Inhibition of PARP1 Increases IRF-dependent Gene Transcription in Jurkat Cells.

Poly(ADP-ribose) polymerase 1 (PARP1) plays important roles in the regulation of transcription factors. Mounting evidence has shown that inhibition of PARP1 influences the expression of genes associated with inflammatory response. Interferon regulatory factor 1 (IRF1) is a critical transcription factor for the development of both the innate and adaptive immune responses against infections. However, the molecular mechanism through which PARP1 mediates the effects has not been clearly demonstrated. Jurkat cells were exposed to dexamethasone (Dex) or PARP1 inhibitor PJ34. The expression levels of IL-12, LMP2, OAS1 and PKR were detected using real-time RT-PCR. The interactions between PARP1 and IRF1 were examined by co-immunoprecipitation (co-IP) assays. We further explored the mechanism of PARP1 suppressing IRF1 by assessing the activities of interferon stimulated response element (ISRE). The mRNA expression of IL-12, LMP2, OAS1 and PKR was obviously suppressed by Dex in Jurkat cells, which could be rescued by PJ34 treatment. Luciferase study revealed that poly(ADP-ribosyl)- ation suppressed IRF1-mediated transcription through preventing the binding of IRF1 to ISREs. PARP1 inhibited IRF1-mediated transcription in Jurkat cells by preventing IRF1 binding to ISREs in the promoters of target genes. It is suggested that PARP1 is a crucial regulator of IRF1-mediated immune response. This study provides experimental evidence for the possible application of PARP1 inhibitors in the treatment of IRF1-related immune anergy.

Overview on poly(ADP-ribose) immuno-biomedicine and future prospects.

Poly(ADP-ribose), identified in 1966 independently by three groups Strassbourg, Kyoto and Tokyo, is synthesized by poly(ADP-ribose) polymerases (PARP) from NAD(+) as a substrate in the presence of Mg(2+). The structure was unique in that it has ribose-ribose linkage. In the early-1970s, however, its function in vivo/in vitro was still controversial and the antibody against it was desired to help clear its significance. Thereupon, the author tried to produce antibody against poly(ADP-ribose) in rabbits and succeeded in it for the first time in the world. Eventually, this success has led to the following two groundbreaking papers in Nature: "Naturally-occurring antibody against poly(ADP-ribose) in patients with autoimmune disease SLE", and "Induction of anti-poly(ADP-ribose) antibody by immunization with synthetic double-stranded RNA, poly(A)·poly(U)".On the way to the publication of the first paper, a reviewer gave me a friendly comment that there is "heteroclitic" fashion as a mechanism of the production of natural antibody. This comment was really a God-send for me, and became a train of power for publication of another paper, as described above. Accordingly, I thought this, I would say, episode is worth describing herein. Because of its importance in biomedical phenomena, a certain number of articles related to "heteroclitic" have become to be introduced in this review, although they were not always directly related to immuno-biological works on poly(ADP-ribose). Also, I tried to speculate on the future prospects of poly(ADP-ribose), product of PARP, as an immuno-regulatory molecule, including either induced or naturally-occurring antibodies, in view of "heteroclitic".

An enzyme-linked immunosorbent assay-based system for determining the physiological level of poly(ADP-ribose) in cultured cells.

PolyADP-ribosylation is mediated by poly(ADP-ribose) (PAR) polymerases (PARPs) and may be involved in various cellular events, including chromosomal stability, DNA repair, transcription, cell death, and differentiation. The physiological level of PAR is difficult to determine in intact cells because of the rapid synthesis of PAR by PARPs and the breakdown of PAR by PAR-degrading enzymes, including poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribosylhydrolase 3. Artifactual synthesis and/or degradation of PAR likely occurs during lysis of cells in culture. We developed a sensitive enzyme-linked immunosorbent assay (ELISA) to measure the physiological levels of PAR in cultured cells. We immediately inactivated enzymes that catalyze the synthesis and degradation of PAR. We validated that trichloroacetic acid is suitable for inactivating PARPs, PARG, and other enzymes involved in metabolizing PAR in cultured cells during cell lysis. The PAR level in cells harvested with the standard radioimmunoprecipitation assay buffer was increased by 450-fold compared with trichloroacetic acid for lysis, presumably because of activation of PARPs by DNA damage that occurred during cell lysis. This ELISA can be used to analyze the biological functions of polyADP-ribosylation under various physiological conditions in cultured cells.

Poly(ADP-ribosyl)ation of FOXP3 Protein Mediated by PARP-1 Protein Regulates the Function of Regulatory T Cells.

Poly(ADP-ribose) polymerase 1 (PARP-1) is an ADP-ribosylating enzyme participating in diverse cellular functions. The roles of PARP-1 in the immune system, however, have not been well understood. Here we find that PARP-1 interacts with FOXP3 and induces its poly(ADP-ribosyl)ation. By using PARP-1 inhibitors, we show that reduced poly(ADP-ribosyl)ation of FOXP3 results in not only FOXP3 stabilization and increased FOXP3 downstream genes but also enhanced suppressive function of regulatory T cells. Our results suggest that PARP-1 negatively regulates the suppressive function of Treg cells at the posttranslational level via FOXP3 poly(ADP-ribosyl)ation. This finding has implications for developing PARP-1 inhibitors as potential agents for the prevention and treatment of autoimmune diseases.

Time profile of oxidative stress and neutrophil activation in ovine acute lung injury and sepsis.

The formation of oxidative stress in the lung and activation of neutrophils are major determinants in the development of respiratory failure after acute lung injury and sepsis. However, the time changes of these pathogenic factors have not been sufficiently described. Twenty-four chronically instrumented sheep were subjected to cotton smoke inhalation injury and instillation of live Pseudomonas aeruginosa into both lungs. The sheep were euthanized at 4, 8, 12, 18, and 24 h after injury. Additional sheep received sham injury and were euthanized after 24 h. Pulmonary function was assessed by determination of oxygenation index and pulmonary shunt fraction. In addition, lung tissue was harvested at the respective time points for the measurement of malondialdehyde, interleukin 6, poly(ADP ribose), myeloperoxidase, and alveolar polymorphonuclear neutrophil score. The injury induced severe respiratory failure that was associated with an early increase in lipid peroxidation and interleukin 6 expression. The injury further led to an increase in poly(ADP ribose) activity that reached its peak at 12 h after injury and declined afterward. In addition, progressive increases in markers of neutrophil accumulation in the lung were observed. The peak of neutrophil accumulation in the lung was associated with a severe depletion of circulating neutrophils. The results from our model may enhance the understanding of the pathophysiological alterations after acute lung injury and sepsis and thus be useful in exploring therapeutic interventions directed at modifying the expression or activation of inflammatory mediators.

Could serum antibody to poly(ADP-ribose) and/or histone H1 be marker for senile dementia of Alzheimer type?

Poly(ADP-ribosyl)ation has been focused on ischemic injury in the brain in relation to Alzheimer's disease (AD). We have measured IgG antibodies against poly adenosine diphosphate-ribose (pADPR) as well as histone H1 (H1) in 26 patients with either AD or with senile dementia of Alzheimer type (SDAT), and found that 80.7% (21/26) were positive for anti-pADPR IgG antibodies. Anti-H1 IgG antibodies were less positive (57.6%) (15/26) than anti-pADPR IgG antibodies, however, titers of both antibodies were well correlated (r = 0.768). Meanwhile, similar studies on 32 patients with systemic lupus erythematosus (SLE) who were positive for anti-pADPR antibody showed poor correlation (r = 0.184) and the difference in the correlation was statistically significant (r < 0.01). It is worthy of remark that anti-double-stranded (ds) DNA antibody, which is the hallmark of SLE, was negative in all dementia patients. Together with the findings that major subclass in dementia is both IgG1 and IgG2 and that in SLE was IgG2, the mode of production of anti-pADPR antibody in AD and SDAT is under different regulation mechanisms from that in SLE. Given the evidence that major target for ADP-ribosylation is H1 molecule, the association between anti-pADPR and anti-H1 in AD/SDAT makes sense and supports the concept that modification of proteins renders them immunogenic. Whatever the regulation is, parallel assay of two antibodies above would be of use not only for monitoring the disease process but also as a prodrome for possible subsets of SDAT and AD.

Essential role for poly (ADP-ribosyl)ation in mouse preimplantation development.

BACKGROUND: Poly (ADP-ribosyl)ation is a covalent modification of many nuclear proteins. It has a strong chromatin modifying potential involved in DNA repair, transcription and replication. Its role during preimplantation development is unknown. RESULTS: We have observed strong but transient synthesis of poly ADP-ribose polymers on decondensing chromosomes of fertilized and parthenogenetically activated mouse oocytes. Inhibition of this transient upregulation with a specific enzyme inhibitor, 3-aminobenzamide, has long-term effects on the postimplantation development of the embryos. In addition, inhibition of poly (ADP-ribosyl)ation at the 4-8 cell stage selectively blocks morula compaction. CONCLUSION: These observations suggest that poly (ADP-ribosyl)ation is involved in the epigenetic chromatin remodeling in the zygote.

Immunological determination and size characterization of poly(ADP-ribose) synthesized in vitro and in vivo.

Poly(ADP-ribose) polymerase is a DNA break detecting enzyme playing a role in the surveillance of genome integrity. Poly(ADP-ribose) is synthesized rapidly and transiently from beta-NAD in response to DNA damaging agents. In order to study the physiological significance of poly(ADP-ribose) metabolism, we have developed immunological methods which enable us to study endogenous poly(ADP-ribose) without interfering with cell metabolism and integrity. For this purpose, we produced a highly specific polyclonal anti-poly(ADP-ribose) antibody which immunoreacts with polymers and oligomers. In addition to the immunodot blot method recently described by us (Affar et al., Anal. Biochem. 259 (1998) 280-283), other applications were investigated in cells: (i) detection of poly(ADP-ribose) by ELISA; (ii) characterization of poly(ADP-ribose) size using high resolution gel electrophoresis of polymers, followed by its transfer onto a positively charged membrane and detection with anti-poly(ADP-ribose) antibody; (iii) immunocytochemistry and flow cytometry analyses allowing poly(ADP-ribose) study at the level of individual cells.

Autoantibody to thioredoxin reductase in an ovarian cancer patient.

In a patient with ovarian cancer, anti-nuclear antibodies were found by indirect immunofluorescence analysis in cultured cells. The patient's serum was used for immunoscreening of a cDNA library. Three overlapping positive clones were partial cDNA clones of thioredoxin reductase (TR), which is a member of the pyridine nucleotide-disulfide oxidoreductase family of flavoenzymes. Human autoantibodies affinity-purified with bacterial recombinant TR reacted to approximately 60-kDa polypeptide in HeLa extract in immunoblotting analysis and strongly stained around chromosome of metaphase of HeLa cells in immunofluorescence studies. Autoantibodies against recombinant TR were not found in our 100 patients with systemic autoimmune diseases. Immunoblotting analysis with bovine purified TR confirmed that the human serum had reactivities to TR. This is the first report of autoantibodies against a redox member in a cancer patient. TR has diverse functions in cell growth, death, and transformation through redox modulation with thioredoxin. Since dysregulation of TR in cancer cells is considered to be involved in some redox states of tumorigenesis, the observed antibody response in this patient could have arisen due to immunoreaction to the abnormally regulated protein.

Autoantibodies reacting with poly(ADP-ribose) and with a zinc-finger functional domain of poly(ADP-ribose) polymerase involved in the recognition of damaged DNA.

Poly(ADP-Ribose) polymerase (PARP) is a chromatin-associated enzyme that specifically binds to DNA strand breaks in a zinc-dependent manner. We describe here the presence of IgG antibodies reacting with recombinant human PARP in the serum of patients with systemic lupus erythematosus (SLE) and primary and secondary Sjögren's syndrome (pSS and sSS). The reactivity of patients' sera was further studied in ELISA with a synthetic peptide of 44 residues corresponding to the second zinc finger (F2) present in the DNA-binding domain of PARP and which was shown to effectively bind 65Zn. Thirty-five percent of SLE sera (n = 97), 42% of pSS sera (n = 67), and 56% of sSS sera (n = 16) were found to contain raised levels of IgG antibodies reacting with peptide F2 which corresponds to the domain in PARP that is directly involved in the specific recognition of single and double strand breaks in DNA. Antibodies reacting with the whole enzyme and/or peptide F2 occurred independently from antibodies reacting with poly(ADP-ribose) which is rapidly synthesized in vivo by PARP from NAD and then degraded in response to DNA strand breaks.

Differences in the poly(ADP-ribosyl)ation patterns of ICP4, the herpes simplex virus major regulatory protein, in infected cells and in isolated nuclei.

Infected-cell protein 4 (ICP4), the major regulatory protein in herpes simplex viruses 1 and 2, was previously reported to accept 32P from [32P]NAD in isolated nuclei. This modification was attributed to poly(ADP-ribosyl)ation (C. M. Preston and E. L. Notarianni, Virology 131:492-501, 1983). We determined that an antibody specific for poly(ADP-ribose) reacts with ICP4 extracted from infected cells, electrophoretically separated in denaturing gels, and electrically transferred to nitrocellulose. Our results indicate that all forms of ICP4 observed in one-dimensional gel electrophoresis are poly(ADP-ribosyl)ated. Poly(ADP-ribose) on ICP4 extracted from infected cells was resistant to cleavage by purified poly(ADP-ribose) glycohydrolase unless ICP4 was in a denatured state. Poly(ADP-ribose) added to ICP4 in isolated nuclei was sensitive to this enzyme. This result indicates that the two processes are distinct and may involve different sites on the ICP4 molecule.

[Antibodies to poly (adenosine diphosphate-ribose) in systemic lupus erythematosus and drug induced lupus].

The difference between anti poly (ADP-ribose) antibodies was studied in patients with systemic lupus erythematosus (SLE), progressive systemic sclerosis (PSS) and drug-induced lupus (DIL). Radioimmunoassay showed that high concentrations of anti poly (ADP-ribose) antibodies (10.3-22.2%) were induced by phenobarbital, phenytoin, valproic acid (anti-epileptic agent) and procainamide (anti-arrhythmic agent). Poly (ADP-ribose) antibodies were separated by hydroxylapatite column chromatography. The average chain length of the polymer consisted of 2.4, 10.8 and 28.2 ADP-ribose units. Anti poly (ADP-ribose) antibodies from patients with SLE and PSS reacted with 2.4, 10.8, and 28.2 ADP-ribose units in RIA, but those from patients with DIL reacted only with 28.2 ADP-ribose units in RIA. The binding specificity of anti poly (ADP-ribose) antibodies from pregnant women was found to be very similar to that of the antibodies from case of DIL. The present results clearly demonstrated that anti poly (ADP-ribose) antibodies found in systemic autoimmune diseases bound not only to poly (ADP-ribose) with an average chain length of more than 20 ADP-ribose units, but also to oligo (ADP-ribose) with an average chain length of about 2 ADP-ribose units. Anti poly (ADP-ribose) antibodies found in DIL cases and pregnant women, however, bound only to poly (ADP-ribose) with an average chain length of more than 20 ADP-ribose units.

Disease specificity of antibodies to poly (ADP-ribose); their relationships to anti-DNA antibodies and to disease activity in lupus.

In this study we have measured the level of anti poly (ADP-ribose) antibodies in the sera of a number of patients with SLE and their relatives, patients with a wide variety of other autoimmune and infectious diseases, and a group of normal healthy controls. It was found that these antibodies were not disease specific but were present in nine out of thirteen groups tested in significant numbers. The levels of anti poly (ADP-ribose) antibodies and anti DNA antibodies in SLE patients bled serially were also measured. The level of these antibodies fluctuated in parallel in many of these patients, although the anti poly (ADP-ribose) antibodies reflected disease activity more accurately in some.

Antibody to poly(ADP-ribose) is an indicator of obstetric complications in pregnant patients with systemic lupus erythematosus.

Anti-poly(ADP-ribose) antibodies were measured by an enzyme-linked immunosorbent assay (ELISA) in 6 pregnant women with systemic lupus erythematosus (SLE), 11 normal pregnant women and 6 randomly selected female SLE patients. Four pregnant SLE patients who had either an abortion after 13 weeks of gestation or a premature delivery showed very high titers of anti-poly(ADP-ribose) antibodies in week 8 of pregnancy. However, the titers of anti-poly(ADP-ribose) antibodies of all the normal pregnant women were similar to those of non-pregnant female SLE patients, being slightly higher than those of normal non-pregnant women. The isotype of anti-poly(ADP-ribose) antibodies in pregnant SLE patients was IgG, which did not crossreact with either DNA or cardiolipin. The number of pregnant SLE patients tested was small, but the coincidence of abortion with high titers of anti-poly (ADP-ribose) antibody was very close. Therefore, a high titer of anti-poly(ADP-ribose) antibody in pregnant SLE patients seems useful as an indicator of abortion or fetal distress.

A dot-blot method for screening polyclonal and monoclonal antisera to poly(ADP-ribose).

ADP-ribosylation reactions play a key role at several points in cellular regulation and repair of DNA damage. The use of polyclonal or monoclonal antisera to poly(ADP-ribose) as probes to localize the site(s) of action of the polymer offers a promising tool for these studies. We report here a simple, sensitive method for detection and titration of these antisera to poly(ADP-ribose) using nitrocellulose membrane (NC) as a support for a dot-blot analysis. We take advantage of the fact that a highly labeled poly(ADP-ribose) preparation can be obtained by incubation of a 0.3 M KCl extract prepared from calf thymus nuclei with 32P-NAD. Such a preparation of labeled antigen is used as a reagent to detect the positive antibody spots on the NC with negligible background. Subsequent titration of the antisera and their semi-quantitative evaluation are also feasible using the dot-blot method. The sensitivity of the assay is only limited by the specific activity that can be achieved for the labeled polymer prepared as the antigen probe. The advantage of this method is that it eliminates the need to prepare pure, highly radiolabeled polymer as well as the fact that several samples can be handled on the membrane simultaneously. We demonstrate application of this technique for screening sera from patients with systemic lupus erythematosus (SLE) for anti-poly(ADP-ribose) antibodies. Further, we also extend the use of these sera for immunoquantitation of ADP-ribosylated proteins in six human tumor cells in tissue culture.