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P Cerutti

Publications and source records attributed to P Cerutti.

At least 55 records · Page 3Linked to original sources
PubMed1989

ADP-ribosylation of ADPR-transferase and topoisomerase I in intact mouse epidermal cells JB6.

Poly(ADP-ribosylation) [poly(ADPR)] is a posttranslational modification of chromosomal proteins that affects the structural and functional properties of chromatin. We have studied poly(ADPR) of ADPR-transferase and topoisomerase I in intact mouse epidermal cells JB6 (clone 41) by a combination of affinity chromatography on phenylboronate and immunoblotting with monoclonal antibodies against poly(ADPR) chains and polyclonal antibodies against ADPR-transferase and topoisomerase I, respectively. Constitutive, steady-state poly(ADPR) substitution of ADPR-transferase was estimated at 4% and that of topoisomerase I at 0.1%. Active oxygen produced extracellularly by xanthine-xanthine oxidase and the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine transiently increased the level of poly(ADPR) substitution of these enzymes by a factor of 6-10. While the poly(ADPR) substitution of ADPR-transferase remained elevated after 60 min of incubation, the poly(ADPR) substitution of topoisomerase I had returned to control values within this time. Benzamide (100 microM) partially prevented the stimulation of poly(ADPR) synthesis by these agents. We speculate that self-inactivation of ADPR-transferase by poly(ADPR) represents a feedback mechanism that has the function to avoid excessive poly(ADPR) synthesis and concomitant NAD and ATP depletion. Inactivation of topoisomerase I in the neighborhood of DNA breakage may temporarily shut down DNA replication and allow DNA repair to occur.

Animals↗
PubMed1989

Effects of tert-butyl hydroperoxide on promotable and non-promotable JB6 mouse epidermal cells.

Oxidants and agents that induce a cellular prooxidant state can act as carcinogens. We compared the effect of tert-butyl hydroperoxide (Bu-OOH) on DNA strand breakage, poly ADP-ribosylation of chromosomal proteins and the expression of the proto-oncogenes c-fos and c-myc between non-promotable clone 30 and promotable clone 41 of mouse epidermal cells JB6. These pathophysiological effects of oxidants are mechanistically related. Bu--OOH caused more DNA-strand breakage at high concentrations and more extensive poly ADP-ribose accumulation in clone 30 than in clone 41, in reactions which require intracellular free iron. Clone 41 exhibited constitutive c-myc expression while c-fos mRNA was very low in untreated cultures of both clones. Low concentrations of Bu-OOH induced c-myc and more strongly c-fos in clone 41. Both proto-oncogenes were strongly induced in clone 30. Our results allow insights into the mechanisms of action of a typical organic hydroperoxide in JB6 cells. However, they do not uncover the reasons for the differential promotability of the two JB6 clones by oxidants beyond the implication of the constitutive expression of c-myc in promotable clone 41.

Animals↗
PubMed1989

Pathophysiological mechanisms of active oxygen.

Besides being toxic, oxidants can induce pathophysiological effects in mammalian cells. For example they can stimulate rather than inhibit cell growth. Since oxidants are ubiquitous they may represent 'natural' tumour promoters. Our work with xanthine/xanthine-oxidase as an extracellular source of active oxygen (AO) and promotable (clone 41) and non-promotable (clone 30) mouse epidermal cells JB6 allows insights into the mechanism of action of oxidant promoters. We found that AO stimulated the growth only of promotable clone 41 after an initial period of moderate inhibition while it was strongly cytostatic for non-promotable clone 30. Active oxygen induced larger amounts of DNA-strand breaks and poly ADP-ribosylation of chromosomal proteins in non-promotable cells. In addition, AO was capable of inducing the growth- and differentiation-related proto-oncogenes c-fos and c-myc in promotable and non-promotable JB6 cells. We speculate that these genes can exert their functions only in the promotable clone 41 because the general cytostatic effects of AO are moderate. A possible explanation for the differences between these 2 clones was discovered when we compared the constitutive activities, protein concentrations and mRNA levels for the antioxidant enzymes catalase (CAT), Cu,Zn-superoxide dismutase (SOD) and glutathione-peroxidase (GPx). We found that CAT and SOD (but not GPx) levels were 2-3-fold higher in the promotable clone 41. We propose that promotable cells possess a superior antioxidant defence which protects them from excessive cytostatic effects of AO.

Animals↗
PubMed1989

Infectivity of vesicles prepared from chilo iridescent virus inner membrane: evidence for recombination between associated DNA fragments.

Treatment of CIV particles with octylglucoside at high ionic strength leads to the solubilization of the inner viral membrane. Incubation of permissive cells (Cf124 cells) with vesicles obtained after dialysis of the detergent shows that this fraction is infectious. This infectivity, which is very low, could only be detected after two serial passages on permissive cells. This phenomenon is, however, reproducible. Isopycnic centrifugation analysis shows that some DNA cosediments with the vesicles. Extraction and purification of this DNA confirm the presence of a large DNA fragment of about 50.10(6) Da. Digestion with restriction endonucleases demonstrated that this DNA did not correspond to a particular fragment but to a population of DNA fragments of homogeneous size arising from various regions of the viral genome. Purified viral DNA was not infectious, the presence of DNA in the vesicles could not account therefore for their infectivity. Experiments of non-genetic reactivation of purified CIV DNA by UV-irradiated virus suggest that one (or several) structural component(s) of CIV particles must be involved in the first stages of the viral replication cycle. In addition, transfection of cells with large overlapping DNA fragments could generate infectious particles when the cells were superinfected with UV-irradiated virus. It can be supposed that the vesicle suspensions, which probably contain the reactivating factor, are composed of a population of vesicles which are all different in their DNA content. Infectivity of such suspensions would be the consequence of a recombination between large overlapping DNA fragments.

Aedes↗
PubMed1988

Oxidants induce phosphorylation of ribosomal protein S6.

We have investigated the phosphorylation of the ribosomal S6 protein which may be on the pathway of mitogenic stimulation in response to oxidants. Mouse epidermal cells JB6 (clone 41) were exposed to active oxygen generated extracellularly by glucose/glucose oxidase (producing H2O2) or xanthine oxidase (producing H2O2 plus superoxide) or active oxygen produced intracellularly by the metabolism of menadione (producing mostly superoxide). All three sources of active oxygen induced rapidly a protein kinase activity which phosphorylated S6 in cellular extracts prepared in the presence of the phosphatase inhibitor beta-glycerophosphate. Maximal activity was reached within 15 min of exposure, and phosphorylation occurred specifically at serine residues. Strong activation of the protein kinase activity was also observed by diamide which selectively oxidizes SH functions. The following observations characterize the reaction: 1) Extracellular addition of catalase but not Cu,Zn-superoxide dismutase was inhibitory, implicating H2O2 rather than superoxide as the active species. 2) Exposure of JB6 cells to reagent H2O2 or H2O2 released by glucose/glucose oxidase resulted in a measurable increase in intracellular free Ca2+. 3) The intracellular Ca2+ complexer quin 2 suppressed the reaction. 4) The calmodulin antagonist trifluoperazine prevented the activation of the protein kinase. 5) Exposure of cells to Mn2+ and La3+, which stimulate calmodulin-dependent activities, potently increased the S6 kinase activity of the cell extracts. 6) Desalted extracts strictly required the addition of Mg2+ and their activity was inhibited by Mn2+. In contrast, the phosphorylation of a 95-kDa protein was strongly stimulated by Mn2+. 7) For several agonists, i.e. active oxygen, phorbol 12-myristate 13-acetate, and serum, tryptic peptide analysis yielded the same phosphopeptides, suggesting that a common S6 kinase is involved in these reactions. From these data we propose that oxidants induce an increase in intracellular free Ca2+ which activates a Ca2+/calmodulin-dependent protein kinase and, as a consequence, an S6 kinase.

Animals↗
PubMed1988

Antioxidant enzymes in xeroderma pigmentosum fibroblasts.

In light of recent studies implicating low catalase activities in the pathogenesis of the cancer-prone disease xeroderma pigmentosum (XP) we have measured catalase activity, protein levels, and mRNA concentrations in six XP fibroblast strains and three normal controls. Only one XP strain of complementation group A (XP1223) possessed significantly lower catalase by all three criteria. The other five XP strains (two XP variants, two strains of complementation group D, and one strain of complementation group C) possessed catalase levels which fell into the range of the interindividual variations of normal controls. We further assessed the total enzymatic antioxidant defense status by measuring the levels of copper, zinc, and manganese superoxide dismutase and glutathione peroxidase. None of these enzymes showed significant deviations from controls in XP cells. Our results do not support the notion that a deficient enzymatic antioxidant defense facilitates the establishment of a prooxidant state in XP upon exposure to near-UV. However, they do not argue against the participation of active oxygen in near-UV-induced carcinogenesis in XP.

Antioxidants↗
PubMed1988

Active oxygen induced DNA strand breakage and poly ADP-ribosylation in promotable and non-promotable JB6 mouse epidermal cells.

The evidence is convincing that oxidants and agents which induce a cellular pro-oxidant state can act as carcinogens, in particular as promoters and progressors. Importantly, infiltrated phagocytes represent a source of oxidants in inflamed tissues. We have studied the mechanism of the promotional action of active oxygen (AO) in mouse epidermal cells JB6 by comparing the non-promotable clone 30 to the promotable clone 41. In order to mimick AO released by phagocytes we used xanthine/xanthine oxidase as a source of extracellular superoxide and hydrogen peroxide. We found that AO stimulated the growth only of promotable clone 41 after an initial period of moderate inhibition while it was strongly cytostatic for non-promotable clone 30. Reasons for the higher cytostatic effect of AO on the non-promotable clone 30 were discovered when we measured DNA strand breakage and poly ADP-ribosylation of chromosomal proteins. At equal doses AO induced 4-5 times more DNA breaks in clone 30 in reactions which required iron--and probably also calcium--ions. The higher amount of DNA breakage in clone 30 was reflected in a higher extent of poly ADP-ribosylation. Excessive DNA breakage and poly ADP-ribosylation which causes the depletion of NAD and ATP may be responsible for the strong cytostatic effect of AO in clone 30. We conclude that differential resistance to the cytostatic/cytotoxic effect of AO in part determines the promotability of mouse epidermal cells JB6.

Animals↗
PubMed1987

Molecular mechanisms involved in T cell activation. I. Evidence for independent signal-transducing pathways in lymphokine production vs proliferation in cloned cytotoxic T lymphocytes.

It is well-established that activated T cells proliferate in response to interleukin 2 (IL 2) and produce various soluble lymphokines such as macrophage-activating factor (MAF) in response to antigen. Prior to investigating the molecular events involved in signaling the initiation of these responses in cloned murine cytotoxic T lymphocytes (CTL), we determined whether these responses could occur independently, and we established for each response the time during which signal transducing mechanisms may function. It was found that this cloned CTL population was in a resting state (G1 phase of cell cycle) 7 days after stimulation with antigen plus IL 2. At this time, the incubation of these resting CTL with IL 2 for 4 to 6 hr resulted in a maximal proliferative response that was not accompanied by the production of MAF. Conversely, the incubation of resting CTL with antigen or lectin (in the absence of IL 2) for at least 8 hr resulted in the maximal production of MAF at 24 hr without inducing a proliferative response. In addition, antigen or lectin, but not IL 2, triggered an immediate (less than 1 min) and sustained (at least 8 hr) mobilization of intracellular calcium. The kinetics of this calcium response paralleled the minimum time (8 hr) that was required for resting CTL to interact with either antigen or lectin in order to produce maximal titers of MAF. These results indicate that proliferation and lymphokine (MAF) production in cloned murine CTL are independent events. In these resting CTL, the signal mechanisms that mediate the production of lymphokines are most likely restricted to the initial 8 hr of stimulation by antigen or lectin and involve the rapid and prolonged mobilization of cytoplasmic calcium. Proliferative signals, however, are probably complete within 4 to 6 hr after stimulation by IL 2 and do not involve readily demonstrable fluxes of cytoplasmic calcium, as determined by the fluorescent calcium probe Quin 2.

Animals↗
PubMed1987

Phorbol ester-induced formation of clastogenic factor from human monocytes.

Phorbol-12-myristate-13-acetate (PMA) acts as a tumor promoter on mouse skin. It induces inflammation and leukocyte-mediated clastogenicity which appears to be related to rapid changes in lipid metabolism. To identify lipids possessing clastogenic and/or tumor-promoting properties, we have characterized the metabolism and release of arachidonic acid (AA) and related lipids during the formation of lipophilic clastogenic factors by PMA-treated human monocytes. In 1 h, [3H]AA-labeled monocytes spontaneously released significant amounts of their total radioactivity (4%) which increased nearly 4-fold (15%) with PMA (30 ng/ml) treatment. Eighty-five per cent of extracellular 3H-label from both control and PMA-treated monocytes was composed of free AA (plus AA-metabolites), while the remaining radioactivity was incorporated in phospholipids and mono- and diacylglycerols. Treated and non-treated cells released essentially the same kind of metabolites but PMA induced a 3- to 4-fold increase in total amounts. The major products consisted of prostaglandins F2 alpha and E2, thromboxane B2, 12-hydroxy-5,8,10-heptadecatrienoic acid and 5-, 11- and 15-hydroxyeicosatetraenoic acids. PMA also induced increases in the levels of three unidentified products. Neither leukotrienes nor 4-hydroxynonenal, a major alkenal degradation product of AA, were found in medium from PMA-treated monocytes. PMA, in contrast to the first-stage tumor promoter calcium ionophore A23187, failed to stimulate the release of platelet activating factor. The increased formation of phorbol ester-induced AA metabolites was proportional to the increase in free extracellular AA. The source of AA from treated and untreated monocytes consisted of cellular phospholipids with phosphatidylcholine and phosphatidylethanolamine accounting for 85%.

Arachidonic Acid↗
PubMed1987

Gene expression rather than the initiation of DNA replication is the principal target of lethal u.v.-induced damage in a regulatory region of SV40 DNA.

The survival of transfected simian virus (SV) 40 DNA is acutely sensitive to damage in a 302-bp regulatory region that governs viral gene expression and the initiation of viral DNA replication. We investigated whether the lethal effect of damage in this region is due to the disruption of gene expression or to the inhibition of DNA replication by comparing the survival of damaged viral DNA in CV-1 and cos-1 African green monkey cells. Viral early sequences integrated into the genomic DNA of cos-1 cells complement the growth of virus with defective early genes and were therefore expected to reverse viral sensitivity to lesions that interfere with early gene expression. Our results indicate that viral sensitivity to damage in the regulatory region is almost completely abolished in cos-1 cells. This finding identifies gene expression rather than the initiation of DNA replication as the major target for lethal damage in that portion of the SV40 genome. Sensitivity to damage in the viral late gene region is the same in CV-1 and cos-1 cells, indicating that cos-1 cells are not merely more proficient in host-cell reactivation. Our results allow us to partition the overall lethal effect of DNA damage into sectors, and to assign each sector to the disruption of a particular genetic function.

Animals↗
PubMed1985

Poly ADP-ribosylation of histones in tumor promoter phorbol-12-myristate-13-acetate treated mouse embryo fibroblasts C3H10T1/2.

The tumor promoter phorbol-12-myristate-13-acetate (PMA) increases the poly ADP-ribosylation of acid extractable (0.2N H2SO4) nuclear proteins in mouse embryo fibroblasts C3H10T1/2. Catalase suppresses the reaction by approximately 50%. Polyacrylamide gel electrophoresis reveals that the core histones H2B, A24 and H3d serve as major poly ADP-ribose acceptors. Smaller amounts of poly ADP-ribose are associated with histones H2A/H3 and H1. Poly ADP-ribosylation of histones may change the nucleosomal structure and function and play a role in PMA induced modulation of gene expression in promotion.

Animals↗
PubMed1985

Tumor promoter phorbol-12-myristate-13-acetate induces poly ADP-ribosylation in human monocytes.

The tumor promoter phorbol-12-myristate-13-acetate (PMA) induces rapid poly ADP-ribosylation and a drop in cellular NAD concentration in human monocytes. The antioxidants CuZn-superoxide dismutase, catalase, glutathione peroxidase and butylated-hydroxytoluene inhibit the reaction indicating that active oxygen species produced in the PMA-induced oxidative burst represent intermediates. The inhibitor of ADP-ribosyl-transferase, 3-amino-benzamide, inhibited poly ADP-ribosylation but did not prevent the drop in NAD-levels. PMA also causes the slow accumulation of DNA strand breaks in monocytes. The difference in the kinetics of poly ADP-ribosylation and DNA breakage argues against a simple relationship between the two reactions.

Antioxidants↗
PubMed1985

Tumor promoter phorbol-12-myristate-13-acetate induces poly(ADP)-ribosylation in fibroblasts.

The tumor promoter phorbol-12-myristate-13-acetate (PMA) causes an increase in pol(ADP)-ribosylation in mouse and human fibroblasts via the intermediate formation of active oxygen. In contrast to poly(ADP)-ribosylation induced by the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine, de novo RNA and protein synthesis are required and the accumulation of the polymer occurs in the absence of detectable DNA strand breakage. Our results suggest a mechanism for PMA-induced modulation of chromatin structure and gene expression.

Animals↗
PubMed1985

Non-histone chromosomal protein acceptors for poly(ADP)-ribose in phorbol-12-myristate-13-acetate treated mouse embryo fibroblasts (C3H10T1/2).

The tumor promoter phorbol-12-myristate-13-acetate (PMA) increases the level of poly ADP-ribosylation of chromosomal proteins in mouse embryo fibroblasts C3H10T1/2. The poly ADP-ribosylated nuclear proteins fall into the following molecular weight classes: 40, 48, 61, 77, 92, 158, 200 kd. Preincubation with catalase reduced the poly ADP-ribose (ADPR) substitution of all these proteins essentially to control levels. Western blot analysis with antibody directed against ADPR transferase indicates that the major acceptors are ADP-ribose transferase (116 kd) itself and its proteolytic degradation products of 20-25, 45 and 72-95 kd. Poly ADP-ribosylation of these proteins is suppressed by cycloheximide, 3-aminobenzamide, antipain and catalase. The latter three inhibitors possess anti-promotional activities in certain in vitro cell culture systems. Auto-poly ADP-ribosylation of ADPR transferase and its proteolytic cleavage as well as the poly ADP-ribosylation of other chromosomal proteins may play a role in the modulation of gene expression by PMA.

Animals↗
PubMed1985

Enhanced formation of benzo(a)pyrene:DNA adducts in monocytes of patients with a presumed predisposition to lung cancer.

Blood monocytes from 45 selected patients with lung cancer and 30 healthy controls were incubated with [G-3H]-benzo(a)pyrene for 30 h, and the formation of covalently bound DNA adducts was determined. The lung cancer patients were either relatively young (below 46 yr), nonsmokers, or had at least one first degree relative with lung cancer. Therefore, they might be considered cancer prone. The DNA adducts were significantly elevated in 22 patients with early age cancer (4.34 fmol/micrograms of DNA; P less than 0.04). In 12 familial cases, the slight elevation (2.77 fmol/micrograms of DNA) was not statistically significant in comparison to healthy controls. Benzo(a)pyrene:DNA adduct levels did not differ significantly between smokers and nonsmokers. Eight of 9 lung cancer patients with DNA adducts equal or above 4.5 fmol/micrograms of DNA but only 16 of 36 with adducts below this value had either oat cell or squamous cell cancer (P less than 0.05). The observed enhanced formation of covalent DNA adducts in blood monocytes exposed to a carcinogenic polycyclic hydrocarbon may be genetically determined and could play a role in the development of lung cancer at an early age.

Adult↗
PubMed1984

Active oxygen acts as a promoter of transformation in mouse embryo C3H/10T1/2/C18 fibroblasts.

There is much evidence from in vivo and in vitro carcinogenesis studies that active oxygen species play a role in tumor promotion. We tested directly whether superoxide produced extracellularly by xanthine-xanthine oxidase (X-XO) has the capacity to promote initiated mouse embryo C3H/10T1/2 fibroblasts. Cell cultures initiated with either 137Cs gamma-rays or benzo[a]pyrene diol epoxide I were found to transform 3-30 times more effectively when subsequently treated daily for 3 weeks with nontoxic doses of X-XO. Scavengers of active oxygen radicals such as superoxide dismutase or superoxide dismutase in combination with catalase reduced the frequency of appearance of transformed foci by 3-25 times when compared to cultures receiving X-XO alone. These results show that active oxygen species such as superoxide and H2O2 can act in a promotional manner that mimics the effects of the mouse skin promoter phorbol 12-myristate 13-acetate in this system. X-XO also acted as a weak complete carcinogen.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1984

Evidence for membrane-mediated chromosomal damage by aflatoxin B1 in human lymphocytes.

The hepatocarcinogen aflatoxin B1 (AFB1) was found to be a potent clastogen for phytohemagglutinin stimulated human lymphocytes. It also induced sister chromatid exchanges. These types of chromosomal damage were induced at very low levels of covalent AFB1 - DNA adducts suggesting that AFB1 operates in part by indirect action because of its membrane-active character. The membrane-active character of AFB1 is documented by the following results: (i) AFB1 stimulated the excretion of hydroxy- and/or hydroperoxy-arachidonic acid (AA) and free AA into the culture medium; (ii) the phospholipase A2 inhibitor p-bromophenacylbromide was anticlastogenic ; (iii) the inhibitors of the oxidative metabolism of AA indomethacin, flufenamic acid, 5,8,11,14-eicosatetraynoic acid, nordihydroguaiaretic acid and BN 1015 were anticlastogenic . These results are compatible with the induction of DNA damage by indirect action or the formation of covalent adducts via metabolic activation by cooxygenation . The observation that CuZn superoxide dismutase was anticlastogenic indicates the intermediacy of superoxide in DNA damage formation and supports the former mechanism.

Aflatoxin B1↗