A phase I study of veliparib with cyclophosphamide and veliparib combined with doxorubicin and cyclophosphamide in advanced malignancies.

PURPOSE: Veliparib (V), an oral poly(ADP-ribose) polymerase (PARP) inhibitor, potentiates effects of alkylating agents and topoisomerase inhibitors in preclinical tumor models. We conducted a phase I trial of V with iv cyclophosphamide (C) and V plus iv doxorubicin (A) and C. METHODS: Objectives were to establish the maximum tolerated dose (MTD) of the combinations, characterize V pharmacokinetics (PK) in the presence and absence of C, measure PAR in peripheral blood mononuclear cells (PBMCs) and γH2AX in circulating tumor cells (CTCs). In Group 1, dose escalations of V from 10 to 50 mg every 12 h Days 1-4 plus C 450 to 750 mg/m2 Day 3 in 21-day cycles were evaluated. In Group 2, V doses ranged from 50 to 150 mg every 12 h Days 1-4 with AC (60/600 mg/m2) Day 3 in 21-day cycles. In Group 3, patients received AC Day 1 plus V Days 1-7, and in Group 4, AC Day 1 plus V Days 1-14 was given in 21-day cycles to evaluate effects on γH2AX foci. RESULTS: Eighty patients were enrolled. MTD was not reached for V and C. MTD for V and AC was V 100 mg every 12 h Days 1-4 with AC (60/600 mg/m2) Day 3 every 21 days. V PK appears to be dose-dependent and has no effect on the PK of C. Overall, neutropenia and anemia were the most common adverse events. Objective response in V and AC treated groups was 22% (11/49). Overall clinical benefit rate was 31% (25/80). PAR decreased in PBMCs. Percentage of γH2AX-positive CTCs increased after treatment with V and AC. CONCLUSION: V and AC can be safely combined. Activity was observed in patients with metastatic breast cancer.

New Insights into the Significance of PARP-1 Activation: Flow Cytometric Detection of Poly(ADP-Ribose) as a Marker of Bovine Intramammary Infection.

Bovine intramammary infections are common diseases affecting dairy cattle worldwide and represent a major focus of veterinary research due to financial losses and food safety concerns. The identification of new biomarkers of intramammary infection, useful for monitoring the health of dairy cows and wellness verification, represents a key advancement having potential beneficial effects on public health. In vitro experiments using bovine peripheral blood mononuclear cells (PBMC), stimulated with the bacterial endotoxin lipopolysaccharide (LPS) enabled a flow cytometric assay in order to evaluate in vivo poly-ADP-ribose (PAR) levels. Results showed a significant increase of PAR after 1 h of treatment, which is consistent with the involvement of PARP activity in the inflammatory response. This study investigated PARP-1 activation in leukocyte subpopulations from bovine milk samples during udder infection. A flow cytometric assay was, therefore, performed to evaluate the PAR content in milk leukocyte subsets of cows with and without intramammary infection (IMI). Results showed that milk lymphocytes and macrophages isolated from cows with IMI had a significant increase of PAR content compared to uninfected samples. These results suggest mastitis as a new model for the study of the role of PARP in zoonotic inflammatory diseases, opening a new perspective to the "One Health" approach.

Automodified Poly(ADP-Ribose) Polymerase Analysisto Monitor DNA Damagein Peripheral Lymphocytes of Floriculturists Occupationally Exposed to Pesticides.

Increased DNA damage and the propension to cancer development, depend on the modulation of the mechanisms to control and maintain genomic integrity. Poly(ADP-Ribose)Polymerase activation and automodification are early responses to genotoxic stress. Upon binding to DNA strand breaks, the enzyme, a molecular DNA nick sensor, is hyperactivated: this is the first step in a series of events leading to either DNA repair or apoptosis. Enzyme hyperactivation and automodification can be easily measured and are widely used to look at DNA damage extent in the cell. We investigated whether these two markers (increased catalytic activity and auto modification), could help to monitor DNA damage in lymphocytes of flower growers from Southern Italy, occupationally exposed to pesticides. Peripheral lymphocyte lysates were analyzed for Poly(ADP-Ribose)Polymerase activity, and by SDS-PAGE and anti-Poly(ADP-Ribose)Polymerase 1-antibodyto measure automodified Poly(ADP-Ribose)Polymerase levels bydensitometry. Poly(ADP-Ribose)Polymerase activity and PARP automodification followed the same trend. Growers daily exposed to pesticides, showed both biomarkers very high, either in the presence or in the absence of pathologies. PARP activity and auto-modification in peripheral blood lymphocytes are possible, non-invasive, androutinartools to monitor the healthy conditions of floricoltorists.

Nicotinic acid supplementation: effects on niacin status, cytogenetic damage, and poly(ADP-ribosylation) in lymphocytes of smokers.

As a substrate for poly(ADP-ribose) polymerase (PARP; EC, 2.4.2.30), an enzyme that is activated by DNA strand breaks and is thought to facilitate efficient DNA repair, NAD+ and its precursor nicotinic acid (niacin) are involved in the cellular defense against DNA damage by genotoxic compounds. In this study, the effect of nicotinic acid supplementation on cytogenetic damage and poly(ADP-ribosylation) was evaluated in a human population that is continuously exposed to genotoxic agents, e.g., smokers. By use of a placebo-controlled intervention design, 21 healthy smokers received supplementary nicotinic acid at 0-100 mg/day for 14 weeks. An increased niacin status, as assessed from blood nicotinamide concentrations and lymphocyte NAD+ concentrations, was observed in groups supplemented with 50 and 100 mg/day. This effect was most pronounced in subjects with lower initial NAD+ levels. An increased niacin status did not result in decreased hypoxanthine guanine phosphoribosyltransferase variant frequencies and micronuclei induction in peripheral blood lymphocytes (PBLs). Sister chromatid exchanges in PBLs, however, were increased after supplementation with nicotinic acid. This increase was positively associated with the daily dose of nicotinic acid. No effects of nicotinic acid supplementation were found for ex vivo (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene-induced poly(ADP-ribosylation), although the small number of samples that could be analyzed (n = 12) does not allow firm conclusions. Because no evidence was found for a decrease in cigarette smoke-induced cytogenetic damage in PBLs of smokers after nicotinic acid supplementation of up to 100 mg/day, it is concluded that supplemental niacin does not contribute to a reduced genetic risk in healthy smokers.

Biochemical characterization of ADP-ribose polymer metabolism in SLE.

The metabolism of poly(ADP-ribose) in peripheral blood mononuclear (PBM) cells was studied in 13 patients with systemic lupus erythematosus (SLE) and in 12 age and sex matched controls. Poly(ADP-ribose) polymerase activity was measured as the net accumulation of ADP-ribose polymers during the conversion of 32P-NAD to poly(ADP-ribose) in PBM cells in vitro. The control population showed a mean activity of 418 +/- 91(s.d.)pmol ADP-ribose/10 min/10(6) cells. The SLE population was more heterogeneous and showed a lower mean of 225 +/- 147(s.d.)pmol ADP-ribose/10 min/10(6) cells. The mechanism of decreased ADP-ribose polymer accumulation was investigated. Measurements of turnover of the ADP-ribose polymers and its substrate, NAD+, showed that diminished ADP-ribose polymer accumulation in SLE subjects resulted from decreased poly(ADP-ribose) synthesis and not from altered rates of polymer turnover or NAD utilization. Western blot analyses of enzyme protein levels, kinetic studies of poly(ADP-ribose) polymerase activity and analyses of polymer size distribution suggested that the mechanisms of poly(ADP-ribose) synthesis in SLE cells is not altered but that the number of active poly(ADP-ribose) polymerase molecules is reduced.

Altered metabolism of poly(ADP-ribose) in the peripheral blood lymphocytes of patients with systemic lupus erythematosus.

A method was developed to measure poly(ADP-ribose) metabolism in peripheral blood lymphocytes. The technique involved the isolation of lymphocytes on Ficoll gradients, followed by lysis with 5M NaCl. The synthesis and degradation of poly(ADP-ribose) in this crude lysate, measured by the incorporation of 3H-labeled NAD into acid-precipitable counts, was compared in 18 patients with systemic lupus erythematosus (SLE), 10 patients with rheumatoid arthritis, and in 10 control patients without rheumatoid arthritis. Patients with SLE showed a 70% decrease in poly(ADP-ribose) synthesis (P less than 0.001); this decreased synthesis persisted even with the addition of histones or DNase. We present possible explanations of the role of poly(ADP-ribose) in SLE.

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.

Unique acceptors for poly(ADP-ribose) in resting, proliferating and DNA-damaged human lymphocytes.

Acceptor proteins for poly(adenosine diphosphoribosyl)ation were determined in resting human lymphocytes, in lymphocytes with N-methyl-N'-nitro-N-nitrosoguanidine-induced DNA damage and in lymphocytes stimulated to proliferate by phytohemagglutinin. Kinetic studies showed that the increase in ADP-ribosylation which occurred in response to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment was greater in magnitude but more transient in duration than that which occurred in phytohemagglutinin-stimulated cells. Gel electrophoretic analyses revealed that MNNG treatment and phytohemagglutinin stimulation both caused an increase in ADP-ribosylation of poly(ADP-ribose) polymerase and core histones. In MNNG-treated cells, an increase in ADP-ribosylation of histone H1 was also observed. In contrast, phytohemagglutinin-stimulated cells showed no increase in ADP-ribosylation of histone H1. In MNNG-treated cells there was also ADP-ribosylation of a protein of molecular weight 62 000, while in phytohemagglutinin-stimulated cells there was a marked increase in ADP-ribosylation of a protein of molecular weight 96 000. MNNG treatment of phytohemagglutinin-stimulated cells produced a pattern of ADP-ribosylation that appeared to be due to the combined effects of the individual treatments. 3-Aminobenzamide effectively inhibited ADP-ribosylation under all treatment conditions.

Cholera toxin-catalysed ADP-ribosylation of erythrocyte proteins: general properties.

Upon incubation of lysed pigeon erythrocytes with NAD, adenosine diphosphate-ribose (ADP-ribose) is incorporated into nuclear poly ADP-ribose and into an unidentified acid-insoluble product of the cytosol. The properties of these incorporations have been examined and a method developed for reducing their amount whilst retaining the sensitivity of the lysate to cholera toxin. This method has allowed the detection and description of a set of cholera toxin-specific ADP-ribose transfers to membrane-bound and soluble proteins under conditions that lead to adenylate cyclase activation.

Synthesis of DNA and poly(adenosine diphosphate ribose) in normal and chronic lymphocytic leukemia lymphocytes.

Peripheral blood lymphocytes were isolated from 9 patients with chronic lymphocytic leukemia (CLL) and 12 normal control donors. The cells were assayed for synthesis of DNA and poly-(adenosine diphosphate ribose) (poly[ADPR]) immediately after isolation and on successive days following their treatment with phytohemagglutinin (PHA). Two different techniques were used to measure DNA synthesis. In the standard technique, DNA synthesis was measured by incubating intact cells with [(3)H]deoxythymidine. In the new technique, the lymphocytes were first rendered permeable to nucleotides, then DNA synthesis was measured by incubating them with [(3)H]deoxythymidine triphosphate in the presence of deoxyATP, deoxyGTP, deoxyCTP, ATP, and Mg(++). Both assays showed the anticipated rise in DNA synthesis after PHA stimulation of normal cells. PHA-stimulated lymphocytes from patients with CLL demonstrated low levels of DNA synthesis in both assay systems. The initial levels of poly(ADPR) synthesis were greater in CLL lymphocytes than in normal cells. Studies with a T-cell-depleted population of normal cells showed the same activity for poly(ADPR) synthesis that was demonstrated by the original population of normal cells. PHA stimulation produced an increase in poly(ADPR) synthesis in both the normal and CLL cells. The increase in poly(ADPR) synthesis in normal cells was coincident with the increase in DNA synthesis. The increase in poly(ADPR) synthesis in the CLL cells was dissociated from the delayed and diminished increase in DNA synthesis. Thus, CLL cells have higher than normal initial levels of poly(ADPR) synthesis. Poly(ADPR) synthesis is dissociated from DNA synthesis in CLL cells whereas it varies directly with DNA synthesis in normal lymphocytes.