocytes, we have investigated whether this event may be connected to the differentiation of U937. We have found that TPA, in the presence of serum, increased the production of thromboxane B2 (TXB2) 4-5 fold, while DMSO, which induced differentiation to neutrophils, was not active. Here we report studies indicating that the effect of protein and RNA synthesis inhibitors on prostanoid production, in cells incubated in the presence of CSF-1 (or FCS), can be correlated with an inductive event carried out by the growth factor, as demonstrated by the use of Western and Northern blotting procedures. However, while in human monocytes PGH-s and its mRNA are absent in controls and are expressed at high levels in CSF-1 stimulated cells, in U937 cells exposed to TPA, PGH-s mRNA was clearly detected by Northern blots, but its translation product was expressed at low level, and cells generated low amounts of TXA2 (13% of maximal production). After incubation with CSF-1 (or FCS) mRNA levels were only slightly modified, but large amounts of TXA2 accumulated in the medium. We have interpreted these findings by suggesting that CSF-1 is capable not only of regulating the expression of the gene encoding PGHs, but also of acing translationally to regulate the expression of its mature mRNA.

Blotting, Western↗
PubMed1994

Thromboxane A2 synthase activity in platelet free human monocytes.

Soon after platelets, the highest amounts of thromboxane A2 (TXA2) can be detected in human monocytes activated by serum. Using platelet-free human monocytes, we have shown that foetal calf serum (FCS) induces prostaglandin H synthase (PGH synthase) after 16 h of incubation, as shown by the use of transcriptional inhibitors and Western blotting. The effect of serum can be in part mimicked by recombinant colony stimulating factor-1 (hr CSF-1). It is not known whether the limiting step leading from arachidonate to TXA2 is represented solely by the level of PGH synthase or also by the level of TXA2 synthase. We approached this problem by using a Western blot specific for the enzyme, as well as by using PGH2 as substrate. The results show that TXA2 synthase is constitutively expressed in monocytes, i.e., its levels were high soon after their isolation, and similar to those observed after 24 h of incubation with serum. However TXA2 failed to be synthesized until at least 3 h of incubation, and the pattern of synthesis was dependent on the kinetics of PGH synthase induction. In any condition in which TXA2 synthase was immunodetectable, using PGH2 as substrate a high rate of conversion to TXB2 could be detected. Experiments with actinomycin D and cycloheximide indicate that the half-life of TXA2 synthase was longer than 16 h, therefore much longer than that of PGH synthase, that the gene coding for it is fully active in resting monocytes, and that the conversion of arachidonate to TXA2 induced by serum or CSF-1 is dependent solely on the de novo synthesis of PGH synthase.

Apoenzymes↗
PubMed1992

Involvement of prostanoids in the regulation of angiogenesis by polypeptide growth factors.

Polypeptide growth factors (PGFs), mainly those of the fibroblast growth factor (FGF) family, have been shown to be capable of regulating angiogenesis. Although many data have been accumulated during this last year on the mechanism of action of PGF, little is known about a possible identification of second messengers signalling to the cell the occupancy of the receptor by its ligand. We have previously proposed that arachidonic acid or its derivatives may play a role as PGF second messengers. In the present paper we described a modification of the chorioallanthoic membrane (CAM) technique, involving the use of labelled sulphate to follow the angiogenic process. Thus we have been able to correlate morphological observation of CAMs development with incorporation of labelled sulphate in a stable form. Here we show that, as expected, PGF as endothelial cell growth factor (ECGS) or basic fibroblast growth factor (bFGF) potentiate the incorporation of radioactivity into CAMs at concentrations which for bFGF are of the order of 1.5 micrograms/egg. This effect can be correlated to the generation of prostanoids by two kinds of approach: A) PGE1 injected into eggs was capable of strongly increasing labelling of CAMs; B) Indomethacin had a dramatic effect on embryo survival as well as on CAM development, decreasing both at very low concentration (50 survival rate observable at 2 micrograms/egg). Finally vanadate, which is known to inhibit tyrosine phosphatase, was capable of potentiating the effect of PGF on angiogenesis. Thus it appears that products of the prostaglandin H synthase pathway behave as mediators of PGF control of angiogenesis.

Allantois↗
PubMed1990

Prostanoids as second messengers of polypeptide growth factors.

Prostaglandin H synthase (PGHs), also known as cyclooxygenase, is an unstable enzyme whose mRNA has an half life of 10 minutes. Some polypeptide factors have been reported to induce the enzyme in target cells. We have purified and characterized a component of animal sera which behaves as a potent inducer of human monocyte PGHs. This factor, called serum monocytotropic factor, has been identified in human platelets and it appears to be structurally and biochemically different from identified platelet factors, such as platelet derived growth factor (PDGF) and transforming growth factor beta (TGF-beta), while showing strong similarities to colony stimulating factor 1 (CSF-1), so far undetected in platelets. Moreover, we have shown, by immunoblot analysis, that CSF-1 behaves as a potent and specific inducer of monocyte PGHs. The hypothesis that prostanoids may be considered as second messengers of platelet CSF-1 like factor, as well as of other growth factors and that PGHs induction plays a pivotal role in this process, will be illustrated.

Animals↗
PubMed1990

Polypeptide growth factors and angiogenesis.

Angiogenesis is the term used to describe the formation and development of blood vessels. The renewed interest in regulation and mechanistic aspects of angiogenesis depends on advances in the comprehension of metastatic dissemination of cancers, ischaemic heart disease and blood-brain barrier formation. Recently, many poly-peptide growth factors have been discovered which regulate the angiogenic process, most of them are stimulators and few inhibitors have been described. There is some evidence that many polypeptide growth factors employ prostanoids as second messengers. If this evidence will be extended to angiogenic factors, it will be possible to use inhibitors of prostaglandin H synthase and/or prostanoid receptor blockers in the control of tumour induced angiogenesis.

Animals↗
PubMed1990

Interleukins and interferons: yin-yang modulators of PGH synthase in human macrophages.

Prostaglandin H synthase (PGHs) not only is an unstable enzyme, mainly when it is challenged with substrate, but its mRNA is one of the shortest lived species so far identified in mammalian cells. Therefore, signals regulating its level are critical for the role it plays in many cells. The expression of genes coding for PGHs appears to be under the control of polypeptide growth factors. This is in accordance with our previous data showing that a colony stimulating factor-1 (CSF-1)-like factor induces the de novo synthesis of PGHs in human monocytes, as demonstrated by immunoblotting. Here we extend this concept by showing that interleukin (IL)-1 alpha behaves as a potent inducer of PGHs in human macrophages, as indicated by the block in its action due to the addition of RNA and protein synthesis inhibitors. Interferons (IFNs) alpha and beta, however, inhibit prostanoid production in a dose-dependent fashion mainly when macrophages activated by serum are tested. Thus, the PGHs system appears to be under a fine control, CSF-1 being the main regulator during the differentiation from pro-monocyte to monocyte and from monocyte to macrophage, and IL-1 (and perhaps IL-2) as well as IFN alpha and beta, the regulators during differentiation and/or proliferation of human macrophages.

Cycloheximide↗
PubMed1989

Prostaglandin and thromboxane biosynthesis in isolated platelet-free human monocytes. III. The induction of cycloxygenase by colony stimulating factor-1.

Previous observations showing the presence in the serum of a component capable of regulating prostanoid biosynthesis in human cultured monocytes, have led us to suspect its presence in human platelets. We have purified this serum monocytotropic factor (SMF) and have shown its identity with a component of platelet membranes. Surprisingly its structure appeared to be very similar to that of a polypeptide growth factor never before identified in platelets: the colony stimulating factor-1 (CSF-1 or M-CSF). Here we show that SMF and CSF-1 have very similar biological properties. Thus, CSF-1 when released from human platelets is capable of triggering the differentiation pathway leading from blood monocytes to resident macrophages. It is likely that cycloxygenase induction plays a pivotal role in these events.

Chromatography, High Pressure Liquid↗
PubMed1989

Immune dysfunction in primary biliary cirrhosis. II. Increased production of prostaglandin E.

In a previous study we observed that after in vitro treatment with indomethacin, lymphocyte response to phytohaemagglutinin (PHA) in primary biliary cirrhosis (PBC) patients was higher than that of controls. We know that indomethacin also inhibits prostanoid production, and thus in the present work we directly measured prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) production by mononuclear cells and monocytes from 12 PBC patients, 11 control subjects, and three control disease patients (alcoholic cirrhosis, AC). PHA-stimulated enriched monocytes from PBC patients produced approximately threefold more PGE2 (after 48 h of culture) than did normal and AC monocytes (P less than 0.05). TXB2 production was similar in all groups studied. We also made cultures in which PBC-purified lymphocytes proliferated better than PBC mononuclear cells (i.e. lymphocytes plus monocytes). Thus, a monocyte population producing PGE2 could be responsible, at least in part, for the hyporesponsiveness to PHA observed in PBC patients.

Humans↗
PubMed1989

A conjugate of prednisolone with albumin is pharmacologically active in macrophages.

In order to selectively deliver prednisolone to macrophages, this drug was coupled to human albumin. In experiments in vitro the conjugate entered into macrophages in much higher amounts than in fibroblasts; in the former cells it was pharmacologically active as demonstrated by the inhibition of prostaglandin E2 and thromboxane A2 production. Prednisolone-albumin conjugate might be useful in research on steroids; moreover in some pathological conditions it might produce the therapeutic effects of these drugs without causing their side effects.

Albumins↗
PubMed1988

Purification and partial characterization of serum monocytotropic factor, a platelet-derived cyclooxygenase-inducing polypeptide.

It has previously been shown that a heat- and acid-stable component of human and animal sera was capable of stimulating prostanoid biosynthesis in human blood monocytes, very probably by a mechanism involving cyclooxygenase induction. Many physico-chemical characteristics of this factor are similar to those of identified platelet factors. Here we show that human platelets are a rich source of this factor (serum monocytotropic factor) and that results from experiments using arachidonic acid or thrombin as releasers are consistent with its presence in platelet membranes. Serum monocytotropic factor has been purified 1500-fold by three chromatographic steps. Purification was more difficult when starting from platelet releasates or lysates. The purified serum monocytotropic factor had an apparent molecular mass of 70,000 as judged by Sephadex G-75 chromatography and by polyacrylamide gel electrophoresis; however, when subjected to HPLC on a gel permeation column in the presence of 6 M urea, one major peak corresponding to a relative molecular mass (Mr) of 30,000-35,000 was observed, which suggests a homodimeric structure. It is therefore very likely that human platelets store, in addition to the two well-identified polypeptide growth factors, platelet-derived growth factor and transforming growth factor-beta, a third polypeptide capable of regulating prostanoid production in monocytes.

Arachidonic Acid↗