DNA damage and autophagy

Both exogenous and endogenous agents are a threat to DNA integrity. Exogenous environmental agents such as ultraviolet (UV) and ionizing radiation, genotoxic chemicals and endogenous byproducts of metabolism including reactive oxygen species can cause alterations in DNA structure (DNA damage). Unrepaired DNA damage has been linked to a variety of human disorders including cancer and neurodegenerative disease. Thus, efficient mechanisms to detect DNA lesions, signal their presence and promote their repair have been evolved in cells. If DNA is effectively repaired, DNA damage response is inactivated and normal cell functioning resumes. In contrast, when DNA lesions cannot be removed, chronic DNA damage triggers specific cell responses such as cell death and senescence. Recently, DNA damage has been shown to induce autophagy, a cellular catabolic process that maintains a balance between synthesis, degradation, and recycling of cellular components. But the exact mechanisms by which DNA damage triggers autophagy are unclear. More importantly, the role of autophagy in the DNA damage response and cellular fate is unknown. In this review we analyze evidence that supports a role for autophagy as an integral part of the DNA damage response.     

Chk1 and p21 cooperate to prevent apoptosis during DNA replication fork stress

Cells respond to DNA replication stress by triggering cell cycle checkpoints, repair, or death. To understand the role of the DNA damage response pathways in determining whether cells survive replication stress or become committed to death, we examined the effect of loss of these pathways on cellular response to agents that slow or arrest DNA synthesis. We show that replication inhibitors such as excess thymidine, hydroxyurea, and camptothecin are normally poor inducers of apoptosis. However, these agents become potent inducers of death in S-phase cells upon small interfering RNA-mediated depletion of the checkpoint kinase Chk1. This death response is independent of p53 and Chk2. p21-deficient cells, on the other hand, produce a more robust apoptotic response upon Chk1 depletion. p21 is normally induced only late after thymidine treatment. In Chk1-depleted cells p21 induction occurs earlier and does not require p53. Thus, Chk1 plays a primary role in the protection of cells from death induced by replication fork stress, whereas p21 mediates through its role in regulating entry into S phase. These findings are of potential importance to cancer therapy because we demonstrate that the efficacy of clinically relevant agents can be enhanced by manipulation of these signaling pathways.     

TGF-β Targets the Wnt Pathway Components,APC and β-catenin,as Mv1Lu Cells Undergo Cell Cycle Arrest

Mammalian cells exhibit complex cellular responses to DNA damage, including cell cycle arrest, DNA repair and apoptosis. Defects in any one of these responses can result in carcinogenesis. Absence of the chromatin remodeling complex Swi/Snf is found in many instances of cancer, and we have investigated its role in the UV damage response. The human carcinoma cell line SW13 is deficient in Swi/Snf and is very sensitive to UV radiation. In contrast, SW13 cells with ectopic Brg1 expression regain active Swi/Snf and become significantly more resistant to UV radiation. Sensitivity to UV light correlates well with dramatic UV induced apoptosis in SW13 cells, but not in SW13 cells expressing Brg1. We show that SW13 cells synchronized at the G1/S border progress into S phase after UV irradiation, and this checkpoint deficiency is corrected after Brg1 expression is restored. Interestingly, Brg1 expression in SW13 cells restores expression of two DNA damage responsive genes, Gadd45a and p21. Furthermore, Gadd45a induction and p21 degradation were observed in the Brg1-expressing SW13 cells after UV irradiation. Our findings demonstrate that Swi/Snf protects cells against deleterious consequences of UV induced DNA damage. These results also indicate that Swi/Snf may play a role in replication checkpoint activation after UV damage via regulation of the two PCNA-binding proteins Gadd45a and p21.     

Nucleolar localization and dynamic roles of flap endonuclease 1 in ribosomal DNA replication and damage repair

Despite the wealth of information available on the biochemical functions and our recent findings of its roles in genome stability and cancer avoidance of the structure-specific flap endonuclease 1 (FEN1), its cellular compartmentalization and dynamics corresponding to its involvement in various DNA metabolic pathways are not yet elucidated. Several years ago, we demonstrated that FEN1 migrates into the nucleus in response to DNA damage and under certain cell cycle conditions. In the current paper, we found that FEN1 is superaccumulated in the nucleolus and plays a role in the resolution of stalled DNA replication forks formed at the sites of natural replication fork barriers. In response to UV irradiation and upon phosphorylation, FEN1 migrates to nuclear plasma to participate in the resolution of UV cross-links on DNA, most likely employing its concerted action of exonuclease and gap-dependent endonuclease activities. Based on yeast complementation experiments, the mutation of Ser(187)Asp, mimicking constant phosphorylation, excludes FEN1 from nucleolar accumulation. The replacement of Ser(187) by Ala, eliminating the only phosphorylation site, retains FEN1 in nucleoli. Both of the mutations cause UV sensitivity, impair cellular UV damage repair capacity, and decline overall cellular survivorship.     

The heterogenic final cell cycle of chicken retinal Lim1 horizontal cells is not regulated by the DNA damage response pathway

Cells with aberrations in chromosomal ploidy are normally removed by apoptosis. However, aneuploid neurons have been shown to remain functional and active both in the cortex and in the retina. Lim1 horizontal progenitor cells in the chicken retina have a heterogenic final cell cycle, producing some cells that enter S-phase without proceeding into M-phase. The cells become heteroploid but do not undergo developmental cell death. This prompted us to investigate if the final cell cycle of these cells is under the regulation of an active DNA damage response. Our results show that the DNA damage response pathway, including γ-H2AX and Rad51 foci, is not triggered during any phase of the different final cell cycles of horizontal progenitor cells. However, chemically inducing DNA adducts or double-strand breaks in Lim1 horizontal progenitor cells activated the DNA damage response pathway, showing that the cells are capable of a functional response to DNA damage. Moreover, manipulation of the DNA damage response pathway during the final cell cycle using inhibitors of ATM/ATR, Chk1/2, and p38MAPK, neither induced apoptosis nor mitosis in the Lim1 horizontal progenitor cells. We conclude that the DNA damage response pathway is functional in the Lim1 horizontal progenitor cells, but that it is not directly involved in the regulation of the final cell cycle that gives rise to the heteroploid horizontal cell population.     

A novel approach for organelle-specific DNA damage targeting reveals different susceptibility of mitochondrial DNA to the anticancer drugs camptothecin and topotecan

DNA is susceptible of being damaged by chemicals, UV light or gamma irradiation. Nuclear DNA damage invokes both a checkpoint and a repair response. By contrast, little is known about the cellular response to mitochondrial DNA damage. We designed an experimental system that allows organelle-specific DNA damage targeting in Saccharomyces cerevisiae. DNA damage is mediated by a toxic topoisomerase I allele which leads to the formation of persistent DNA single-strand breaks. We show that organelle-specific targeting of a toxic topoisomerase I to either the nucleus or mitochondria leads to nuclear DNA damage and cell death or to loss of mitochondrial DNA and formation of respiration-deficient ‘petite’ cells, respectively. In wild-type cells, toxic topoisomerase I–DNA intermediates are formed as a consequence of topoisomerase I interaction with camptothecin-based anticancer drugs. We reasoned that targeting of topoisomerase I to the mitochondria of top1Δ cells should lead to petite formation in the presence of camptothecin. Interestingly, camptothecin failed to generate petite; however, its derivative topotecan accumulates in mitochondria and induces petite formation. Our findings demonstrate that drug modifications can lead to organelle-specific DNA damage and thus opens new perspectives on the role of mitochondrial DNA-damage in cancer treatment.     

Activation of protein kinase A and clustering of cell surface receptors by N-methyl-N'-nitro-N-nitrosoguanidine are independent of genomic DNA damage

Alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induces cellular stress leading to chromosomal aberrations, mutations and cell death. Previous reports from our laboratory have shown that low concentration of MNNG induces untargeted mutation (UTM), which occurs on intact DNA in mammalian cells through changes in gene expression profile. It also causes the activation of cAMP-protein kinase A (PKA) and up-regulation of POL-beta, which is demonstrated to play a role in DNA repair system. In order to find out the possible initial signal involved in UTM, we try to investigate whether the activation of PKA-CREB signal pathway is closely related to DNA damage. Our data shows that the treatment of low concentration MNNG (0.2 microM) activates PKA-CREB pathway in a comparable level both in a nuclear and enucleated cell system. And similar to the cell response caused by UV, the clustering of cell surface receptors of epidermal growth factor (EGF) and tumor necrosis factor alpha (TNFalpha) was also observed in cells exposed to MNNG. It was further demonstrated that the clustering of the surface receptors is independent of the genomic DNA damage, as this phenomenon was also observed in enucleated cells. These observations indicate that the initiation of signal cascades induced by low concentration of MNNG might be associated with its interaction with cell surface receptors and/or direct activation of related signal proteins but not its DNA damaging property.     

RNF8-dependent and RNF8-independent regulation of 53BP1 in response to DNA damage

The DNA damage surveillance network orchestrates cellular responses to DNA damage through the recruitment of DNA damage-signaling molecules to DNA damage sites and the concomitant activation of protein phosphorylation cascades controlled by the ATM (ataxia-telangiectasia-mutated) and ATR (ATM-Rad3-related) kinases. Activation of ATM/ATR triggers cell cycle checkpoint activation and adaptive responses to DNA damage. Recent studies suggest that protein ubiquitylation or degradation plays an important role in the DNA damage response. In this study, we examined the potential role of the proteasome in checkpoint activation and ATM/ATR signaling in response to UV light-induced DNA damage. HeLa cells treated with the proteasome inhibitor MG-132 showed delayed phosphorylation of ATM substrates in response to UV light. UV light-induced phosphorylation of 53BP1, as well as its recruitment to DNA damage foci, was strongly suppressed by proteasome inhibition, whereas the recruitment of upstream regulators of 53BP1, including MDC1 and H2AX, was unaffected. The ubiquitin-protein isopeptide ligase RNF8 was critical for 53BP1 focus targeting and phosphorylation in ionizing radiation-damaged cells, whereas UV light-induced 53BP1 phosphorylation and targeting exhibited partial dependence on RNF8 and the ubiquitin-conjugating enzyme UBC13. Suppression of RNF8 or UBC13 also led to subtle defects in UV light-induced G2/M checkpoint activation. These findings are consistent with a model in which RNF8 ubiquitylation pathways are essential for 53BP1 regulation in response to ionizing radiation, whereas RNF8-independent pathways contribute to 53BP1 targeting and phosphorylation in response to UV light and potentially other forms of DNA replication stress.     

Enhancement of UV-induced cytotoxicity by the adeno-associated virus replication proteins

Adeno-associated virus (AAV) normally requires co-infection of a helper virus to complete its life cycle. However, under conditions of cellular stress, such as treatment with carcinogens or ultraviolet (UV) light, a permissive intracellular environment is established and AAV completes its replicative cycle producing low levels of progeny virus. AAV DNA replication is dependent upon viral replication proteins, Rep78 and Rep68. The detailed mechanism by which these proteins interact with host cell factors is unknown. We have used a cell line (Neo6) that inducibly expresses the AAV Rep proteins to study their effects on cells that have undergone UV-induced DNA damage. Induction of Rep protein expression immediately after a sub-lethal dose of UV irradiation resulted in rapid cell killing. Those cells that die had chromatin condensation while cellular membranes remained intact, suggesting that concurrent Rep expression and UV damage induces an apoptosis-like response. However, we did not observe any DNA degradation. Thus we believe that the combination of Rep expression and UV irradiation induces cell death that shares some of the characteristics of apoptosis. UV irradiation and Rep expression induced an increase in the level of the CDK inhibitor, p21Cip, and the appearance of modified forms of both p21Cip and Bcl-2. Alteration of normal expression of these cytostatic/apoptotic proteins provides insight into the intracellular targets of the AAV replication proteins.     

Cell cycle inhibitor, p19INK4d, promotes cell survival and decreases chromosomal aberrations after genotoxic insult due to enhanced DNA repair

Genome integrity and cell proliferation and survival are regulated by an intricate network of pathways that includes cell cycle checkpoints, DNA repair and recombination, and programmed cell death. It makes sense that there should be a coordinated regulation of these different processes, but the components of such mechanisms remain unknown. In this report, we demonstrate that p19INK4d expression enhances cell survival under genotoxic conditions. By using p19INK4d-overexpressing clones, we demonstrated that p19INK4d expression correlates with the cellular resistance to UV treatment with increased DNA repair activity against UV-induced lesions. On the contrary, cells transfected with p19INK4d antisense cDNA show reduced ability to repair DNA damage and increased sensitivity to genotoxic insult when compared with their p19INK4d-overexpressing counterparts. Consistent with these findings, our studies also show that p19INK4d-overexpressing cells present not only a minor accumulation of UV-induced chromosomal aberrations but a lower frequency of spontaneous chromosome abnormalities than p19INK4d-antisense cells. Lastly, we suggest that p19INK4d effects are dissociated from its role as CDK4/6 inhibitor. The results presented herein support a crucial role for p19INK4d in regulating genomic stability and overall cell viability under conditions of genotoxic stress. We propose that p19INK4d would belong to a protein network that would integrate DNA repair, apoptotic and checkpoint mechanisms in order to maintain the genomic integrity.     

Evidence that increased 12-lipoxygenase activity induces apoptosis in fibroblasts

The 12-lipoxygenase (LO) enzyme has been implicated in playing a role in pancreatic beta cell inflammatory damage and atherosclerosis. 12-LO reacts with fatty acids to form hydroperoxides which may alter cellular growth. In this study we investigated the direct effect of mouse leukocyte type 12-LO cDNA overexpression on apoptosis in Chinese hamster ovary fibroblast cells that also stably overexpress the angiotensin II type 1a receptor. CHO-AT1a cells expressing background levels of 12-LO exhibited clear increases in growth in response to angiotensin II. In contrast, the new 12-LO transfected cells (CHO-AT1a/ML12-LO cells) displayed reduced basal and angiotensin Il-induced growth compared to CHO-AT1a cells. Furthermore, serum-deprivation resulted in a significantly greater number of non-viable cells in clones having the greatest magnitude of 12-LO overexpression. These results suggested that reduction of the proliferation rate of CHO-AT1a/ML12-LO cells was due to an increasing rate of cell death. To determine whether the increase in cell death was due to apoptosis, we evaluated nuclear DNA fragmentation, cell morphologic changes, and activation of caspase-3. Cells overexpressing 12-LO cDNA displayed all these changes characteristic of apoptosis. In addition the 12-LO product, 12-hydroperoxyeicosatetraenoic acid (12-HPETE), directly induced apoptosis in CHO-AT1a cells. These results demonstrate for the first time that 12-LO activation can lead to apoptosis in fibroblasts, suggesting a role of 12-LO in leading to inflammatory mediated cellular damage.     

Activation of BMP-Smad1/5/8 signaling promotes survival of retinal ganglion cells after damage in vivo

While the essential role of bone morphogenetic protein (BMP) signaling in nervous system development is well established, its function in the adult CNS is poorly understood. We investigated the role of BMP signaling in the adult mouse retina following damage in vivo. Intravitreal injection of N-methyl-D-aspartic acid (NMDA) induced extensive retinal ganglion cell death by 2 days. During this period, BMP2, -4 and -7 were upregulated, leading to phosphorylation of the downstream effector, Smad1/5/8 in the inner retina, including in retinal ganglion cells. Expression of Inhibitor of differentiation 1 (Id1; a known BMP-Smad1/5/8 target) was also upregulated in the retina. This activation of BMP-Smad1/5/8 signaling was also observed following light damage, suggesting that it is a general response to retinal injuries. Co-injection of BMP inhibitors with NMDA effectively blocked the damage-induced BMP-Smad1/5/8 activation and led to further cell death of retinal ganglion cells, when compared with NMDA injection alone. Moreover, treatment of the retina with exogenous BMP4 along with NMDA damage led to a significant rescue of retinal ganglion cells. These data demonstrate that BMP-Smad1/5/8 signaling is neuroprotective for retinal ganglion cells after damage, and suggest that stimulation of this pathway can serve as a potential target for neuroprotective therapies in retinal ganglion cell diseases, such as glaucoma.     

Checkpoint silencing during the DNA damage response in Caenorhabditis elegans embryos

In most cells, the DNA damage checkpoint delays cell division when replication is stalled by DNA damage. In early Caenorhabditis elegans embryos, however, the checkpoint responds to developmental signals that control the timing of cell division, and checkpoint activation by nondevelopmental inputs disrupts cell cycle timing and causes embryonic lethality. Given this sensitivity to inappropriate checkpoint activation, we were interested in how embryos respond to DNA damage. We demonstrate that the checkpoint response to DNA damage is actively silenced in embryos but not in the germ line. Silencing requires rad-2, gei-17, and the polh-1 translesion DNA polymerase, which suppress replication fork stalling and thereby eliminate the checkpoint-activating signal. These results explain how checkpoint activation is restricted to developmental signals during embryogenesis and insulated from DNA damage. They also show that checkpoint activation is not an obligatory response to DNA damage and that pathways exist to bypass the checkpoint when survival depends on uninterrupted progression through the cell cycle.     

Radiation-induced epigenetic DNA methylation modification of radiation-response pathways

DNA methylation can regulate gene expression and has been shown to modulate cancer cell biology and chemotherapy resistance. Therapeutic radiation results in a biological response to counter the subsequent DNA damage and genomic stress in order to avoid cell death. In this study, we analyzed DNA methylation changes at >450,000 loci to determine a potential epigenetic response to ionizing radiation in MDA-MB-231 cells. Cells were irradiated at 2 and 6 Gy and analyzed at 7 time points from 1–72 h. Significantly differentially methylated genes were enriched in gene ontology categories relating to cell cycle, DNA repair, and apoptosis pathways. The degree of differential methylation of these pathways varied with radiation dose and time post-irradiation in a manner consistent with classical biological responses to radiation. A cell cycle arrest was observed 24 h post-irradiation and DNA damage, as measured by γH2AX, resolved at 24 h. In addition, cells showed low levels of apoptosis 2–48 h post-6 Gy and cellular senescence became significant at 72 h post-irradiation. These DNA methylation changes suggest an epigenetic role in the cellular response to radiation.     

Differential role of RB in response to UV and IR damage

The retinoblastoma tumor suppressor (RB) is functionally inactivated in the majority of cancers and is a critical mediator of DNA damage checkpoints. Despite the critical importance of RB function in tumor suppression, the coordinate impact of RB loss on the response to environmental and therapeutic sources of damage has remained largely unexplored. Here, we utilized a conditional knockout system to ablate RB in adult fibroblasts. This model system enabled us to investigate the temporal role of RB loss on cell cycle checkpoints and DNA damage repair following ultraviolet (UV) and ionizing radiation (IR) damage. We demonstrate that RB loss compromises rapid cell cycle arrest following UV and IR exposure in adult primary cells. Detailed kinetic analysis of the checkpoint response revealed that disruption of the checkpoint is concomitant with RB target gene deregulation, and is not simply a manifestation of chronic RB loss. RB loss had a differential effect upon repair of the major DNA lesions induced by IR and UV. Whereas RB did not affect resolution of DNA double-strand breaks, RB-deficient cells exhibited accelerated repair of pyrimidine pyrimidone photoproducts (6-4 PP). In parallel, this repair was coupled with enhanced expression of specific factors and the behavior of proliferating cell nuclear antigen (PCNA) recruitment to replication and repair foci. Thus, RB loss and target gene deregulation hastens the repair of specific lesions distinct from its ubiquitous role in checkpoint abrogation.