Poly(ADP-ribose) polymerase inhibition enhances p53-dependent and -independent DNA damage responses induced by DNA damaging agent

Targeting DNA repair with poly(ADP-ribose) polymerase (PARP) inhibitors has shown a broad range of anti-tumor activity in patients with advanced malignancies with and without BRCA deficiency. It remains unclear what role p53 plays in response to PARP inhibition in BRCA-proficient cancer cells treated with DNA damaging agents. Using gene expression microarray analysis, we find that DNA damage response (DDR) pathways elicited by veliparib (ABT-888), a PARP inhibitor, plus topotecan comprise the G1/S checkpoint, ATM, and p53 signaling pathways in p53-wildtype cancer cell lines and BRCA1, BRCA2 and ATR pathway in p53-mutant lines. In contrast, topotecan alone induces the G1/S checkpoint pathway in p53-wildtype lines and not in p53-mutant cells. These responses are coupled with G2/G1 checkpoint effectors p21CDKN1A upregulation, and Chk1 and Chk2 activation. The drug combination enhances G2 cell cycle arrest, apoptosis and a marked increase in cell death relative to topotecan alone in p53-wildtype and p53-mutant or -null cells. We also show that the checkpoint kinase inhibitor UCN-01 abolishes the G2 arrest induced by the veliparib and topotecan combination and further increases cell death in both p53-wildtype and -mutant cells. Collectively, PARP inhibition by veliparib enhances DDR and cell death in BRCA-proficient cancer cells in a p53-dependent and -independent fashion. Abrogating the cell-cycle arrest induced by PARP inhibition plus chemotherapeutics may be a strategy in the treatment of BRCA-proficient cancer.     

Chemotherapy-induced p53-dependent and -independent DNA damage responses are enhanced by poly(ADP-ribose) polymerase (PARP) inhibition in BRCA-proficient cancer cells

Comment on: Nguyen D, et al. Cell Cycle 2011; 10:4074-82.     

DNA damage induces MDMX nuclear translocation by p53-dependent and -independent mechanisms

The MDM2 homolog MDMX is an important regulator of p53 activity during embryonic development. MDMX inactivation in mice results in embryonic lethality in a p53-dependent fashion. The expression level of MDMX is not induced by DNA damage, and its role in stress response is unclear. We show here that ectopically expressed MDMX is mainly localized in the cytoplasm. DNA damage promotes nuclear translocation of MDMX in cells with or without p53. Coexpression of MDM2 or p53 is sufficient to induce MDMX nuclear translocation, suggesting that activation of p53 and induction of MDM2 expression can contribute to this process. Stable transfection of MDMX into U2OS cells does not alter p53 level but results in reduced p53 DNA-binding activity and reduced MDM2 expression. The ability of ARF (alternate reading frame of INK4a) to activate p53 is also significantly inhibited by expression of MDMX. These results suggest that MDMX function may be regulated by DNA damage. Furthermore, MDMX may complement MDM2 in regulating p53 during embryonic development due to its ability to inhibit p53 in the presence of ARF.     

Poly(ADP-ribose) polymerase forms loops with DNA

The interaction between highly purified poly(ADP-ribose) polymerase from calf thymus and different topological forms of pBR322 DNA has been studied by gel retardation electrophoresis and electron microscopy. We show that: (i) in the absence of nicks on DNA the enzyme has a marked affinity for supercoiled (form I) DNA, (ii) in the presence of single stranded breaks poly(ADP-ribose) polymerase preferentially binds to form II, (iii) in all cases enzyme molecules are frequently located at DNA intersections, (iv) a cooperative binding of the enzyme on DNA occurs.     

Survival and proliferation of cells expressing caspase-uncleavable Poly(ADP-ribose) polymerase in response to death-inducing DNA damage by an alkylating agent

To determine whether caspase-3-induced cleavage of poly(ADP-ribose) polymerase (PARP), a DNA damage-sensitive enzyme, alters the balance between survival and death of the cells following DNA damage, we created stable cell lines that express either caspase-uncleavable mutant or wild type PARP in the background of PARP (-/-) fibroblasts. The survival and apoptotic responses of these cells were compared after exposure to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a DNA-damaging agent that activates PARP, or to tumor necrosis factor-alpha, which causes apoptosis without initial DNA damage. In response to MNNG, the cells with caspase-uncleavable PARP were very resistant to loss of viability or induction of apoptosis. Most significantly, approximately 25% of these cells survived and retained clonogenicity at a level of DNA damage that eliminated the cells with wild type PARP or PARP (-/-) cells. Expression of caspase-uncleavable PARP could not protect the cells from death induced by tumor necrosis factor, although there was a slower progression of apoptotic events in these cells. Therefore, one of the functions for cleavage of PARP during apoptosis induced by alkylating agents is to prevent survival of the extensively damaged cells.     

Nicotinamide- and caspase-mediated inhibition of poly(ADP-ribose) polymerase are associated with p53-independent cell cycle (G2) arrest and apoptosis

Poly(ADP-ribose) polymerase (PARP), which is activated by DNA strand breaks, is involved in DNA repair and replication but, during apoptosis, undergoes early caspase-mediated cleavage. Activation of programmed cell death in response to DNA damage may rely on functional p53 protein. Tumor cells are commonly deficient in this oncogene product resulting in resistance to many cytostatic drugs. Here we report that nicotinamide-induced inhibition of poly(ADP-ribosyl)ation and cytokine-induced nitric oxide production both result in a transient increase in p53 levels in pancreatic tumor RINm5F cells. These treatments also induce disruption of the mitochondrial membrane potential (_m), as revealed using the mitochondrial probe JC-1, followed by PARP cleavage and apoptosis all of which are inhibited by the anti-apoptotic protein Bcl-2. Moreover, PARP-inhibition by nicotinamide or 3-aminobenzamide induces apoptosis and/or cell cycle arrest at the G2 checkpoint in all of four tested tumor cell lines of both mesenchymal and epithelial origin including mouse NIH-3T3 cells and p53 deficient human HeLa and Jurkat cells. Bcl-2 counteracts cytokine-, but not nicotinamide-induced G2 arrest. These findings indicate that both chemical and caspase-mediated inhibition of PARP activity, possibly by interfering with DNA replication and repair, may promote a p53-independent G2 arrest and apoptosis.     

Comparison of checkpoint responses triggered by DNA polymerase inhibition versus DNA damaging agents

To better understand the different cellular responses to replication fork pausing versus blockage, early DNA damage response markers were compared after treatment of cultured mammalian cells with agents that either inhibit DNA polymerase activity (hydroxyurea (HU) or aphidicolin) or selectively induce S-phase DNA damage responses (the DNA alkylating agents, methyl methanesulfonate (MMS) and adozelesin). These agents were compared for their relative abilities to induce phosphorylation of Chk1, H2AX, and replication protein A (RPA), and intra-nuclear focalization of gamma-H2AX and RPA. Treatment by aphidicolin and HU resulted in phosphorylation of Chk1, while HU, but not aphidicolin, induced focalization of gamma-H2AX and RPA. Surprisingly, pre-treatment with aphidicolin to stop replication fork progression, did not abrogate HU-induced gamma-H2AX and RPA focalization. This suggests that HU may act on the replication fork machinery directly, such that fork progression is not required to trigger these responses. The DNA-damaging fork-blocking agents, adozelesin and MMS, both induced phosphorylation and focalization of H2AX and RPA. Unlike adozelesin and HU, the pattern of MMS-induced RPA focalization did not match the BUdR incorporation pattern and was not blocked by aphidicolin, suggesting that MMS-induced damage is not replication fork-dependent. In support of this, MMS was the only reagent used that did not induce phosphorylation of Chk1. These results indicate that induction of DNA damage checkpoint responses due to adozelesin is both replication fork and fork progression dependent, induction by HU is replication fork dependent but progression independent, while induction by MMS is independent of both replication forks and fork progression.     

Poly(ADP-ribose)polymerase: a perplexing participant in cellular responses to DNA breakage

Poly(ADP-ribose) polymerase is a major nuclear protein of 116 kd, coded by a gene on chromosome 1, that plays a role in cellular responses to DNA breakage. The polymerase binds to DNA at single- and double-strand breaks and synthesizes long branched chains of poly(ADP-ribose), which covalently, but transiently, modifies itself and numerous other cellular proteins and depletes cells of NAD+. This much is known, but the physiological role of the polymerization-degradation cycle is still unclear. Poly(ADP-ribosyl)ation of proteins generally inhibits their function and can dissociated chromatin proteins from DNA. Inhibition of poly(ADP-ribose) polymerase increases to toxicity of alkylating agents and some other DNA-damaging agents and increases sister-chromatid exchange frequencies. During repair of alkylation damage, inhibition of poly(ADP-ribose) polymerase makes no change in excision of damaged products. increases the total number of repair patches, accelerates the rejoining of DNA breaks, and makes variable increases or decreases in net break frequencies. The polymerization cycle consequently is a major player in the response of cells to DNA breakage, but the game it plays is yet to be explained.     

Poly(ADP-ribose) in the cellular response to DNA damage

Poly(ADP-ribose) polymerase is a chromatin-bound enzyme which, on activation by DNA strand breaks, catalyzes the successive transfer of ADP-ribose units from NAD to nuclear proteins. Poly(ADP-ribose) synthesis is stimulated by DNA strand breaks, and the polymer may alter the structure and/or function of chromosomal proteins to facilitate the DNA repair process. Electronmicroscopic studies show that poly(ADP-ribose) unwinds the tightly packed nucleosomal structure of isolated chromatin. Recent studies also show that the presence of poly(ADP-ribose) enhances the activity of DNA ligase. This may increase the capacity of the cell to complete DNA repair. Inhibitors of poly(ADP-ribose) polymerase or deficiencies of the substrate, NAD, lead to retardation of the DNA repair process. When DNA strand breaks are extensive or when breaks fail to be repaired, the stimulus for activation of poly(ADP-ribose) persists and the activated enzyme is capable of totally consuming cellular pools of NAD. Depletion of NAD and consequent lowering of cellular ATP pools, due to activation of poly(ADP-ribose) polymerase, may account for rapid cell death before DNA repair takes place and before the genetic effects of DNA damage become manifest.     

Thrombocytopenia induced by the histone deacetylase inhibitor abexinostat involves p53-dependent and -independent mechanisms

Abexinostat is a pan histone deacetylase inhibitor (HDACi) that demonstrates efficacy in malignancy treatment. Like other HDACi, this drug induces a profound thrombocytopenia whose mechanism is only partially understood. We have analyzed its effect at doses reached in patient plasma on in vitro megakaryopoiesis derived from human CD34⁺ cells. When added at day 0 in culture, abexinostat inhibited CFU-MK growth, megakaryocyte (MK) proliferation and differentiation. These effects required only a short incubation period. Decreased proliferation was due to induction of apoptosis and was not related to a defect in TPO/MPL/JAK2/STAT signaling. When added later (day 8), the compound induced a dose-dependent decrease (up to 10-fold) in proplatelet (PPT) formation. Gene profiling from MK revealed a silencing in the expression of DNA repair genes with a marked RAD51 decrease at protein level. DNA double-strand breaks were increased as attested by elevated γH2AX phosphorylation level. Moreover, ATM was phosphorylated leading to p53 stabilization and increased BAX and p21 expression. The use of a p53 shRNA rescued apoptosis, and only partially the defect in PPT formation. These results suggest that HDACi induces a thrombocytopenia by a p53-dependent mechanism along MK differentiation and a p53-dependent and -independent mechanism for PPT formation.     

Poly(ADP-ribose) polymerase activity prevents signaling pathways for cell cycle arrest after DNA methylating agent exposure

Mouse fibroblasts, deficient in DNA polymerase beta, are hypersensitive to monofunctional DNA methylating agents such as methyl methanesulfonate (MMS). Both wild-type and, in particular, repair-deficient DNA polymerase beta null cells are highly sensitized to the cytotoxic effects of MMS by 4-amino-1,8-naphthalimide (4-AN), an inhibitor of poly(ADP-ribose) polymerase (PARP) activity. Experiments with synchronized cells suggest that exposure during S-phase of the cell cycle is required for the 4-AN effect. 4-AN elicits a similar extreme sensitization to the thymidine analog, 5-hydroxymethyl-2'-deoxyuridine, implicating the requirement for an intermediate of DNA repair. In PARP-1-expressing fibroblasts treated with a combination of MMS and 4-AN, a complete inhibition of DNA synthesis is apparent after 4 h, and by 24 h, all cells are arrested in S-phase of the cell cycle. Continuous incubation with 4-AN is required to maintain the cell cycle arrest. Caffeine, an inhibitor of the upstream checkpoint kinases ATM (ataxia telangiectasia-mutated) and ATR (ATM and Rad3-related), has no effect on the early inhibition of DNA synthesis, but cells are no longer able to maintain the block after 8 h. Instead, the addition of caffeine leads to arrest of cells in G(2)/M rather than S-phase after 24 h. Analysis of signaling pathways in cell extracts reveals an activation of Chk1 after treatment with MMS and 4-AN, which can be suppressed by caffeine. Our results suggest that inhibition of PARP activity results in sensitization to MMS through maintenance of an ATR and Chk1-dependent S-phase checkpoint.     

A role of the Ca2+/Mg2+-dependent endonuclease in apoptosis and its inhibition by Poly(ADP-ribose) polymerase

Apoptosis is characterized by various cell morphological and biochemical features, one of which is the internucleosomal degradation of genomic DNA. The role of the human chromatin-bound Ca(2+)- and Mg(2+)-dependent endonuclease (CME) DNAS1L3 and its inhibition by poly(ADP-ribosyl)ation in the DNA degradation that accompanies apoptosis was investigated. The nuclear localization of this endonuclease is the unique feature that distinguishes it from other suggested apoptotic nucleases. Purified recombinant DNAS1L3 was shown to cleave nuclear DNA into both high molecular weight and oligonucleosomal fragments in vitro. Furthermore, exposure of mouse skin fibroblasts expressing DNAS1L3 to inducers of apoptosis resulted in oligonucleosomal DNA fragmentation, an effect not observed in cells not expressing this CME, as well as in a decrease in cell viability greater than that apparent in the control cells. Recombinant DNAS1L3 was modified by recombinant human poly(ADP-ribose) polymerase (PARP) in vitro, resulting in a loss of nuclease activity. The DNAS1L3 protein also underwent poly(ADP-ribosyl)ation in transfected mouse skin fibroblasts in response to inducers of apoptosis. The cleavage and inactivation of PARP by a caspase-3-like enzyme late in apoptosis were associated with a decrease in the extent of DNAS1L3 poly(ADP-ribosyl)ation, which likely releases DNAS1L3 from inhibition and allows it to catalyze the degradation of genomic DNA.     

Caspase-dependent apoptosis and -independent poly(ADP-ribose) polymerase cleavage induced by transforming growth factor beta1

Apoptosis is an important cell suicide program which involves the caspases activation and is implicated in physiological and pathological processes. Poly(ADP-ribose) polymerase (PARP) cleavage is often associated with apoptosis and has been served as one hallmark of apoptosis and caspase activation. In this study, we aimed to determine TGF-beta1-induced apoptosis and to examine the involvement of caspases and its relationship with PARP cleavage. TGF-beta1 induces strong apoptosis of AML-12 cells which can be detected by DNA fragmentation, FACS, and morphological assays. Z-VAD-fmk, a selective caspase inhibitor, partially inhibits the TGF-beta1-induced apoptosis; but has no effect on TGF-beta1-induced DNA fragmentation and PARP cleavage. However, BD-fmk, a broad-spectrum caspase inhibitor, completely suppresses TGF-beta1-induced apoptosis, but unexpectedly does not inhibit TGF-beta1-induced PARP cleavage. Furthermore, Z-VAD-fmk treatment is able to completely inhibit the daunorubicin-induced apoptosis in A-431 cells, but only slightly blocks the daunorubicin-induced PARP cleavage, whereas BD-fmk can inhibit both daunorubicin-induced apoptosis and PARP cleavage completely. In addition, we observed that both TGF-beta1-induced apoptosis and PARP degradation in AML-12 cells can be completely blocked by inhibiting the protein synthesis with cycloheximide. These results demonstrate for the first time that TGF-beta1-induced caspase-dependent apoptosis is associated with caspase-independent PARP cleavage that requires the TGF-beta1-induced synthesis of new proteins. The results indicate that caspase-3 is not a major caspase involved in TGF-beta1-induced apoptosis in AML-12 cells, and is not required for apoptosis-associated DNA fragmentation. The results also suggest that PARP cleavage may occur as an independent event that can be disassociated with cell apoptosis.     

Poly(ADP-ribose) polymerase (PARP-1) and p53 independently function in regulating double-strand break repair in primate cells

PARP-1 is rapidly activated by DNA strand breaks, which finally leads to the modulation of multiple protein activities in DNA replication, DNA repair and checkpoint control. PARP-1 may be involved in homologous recombination, and poly(ADP-ribosyl)ation of p53 represents one possible mechanism that activates p53 as a recombination surveillance factor. Here, we examined the influence of PARP-1 on homology-directed double-strand break (DSB) repair by use of a fluorescence- and I-SceI- meganuclease-based assay with either episomal or chromosomally integrated DNA substrates. Surprisingly, the transient expression of both full-length PARP-1 and of a dominant negative mutant, retaining the DNA-binding but lacking the catalytic domain, down-regulated DSB repair in a dose-dependent manner. This effect was seen regardless of p53 status, however, with enhanced inhibition in the presence of wild-type p53. Taken together, our data reveal that PARP-1 overexpression counteracts DSB repair independently of its enzymatic activity and of poly(ADP-ribosyl)ation of p53 in particular, but synergizes with p53 in suppressing chromosomal rearrangements.     

p53-dependent apoptosis induced by proteasome inhibition in mammary epithelial cells

We have examined the effects of inhibition of the 26S proteasome in a murine mammary cell line, KIM-2 cells using the peptide aldehyde inhibitor MG132. These studies have demonstrated a clear requirement for proteasome function in cell viability. Induction of apoptosis was observed following MG132 treatment in KIM-2 cells and this death was shown to be dependent on the cell actively traversing the cell cycle. KIM-2 cells were generated using a temperature sensitive T-antigen (Tag) and studies at the permissive temperature (33 degrees C) have shown that a Tag binding protein was essential for this apoptotic response. Studies in two additional cell lines, HC11, which is a mammary epithelial cell line carrying mutant p53 alleles and p53 null ES cells suggest that p53 is actively required for the apoptosis induced as a consequence of proteasome inhibition. These results suggest a pivotal role for the 26S proteasome degradation pathway in progression through the cell cycle in proliferating cells.