Poly(ADP-ribose) polymerase-1 (PARP-1) controls lung cell proliferation and repair after hyperoxia-induced lung damage

Oxygen-based therapies expose lung to elevated levels of ROS and induce lung cell damage and inflammation. Injured cells are replaced through increased proliferation and differentiation of epithelial cells and fibroblasts. Failure to modulate these processes leads to excessive cell proliferation, collagen deposition, fibrosis, and chronic lung disease. Poly(ADP-ribose) polymerase-1 (PARP-1) is activated in response to DNA damage and participates in DNA repair, genomic integrity, and cell death. In this study, we evaluated the role of PARP-1 in lung repair during recovery after acute hyperoxia exposure. We exposed PARP-1 -/- and wild-type mice for 64 h to 100% hyperoxia and let them recover in air for 5-21 days. PARP-1-deficient mice exhibited significantly higher lung cell hyperplasia and proliferation than PARP-1 +/+ animals after 5 and 10 days of recovery. This was accompanied by an increased inflammatory response in PARP-1 -/- compared with wild-type animals, characterized by neutrophil infiltration and increased IL-6 levels in bronchoalveolar lavages. These lesions were reversible, since the extent of the hyperplastic regions was reduced after 21 days of recovery and did not result in fibrosis. In vitro, lung primary fibroblasts derived from PARP-1 -/- mice showed a higher proliferative response than PARP-1 +/+ cells during air recovery after hyperoxia-induced growth arrest. Altogether, these results reveal an essential role of PARP-1 in the control of cell repair and tissue remodeling after hyperoxia-induced lung injury.     

A Stronger DNA Damage-Induced G2 Checkpoint Due to Over-Activated CHK1 in the Absence of PARP-1

Poly(ADP-ribose) polymerase-1 (PARP-1) is involved in multi-pathways to respond to DNA damage. Lack of or inhibition of PARP-1 activity leads to slow progress of cell cycle and sensitization of cells to different stresses. Recently, it was reported that besides the Ku- dependent main non-homologous end joining (NHEJ) pathway, there is a PARP-1-dependent complementary NHEJ pathway to repair DNA double strand break (DSB). Here we show that compared with PARP-1+/+ cells, PARP-1-/- cells display a much stronger G2 checkpoint response following ionizing radiation (IR). Treatment with Chk1 siRNA abolishes the stronger G2 checkpoint response and sensitizes PARP-1-/- cells to IR. These data indicate that the stronger G2 checkpoint response in PARP-1-/- cells is CHK1-dependent, which protects cells from IR-induced killing. We also show that 4-Amino-1,8-naphthalimide (4-AN, inhibitor of PARP) but not methoxyamine (inhibitor of base excision repair (BER)), affects IR-induced G2 arrest and cell sensitivity in PARP-1+/+ cells, resulting in the phenotypes similar to those of PARP-1-/- cells. These results indicate that DSB repair from the complementary NHEJ pathway of PARP-1, but not single strand break (SSB) repair from the BER function of PARP-1, may play an essential role in the over-activated CHK1 regulated G2 checkpoint response and radiosensitivity in PARP-1-/- cells.     

Defective control of mitotic and post-mitotic checkpoints in poly(ADP-ribose) polymerase-1(-/-) fibroblasts after mitotic spindle disruption

Poly(ADP-ribose) polymerase-1 (PARP), a DNA damage-responsive nuclear enzyme present in higher eukaryotes, is well-known for its roles in protecting the genome after DNA damage. However, even without exogenous DNA damage, PARP may play a role in stabilizing the genome because cells or mice deficient in PARP exhibit various signs of genomic instability, such as tetraploidy, aneuploidy, chromosomal abnormalities and susceptibility to spontaneous carcinogenesis. Normally, cell cycle checkpoints ensure elimination of cells with genomic abnormalities. Therefore, we examined efficiency of mitotic and post-mitotic checkpoints in PARP-/- and PARP+/+ mouse embryonic fibroblasts treated with mitotic spindle disrupting agent colcemid. PARP+/+ cells, like most mammalian cells, eventually escaped from spindle disruption-induced mitotic checkpoint arrest by 60 h. In contrast, PARP-/- cells rapidly escaped from mitotic arrest within 24 h by downregulation of cyclin B1/CDK-1 kinase activity. After escaping from mitotic arrest; both the PARP genotypes arrive in G1 tetraploid state, where they face post-mitotic checkpoints which either induce apoptosis or prevent DNA endoreduplication. While all the G1 tetraploid PARP+/+ cells were eliminated by apoptosis, the majority of the G1 tetraploid PARP-/- cells became polyploid by resisting apoptosis and carrying out DNA endoreduplication. Introduction of PARP in PARP-/- fibroblasts partially increased the stringency of mitotic checkpoint arrest and fully restored susceptibility to G1 tetraploidy checkpoint-induced apoptosis; and thus prevented formation of polyploid cells. Our results suggest that PARP may serve as a guardian angel of the genome even without exogenous DNA damage through its role in mitotic and post-mitotic G1 tetraploidy checkpoints.     

Poly(ADP-ribose) polymerase-1 inhibition prevents eosinophil recruitment by modulating Th2 cytokines in a murine model of allergic airway inflammation: a potential specific effect on IL-5

We recently used a murine model of allergic airway inflammation to show that poly(ADP-ribose) polymerase-1 (PARP-1) plays an important role in the pathogenesis of asthma-related lung inflammation. In this study, we show that PARP-1 inhibition, by a novel inhibitor (TIQ-A) or by gene deletion, prevented eosinophilic infiltration into the airways of OVA-challenged mice. Such impairment of eosinophil recruitment appeared to take place after IgE production. OVA challenge of wild-type mice resulted in a significant increase in IL-4, IL-5, IL-10, IL-13, and GM-CSF secretions. Although IL-4 production was moderately affected in OVA-challenged PARP-1(-/-) mice, the production of IL-5, IL-10, IL-13, and GM-CSF was completely inhibited in ex vivo OVA-challenged lung cells derived from these animals. A single TIQ-A injection before OVA challenge in wild-type mice mimicked the latter effects. The marked effect PARP-1 inhibition exerted on mucus production corroborated the effects observed on the Th2 response. Although PARP-1 inhibition by gene knockout increased the production of the Th1 cytokines IL-2 and IL-12, the inhibition by TIQ-A exerted no effect on these two cytokines. The failure of lung cells derived from OVA-challenged PARP-1(-/-) mice to synthesize GM-CSF, a key cytokine in eosinophil recruitment, was reestablished by replenishment of IL-5. Furthermore, intranasal administration of IL-5 restored the impairment of eosinophil recruitment and mucus production in OVA-challenged PARP-1(-/-) mice. The replenishment of either IL-4 or IgE, however, did not result in such phenotype reversals. Altogether, these results suggest that PARP-1 plays a critical role in eosinophil recruitment by specifically regulating the cascade leading to IL-5 production.     

PARP-1-dependent 3-nitrotyrosine protein modification after DNA damage

3-nitrotyrosine (NO2-Tyr) is thought to be a specific marker of cell injury during oxidative damage. We have evaluated the role of poly(ADP-ribose)polymerase-1 (PARP-1) in protein nitration after treatment of immortalized fibroblasts parp-1+/+ and parp-1-/- with the alkylating agent 2'-methyl-2'-nitroso-urea (MNU). Both cell lines showed increased iNOS expression following MNU treatment in parallel with a selective induction of tyrosine nitration of different proteins. PARP-1 deficient cells displayed a delayed iNOS accumulation, reduced number of nitrated proteins, and a lower global nitrotyrosine "footprint." We have identified the mitochondrial compartment as the major site of oxidative stress during DNA damage, being MnSOD one of the NO2-Tyr-modified proteins, but not in parp-1-/- cells. These results suggest that NO-derived injury can be modulated by proteins involved in the response to genotoxic damage, such as PARP-1, and may account for the limited oxidative injury in parp-1 knockout mice during carcinogenesis and inflammation.     

Simian virus 40-transformed metallothionein null cells showed increased sensitivity to cadmium but not to zinc, copper, mercury or nickel

Primary cultured embryonic cells derived from mice with disrupted metallothionein (MT) I and II genes and from control mice were transformed with a plasmid encoding the simian virus 40 (SV40) large T antigen. The resulting MT-/- and MT+/+ cell strains showed similar cell morphology, cell cycle and no significant differences in glutathione levels or in the activities of glutathione-related enzymes and antioxidant enzymes. The MT-/- cells were more sensitive to Cd than MT+/+ cells, though no increase in the sensitivity to Zn, Cu, Hg or Ni were observed in MT-/- cells. MT+/+ cells accumulated more Cd than MT-/- cells but showed less lesion, suggesting the role of MT induced by Cd in MT+/+ cells as a scavenger of toxic Cd ion. These results suggest a dominant protective role of MT against Cd compared with other metals. SV40-transformed MT-/- cells seem to be a useful tool for the investigation of cellular function of MT.     

Heterozygosity of p21WAF1/CIP1 enhances tumor cell proliferation and cyclin D1-associated kinase activity in a murine mammary cancer model.

The p21(WAF1/cIP1) cyclin-dependent kinase (cdk) inhibitor is a regulator of the G(1)-S cell cycle checkpoint. Despite the importance of p21 in cell cycle inhibition, its role as a tumor suppressor is uncertain. p21 mutations are infrequent in human tumors, and p21 null mice exhibit no increased tumor incidence. To ascertain whether p21 could influence tumor formation or progression in the context of other oncogenic stimuli, we crossed p21-deficient mice with mammary tumor susceptible Wnt-1 transgenic mice. The p21+/+, p21+/-, and p21-/- Wnt-1 transgenic female offspring were monitored for mammary tumor incidence and growth rates. p21 status had no effect on the age at which mammary tumors formed. However, p21+/- mammary tumors grew significantly faster than p21+/+ and p21-/- mammary tumors. The increased growth rates were confirmed by mitotic index counts and by BrdUrd labelling assays, indicating that a significantly higher percentage of p21+/- tumor cells were in S phase and mitosis than their p21+/+ and p21-/- counterparts. Moreover, cyclin D1-associated phosphorylation of retinoblastoma protein was significantly increased in p21+/- tumor lysates compared with p21+/+ and p21-/- lysates. These results are consistent with data indicating that reduced levels of p21 can facilitate cyclin/cdk complex formation while enhancing cdk activity. Thus, a reduction of p21 dosage may promote tumor progression in the presence of other oncogenic initiators. The dependence of p21 on prior oncogenic stimuli for its tumor-promoting activities suggests that it may behave as a tumor modifier gene rather than as a tumor suppressor gene.     

The inhibition and treatment of breast cancer with poly (ADP-ribose) polymerase (PARP-1) inhibitors

BRCA1 and BRCA2 mutations are responsible for most familial breast carcinomas. Recent reports carried out in non-cancerous mouse BRCA1- or BRCA2-deficient embryonic stem (ES) cells, and hamster BRCA2-deficient cells have demonstrated that the targeted inhibition of poly(ADP-ribose) polymerase (PARP-1) kills BRCA mutant cells with high specificity. Although these studies bring hope for BRCA mutation carriers, the effectiveness of PARP-1 inhibitors for breast cancer remains elusive. Here we present the first in vivo demonstration of PARP-1 activity in BRCA1-deficient mammary tumors and describe the effects of PARP-1 inhibitors (AG14361, NU1025, and 3-aminobenzamide) on BRCA1-deficient ES cells, mouse and human breast cancer cells. AG14361 was highly selective for BRCA1-/- ES cells; however, NU1025 and 3-aminobenzamide were relatively non-selective. In allografts of na?ve ES BRCA1-/- cells there was either partial or complete remission of tumors. However, in allografts of mouse, BRCA1-/- mammary tumors, there was no tumor regression or remission although a partial inhibition of tumor growth was observed in both the BRCA1-/- and BRCA1+/+ allografts. In human tumor cells, PARP-1 inhibitors showed no difference in vitro in limiting the growth of mammary tumors irrespective of their BRCA1 status. These results suggest that PARP-1 inhibitors may non-specifically inhibit the growth of mammary tumors.     

The magnitude of methylmercury-induced cytotoxicity and cell cycle arrest is p53-dependent

BACKGROUND: Methylmercury (MeHg), a ubiquitous environmental contaminant, is a known potent teratogen selectively affecting the developing central nervous system. While a definitive mechanism for MeHg-induced developmental neurotoxicity remains elusive, in utero exposure has been associated with reduced brain weight and reduction in cell number. This suggests early toxicant interference with critical molecular signaling events controlling cell behavior, i.e., proliferation. METHODS: To examine the role of p53, a major regulator of the G(1)/S and G(2)/M cell cycle checkpoints, in MeHg toxicity, we isolated GD 14 primary embryonal fibroblasts from homozygous wild-type p53 (p53+/+) and homozygous null p53 (p53-/-) mice. Cells were treated at passages 4-7 for 24 or 48 hr with 0, 1.0, or 2.5 microM MeHg and analyzed for effects on viability, cell cycle progression (using BrdU-Hoechst flow cytometric analysis), and apoptosis via annexin V-FITC and propidium iodide (PI) staining. RESULTS: The p53+/+ cells are more sensitive than p53-/- cells to MeHg-induced cytotoxicity, cell cycle inhibition, and induction of apoptosis: at 24 hr, 2.5 microM MeHg reduced p53+/+ cell viability to 72.6% +/- 3.2%, while p53-/- viability was 94.6% +/- 0.4%. The p53-/- cells underwent less necrosis and less apoptosis following MeHg treatment. MeHg (2.5 microM) also halted all cycling in the p53+/+ cells, while 42.6% +/- 7.2% of p53-/- cells were able to reach a new G(0)/G(1) in 48 hr. Time- and dose-dependent accumulation of cells in G(2)/M phase (1.0 and 2.5 microM MeHg) was observed independent of the p53 genotype; however, the magnitude of change was p53-dependent. CONCLUSIONS: These studies suggest that MeHg-induced cell cycle arrest occurs via both p53-dependent and -independent pathways in our model system; however, cell death resulting from MeHg exposure is highly dependent on p53.     

CD1-dependent regulation of chronic central nervous system inflammation in experimental autoimmune encephalomyelitis

The existence of T cells restricted for the MHC I-like molecule CD1 is well established, but the function of these cells is still obscure; one implication is that CD1-dependent T cells regulate autoimmunity. In this study, we investigate their role in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, using CD1-deficient mice on a C57BL/6 background. We show that CD1-/- mice develop a clinically more severe and chronic EAE compared with CD1+/+ C57BL/6 mice, which was histopathologically confirmed with increased demyelination and CNS infiltration in CD1-/- mice. Autoantigen rechallenge in vitro revealed similar T cell proliferation in CD+/+ and CD1-/- mice but an amplified cytokine response in CD1-/- mice as measured by both the Th1 cytokine IFN-gamma and the Th2 cytokine IL-4. Investigation of cytokine production at the site of inflammation showed a CNS influx of TGF-beta1-producing cells early in the disease in CD1+/+ mice, which was absent in the CD1-/- mice. Passive transfer of EAE using an autoreactive T cell line induced equivalent disease in both groups, which suggested additional requirements for activation of the CD1-dependent regulatory pathway(s). When immunized with CFA before T cell transfer, the CD1-/- mice again developed an augmented EAE compared with CD1+/+ mice. We suggest that CD1 exerts its function during CFA-mediated activation, regulating development of EAE both through enhancing TGF-beta1 production and through limiting autoreactive T cell activation, but not necessarily via effects on the Th1/Th2 balance.     

Poly(ADP-ribose) polymerase triggers the microvascular mechanisms of hepatic ischemia-reperfusion injury

Activation of poly(ADP-ribose) polymerase (PARP) mediates oxidative stress-induced cell injury. We tested the hypothesis that PARP contributes to ischemia-reperfusion (I/R) damage of the liver by triggering the mechanisms of microcirculatory failure. Leukocyte- and platelet-endothelial cell interactions as well as sinusoidal perfusion were analyzed by intravital fluorescence microscopy after lobar hepatic I/R (90 min/30 min) in C57BL/6 x 129/Sv wild-type (PARP+/+) and PARP-deficient (PARP-/-) mice. Hepatic I/R induced leukocyte/platelet-endothelial cell interactions and tissue injury in PARP+/+ mice, as indicated by impaired sinusoidal perfusion and increased alanine aminotransferase (ALT)/aspartate aminotransferase (AST) serum activities. In PARP-/- mice, however, the postischemic increase in the numbers of rolling/adherent leukocytes and platelets was significantly lower. In addition, I/R-induced translocation of CD62P as well as mRNA expression of CD62E, CD54, and CD106 were attenuated. The degree of perfusion failure was reduced and the increase in the ALT/AST activities was lower in PARP-/- mice compared with PARP+/+ mice. We conclude that PARP contributes to hepatic microvascular injury by triggering the expression/translocation of adhesion molecules and modulating leukocyte/platelet-endothelial cell interactions.     

Alveolar epithelial cell inhibition of fibroblast proliferation is regulated by MCP-1/CCR2 and mediated by PGE2

CC chemokine receptor 2 (CCR2) -/- mice are protected from experimental pulmonary fibrosis, a disease increasingly recognized as being mediated by dysfunctional interactions between epithelial cells and fibroblasts. We have sought to investigate the interactions between alveolar epithelial cells (AECs) and fibroblasts in these fibrosis-resistant (CCR2 -/-) and fibrosis-sensitive (CCR2 +/+) mice. AECs from CCR2 -/- mice suppress fibroblast proliferation more than AECs from CCR2 +/+ mice (77 vs. 43%). Exogenous administration of the CCR2 ligand monocyte chemoattractant protein-1 (MCP-1) to the fibroblast-AEC cocultures reverses the suppression mediated by CCR2 +/+ AECs but has no effect with CCR2 -/- AECs. MCP-1 regulates AEC function but not fibroblast function. AEC inhibition of fibroblast proliferation was mediated by a soluble, aspirin-sensitive factor. Accordingly, AECs from CCR2 -/- mice produce greater quantities of PGE(2) than do AECs from CCR2 +/+ mice, and MCP-1 inhibits AEC-derived PGE(2) synthesis. Diminished PGE(2) production by AECs results in enhanced fibroproliferation. Thus an important profibrotic mechanism of MCP-1/CCR2 interactions is to limit PGE(2) production in AECs after injury, thus promoting fibrogenesis.     

Role of ICAM-1 (CD54) in the development of murine cerebral malaria

In susceptible mouse strains, infection of mice with Plasmodium berghei ANKA (PbA) results in a lethal complication, cerebral malaria. Cerebral malaria is due to the immune response induced by the parasite, which results in an increased production of TNF, known to increase the expression of adhesion molecules on the endothelia. To investigate the role of the adhesion molecule ICAM-1 (CD54), we infected wild-type (+/+) and ICAM-1-deficient (-/-) mice with PbA. While +/+ mice died 6-8 days after infection, -/- mice survived > 15 days. Parasitaemia was similar in +/+ and -/- mice. Serum TNF concentration was increased by the infection and was significantly higher in infected +/+ than in -/- mice. However, TNF mRNA levels in spleen, lungs, and brain were elevated in both infected +/+ and -/- mice. For IFN-gamma, serum levels were similar in both groups. A breakdown of the blood-brain barrier was evident in infected +/+ mice only. Interestingly, thrombocytopenia was profound in infected +/+, but practically absent in -/- mice. Moreover, macrophage sequestration was evident in brain venules and lung capillaries of +/+ mice and was significantly less important in the alveolar capillaries of infected -/- mice. In contrast, neutrophil sequestration in the lung was similar in both +/+ and -/- mice. Sequestration of parasitized red blood cells was significantly greater in the alveolar capillaries from +/+ than -/- mice. These results indicate that while the immune response is similar in both +/+ and ICAM-1(-/-) mice, the absence of mortality in ICAM(-/-) mice correlates with a decrease of macrophage and parasitized RBC trapping and a less severe thrombocytopenia.     

HMGN1 Protein Regulates Poly(ADP-ribose) Polymerase-1 (PARP-1) Self-PARylation in Mouse Fibroblasts

In mammalian cells, the nucleosome-binding protein HMGN1 (high mobility group N1) affects the structure and function of chromatin and plays a role in repair of damaged DNA. HMGN1 affects the interaction of DNA repair factors with chromatin and their access to damaged DNA; however, not all of the repair factors affected have been identified. Here, we report that HMGN1 affects the self-poly(ADP-ribosyl)ation (i.e., PARylation) of poly(ADP-ribose) polymerase-1 (PARP-1), a multifunctional and abundant nuclear enzyme known to recognize DNA lesions and promote chromatin remodeling, DNA repair, and other nucleic acid transactions. The catalytic activity of PARP-1 is activated by DNA with a strand break, and this results in self-PARylation and PARylation of other chromatin proteins. Using cells obtained from Hmgn1(-/-) and Hmgn1(+/+) littermate mice, we find that in untreated cells, loss of HMGN1 protein reduces PARP-1 self-PARylation. A similar result was obtained after MMS treatment of these cells. In imaging experiments after low energy laser-induced DNA damage, less PARylation at lesion sites was observed in Hmgn1(-/-) than in Hmgn1(+/+) cells. The HMGN1 regulation of PARP-1 activity could be mediated by direct protein-protein interaction as HMGN1 and PARP-1 were found to interact in binding assays. Purified HMGN1 was able to stimulate self-PARylation of purified PARP-1, and in experiments with cell extracts, self-PARylation was greater in Hmgn1(+/+) than in Hmgn1(-/-) extract. The results suggest a regulatory role for HMGN1 in PARP-1 activation.