Implication of the VRK1 chromatin kinase in the signaling responses to DNA damage: a therapeutic target?

DNA damage causes a local distortion of chromatin that triggers the sequential processes that participate in specific DNA repair mechanisms. This initiation of the repair response requires the involvement of a protein whose activity can be regulated by histones. Kinases are candidates to regulate and coordinate the connection between a locally altered chromatin and the response initiating signals that lead to identification of the type of lesion and the sequential steps required in specific DNA damage responses (DDR). This initiating kinase must be located in chromatin, and be activated independently of the type of DNA damage. We review the contribution of the Ser-Thr vaccinia-related kinase 1 (VRK1) chromatin kinase as a new player in the signaling of DNA damage responses, at chromatin and cellular levels, and its potential as a new therapeutic target in oncology. VRK1 is involved in the regulation of histone modifications, such as histone phosphorylation and acetylation, and in the formation of γH2AX, NBS1 and 53BP1 foci induced in DDR. Induction of DNA damage by chemotherapy or radiation is a mainstay of cancer treatment. Therefore, novel treatments can be targeted to proteins implicated in the regulation of DDR, rather than by directly causing DNA damage.     

The nuclear γ-H2AX apoptotic ring: implications for cancers and autoimmune diseases

Apoptosis is a fundamental process for metazoan development. It is also relevant to the pathophysiology of immune diseases and cancers and to the outcome of cancer chemotherapies, as well as being a target for cancer therapies. Apoptosis involves intrinsic pathways typically initiated by DNA damaging agents and engaging mitochondria, and extrinsic pathways typically initiated by “death receptors” and their ligands TRAIL and TNF at the cell surface. Recently, we discovered the apoptotic ring, which microscopically looks like a nuclear annular staining early in apoptosis. This ring is, in three-dimensional space, a thick intranuclear shell consisting of epigenetic modifications including histone H2AX and DNA damage response (DDR) proteins. It excludes the DNA repair factors usually associated with γ-H2AX in the DDR nuclear foci. Here, we summarize our knowledge of the apoptotic ring, and discuss its biological and pathophysiological relevance, as well as its value as a potential pharmacodynamic biomarker for anticancer therapies.     

Influenza infection induces host DNA damage and dynamic DNA damage responses during tissue regeneration

Influenza viruses account for significant morbidity worldwide. Inflammatory responses, including excessive generation of reactive oxygen and nitrogen species (RONS), mediate lung injury in severe influenza infections. However, the molecular basis of inflammation-induced lung damage is not fully understood. Here, we studied influenza H1N1 infected cells in vitro, as well as H1N1 infected mice, and we monitored molecular and cellular responses over the course of 2 weeks in vivo. We show that influenza induces DNA damage to both, when cells are directly exposed to virus in vitro (measured using the comet assay) and also when cells are exposed to virus in vivo (estimated via γH2AX foci). We show that DNA damage, as well as responses to DNA damage persist in vivo until long after virus has been cleared, at times when there are inflammation associated RONS (measured by xanthine oxidase activity and oxidative products). The frequency of lung epithelial and immune cells with increased γH2AX foci is elevated in vivo, especially for dividing cells (Ki-67-positive) exposed to oxidative stress during tissue regeneration. Additionally, we observed a significant increase in apoptotic cells as well as increased levels of DNA double strand break (DSB) repair proteins Ku70, Ku86 and Rad51 during the regenerative phase. In conclusion, results show that influenza induces DNA damage both in vitro and in vivo, and that DNA damage responses are activated, raising the possibility that DNA repair capacity may be a determining factor for tissue recovery and disease outcome.     

The opportunistic pathogen Pseudomonas aeruginosa activates the DNA double-strand break signaling and repair pathway in infected cells

Highly hazardous DNA double-strand breaks can be induced in eukaryotic cells by a number of agents including pathogenic bacterial strains. We have investigated the genotoxic potential of Pseudomonas aeruginosa, an opportunistic pathogen causing devastating nosocomial infections in cystic fibrosis or immunocompromised patients. Our data revealed that infection of immune or epithelial cells by P. aeruginosa triggered DNA strand breaks and phosphorylation of histone H2AX (γH2AX), a marker of DNA double-strand breaks. Moreover, it induced formation of discrete nuclear repair foci similar to gamma-irradiation-induced foci, and containing γH2AX and 53BP1, an adaptor protein mediating the DNA-damage response pathway. Gene deletion, mutagenesis, and complementation in P. aeruginosa identified ExoS bacterial toxin as the major factor involved in γH2AX induction. Chemical inhibition of several kinases known to phosphorylate H2AX demonstrated that Ataxia Telangiectasia Mutated (ATM) was the principal kinase in P. aeruginosa-induced H2AX phosphorylation. Finally, infection led to ATM kinase activation by an auto-phosphorylation mechanism. Together, these data show for the first time that infection by P. aeruginosa activates the DNA double-strand break repair machinery of the host cells. This novel information sheds new light on the consequences of P. aeruginosa infection in mammalian cells. As pathogenic Escherichia coli or carcinogenic Helicobacter pylori can alter genome integrity through DNA double-strand breaks, leading to chromosomal instability and eventually cancer, our findings highlight possible new routes for further investigations of P. aeruginosa in cancer biology and they identify ATM as a potential target molecule for drug design.     

γ-radiation-induced γH2AX formation occurs preferentially in actively transcribing euchromatic loci

The central dogma in radiation biology is that nuclear DNA is the critical target with respect to radiosensitivity. In accordance with the theoretical expectations, and in the absence of a conclusive model, the general consensus in the field has been to view chromatin as a homogeneous template for DNA damage and repair. This paradigm has been called into question by recent findings indicating a disparity in γ-irradiation-induced γH2AX foci formation in euchromatin and heterochromatin. Here, we have extended those studies and provide evidence that γH2AX foci form preferentially in actively transcribing euchromatin following γ-irradiation.     

Checkpoint kinase 1 in DNA damage response and cell cycle regulation

Originally identified as a mediator of DNA damage response (DDR), checkpoint kinase 1 (Chk1) has a broader role in checkpoint activation in DDR and normal cell cycle regulation. Chk1 activation involves phosphorylation at conserved sites. However, recent work has identified a splice variant of Chk1, which may regulate Chk1 in both DDR and normal cell cycle via molecular interaction. Upon activation, Chk1 phosphorylates a variety of substrate proteins, resulting in the activation of DNA damage checkpoints, cell cycle arrest, DNA repair, and/or cell death. Chk1 and its related signaling may be an effective therapeutic target in diseases such as cancer.     

Eukaryotic DNA damage checkpoint activation in response to double-strand breaks

Double-strand breaks (DSBs) are the most detrimental form of DNA damage. Failure to repair these cytotoxic lesions can result in genome rearrangements conducive to the development of many diseases, including cancer. The DNA damage response (DDR) ensures the rapid detection and repair of DSBs in order to maintain genome integrity. Central to the DDR are the DNA damage checkpoints. When activated by DNA damage, these sophisticated surveillance mechanisms induce transient cell cycle arrests, allowing sufficient time for DNA repair. Since the term “checkpoint” was coined over 20 years ago, our understanding of the molecular mechanisms governing the DNA damage checkpoint has advanced significantly. These pathways are highly conserved from yeast to humans. Thus, significant findings in yeast may be extrapolated to vertebrates, greatly facilitating the molecular dissection of these complex regulatory networks. This review focuses on the cellular response to DSBs in Saccharomyces cerevisiae, providing a comprehensive overview of how these signalling pathways function to orchestrate the cellular response to DNA damage and preserve genome stability in eukaryotic cells.     

Karyotype and chromosome banding in the reed frog Hyperolius viridiflavus ommatostictus (Amphibia, Anura, Hyperoliidae)

C-banding and mithramycin staining were used to characterize the karyotypes of 10 specimens of the African reed frog Hyperolius viridiflavus ommatostictus from Tanzania. The diploid chromosome number is 2n = 24. Although no heteromorphic sex chromosomes were present in the mitotic karyotypes, in many diakineses of male meiosis one or two bivalents exhibited an end-to-end arrangement. In the laboratory 7 out of 24 females changed sex spontaneously. This indicates that an XY/XX system of sex determination operates in H. viridiflavus ommatostictus.     

Karyotype and chromosome banding in the reed frogHyperolius viridiflavus ommatostictus (Amphibia,Anura, Hyperoliidae)

Summary C-banding and mithramycin staining were used to characterize the karyotypes of 10 specimens of the African reed frogHyperolius viridiflavus ommatostictus from Tanzania. The diploid chromosome number is 2n=24. Although no heteromorphic sex chromosomes were present in the mitotic karyotypes, in many diakineses of male meiosis one or two bivalents exhibited an end-to-end arrangement. In the laboratory 7 out of 24 females changed sex spontaneously. This indicates that an XY/XX system of sex determination operates inH. viridiflavus ommatostictus.This study was supported by the Deutsche Forschungsgemeinschaft (Schm 484/2-4). We thank K. E. Linsenmair and C. M. Richards for helpful comments on an earlier draft.     

Regulation of male sex determination: genital ridge formation and Sry activation in mice

Sex determination is essential for the sexual reproduction to generate the next generation by the formation of functional male or female gametes. In mammals, primary sex determination is commenced by the presence or absence of the Y chromosome, which controls the fate of the gonadal primordium. The somatic precursor of gonads, the genital ridge is formed at the mid-gestation stage and gives rise to one of two organs, a testis or an ovary. The fate of the genital ridge, which is governed by the differentiation of somatic cells into Sertoli cells in the testes or granulosa cells in the ovaries, further determines the sex of an individual and their germ cells. Mutation studies in human patients with disorders of sex development and mouse models have revealed factors that are involved in mammalian sex determination. In most of mammals, a single genetic trigger, the Y-linked gene Sry (sex determination region on Y chromosome), regulates testicular differentiation. Despite identification of Sry in 1990, precise mechanisms underlying the sex determination of bipotential genital ridges are still largely unknown. Here, we review the recent progress that has provided new insights into the mechanisms underlying genital ridge formation as well as the regulation of Sry expression and its functions in male sex determination of mice.     

Somatic chromosomes of black-headed oriole,Oriolus xanthornus (Linn.): A probable case of translocation heterozygosity

Summary 2n=80. There are 7 pairs of macrochromosomes in the male karyotype. In 2 females, besides the sex chromosomes, chromosome 3 and the largest microchromosome are unpaired and there is an additional large unpaired macrochromosome. This aberrant karyotype is best interpreted in terms of a reciprocal translocation heterozygosity, in all likelihood, at the population level.Acknowledgment. We thank Prof. U.S. Srivastava, Zoology Department, Allahabad University, for providing facilities. Financial assistance from CSIR, India in the form of a Senior Research Fellowship to one of us (H.A.A.) is thankfully acknowledged.     

Histone deacetylase inhibitors augment doxorubicin-induced DNA damage in cardiomyocytes

Histone deacetylase inhibitors have emerged as a new class of anticancer therapeutics with suberoylanilide hydroxamic acid (Vorinostat) and depsipeptide (Romidepsin) already being approved for clinical use. Numerous studies have identified that histone deacetylase inhibitors will be most effective in the clinic when used in combination with conventional cancer therapies such as ionizing radiation and chemotherapeutic agents. One promising combination, particularly for hematologic malignancies, involves the use of histone deacetylase inhibitors with the anthracycline, doxorubicin. However, we previously identified that trichostatin A can potentiate doxorubicin-induced hypertrophy, the dose-limiting side-effect of the anthracycline, in cardiac myocytes. Here we have the extended the earlier studies and evaluated the effects of combinations of the histone deacetylase inhibitors, trichostatin A, valproic acid and sodium butyrate on doxorubicin-induced DNA double-strand breaks in cardiomyocytes. Using γH2AX as a molecular marker for the DNA lesions, we identified that all of the broad-spectrum histone deacetylase inhibitors tested augment doxorubicin-induced DNA damage. Furthermore, it is evident from the fluorescence photomicrographs of stained nuclei that the histone deacetylase inhibitors also augment doxorubicin-induced hypertrophy. These observations highlight the importance of investigating potential side-effects, in relevant model systems, which may be associated with emerging combination therapies for cancer.     

S-phase checkpoint regulations that preserve replication and chromosome integrity upon dNTP depletion

DNA replication stress, an important source of genomic instability, arises upon different types of DNA replication perturbations, including those that stall replication fork progression. Inhibitors of the cellular pool of deoxynucleotide triphosphates (dNTPs) slow down DNA synthesis throughout the genome. Following depletion of dNTPs, the highly conserved replication checkpoint kinase pathway, also known as the S-phase checkpoint, preserves the functionality and structure of stalled DNA replication forks and prevents chromosome fragmentation. The underlying mechanisms involve pathways extrinsic to replication forks, such as those involving regulation of the ribonucleotide reductase activity, the temporal program of origin firing, and cell cycle transitions. In addition, the S-phase checkpoint modulates the function of replisome components to promote replication integrity. This review summarizes the various functions of the replication checkpoint in promoting replication fork stability and genome integrity in the face of replication stress caused by dNTP depletion.     

The common fragile site FRA16D gene product WWOX: roles in tumor suppression and genomic stability

The fragile WWOX gene, encompassing the chromosomal fragile site FRA16D, is frequently altered in human cancers. While vulnerable to DNA damage itself, recent evidence has shown that the WWOX protein is essential for proper DNA damage response (DDR). Furthermore, the gene product, WWOX, has been associated with multiple protein networks, highlighting its critical functions in normal cell homeostasis. Targeted deletion of Wwox in murine models suggests its in vivo requirement for proper growth, metabolism, and survival. Recent molecular and biochemical analyses of WWOX functions highlighted its role in modulating aerobic glycolysis and genomic stability. Cumulatively, we propose that the gene product of FRA16D, WWOX, is a functionally essential protein that is required for cell homeostasis and that its deletion has important consequences that contribute to the neoplastic process. This review discusses the essential role of WWOX in tumor suppression and genomic stability and how its alteration contributes to cancer transformation.     

Reestablishment of the inactive X chromosome to the ground state through cell fusion-induced reprogramming

The restricted gene expression pattern of a differentiated cell can be reversed by fusion of the somatic cell with a more developmentally potent cell type, such as an embryonic stem (ES) cell. During this reprogramming process, somatic cells obtain most of the characteristics of pluripotent cells. Reactivation of an inactive X chromosome (Xi) is an important epigenetic marker confirming the pluripotent reprogramming of somatic cells. Female somatic cells contain one active X chromosome (Xa) and one Xi, and following the fusion of these cells with male ES cells, the Xi becomes activated, resulting in XaXaXaY fusion hybrid cells. To monitor Xi reactivation, transgenic female neural stem cells (fNSCs) carrying a green fluorescent protein (GFP) reporter gene expressed on the Xa (X-GFP), but not on the Xi, were used for reprogramming. XaXi^GFP NSCs, whose GFP reporter was silenced, were fused with HM1 ES cells (XY) to induce pluripotent reprogramming. The Xi^GFP of NSCs were found to be activated on day 4 post-fusion, indicating reactivation of the Xi. Hybrid cells showed pluripotent cell-specific characteristics cells including inactivation of the NSC marker Nestin, DNA demethylation of Oct4, DNA methylation of Nestin, and reactivation of the Xi. Following differentiation of the (GFP-positive) hybrid cells through embryoid body formation, the proportion of GFP-negative cells was found to be approximately 26?%, indicating that there was random inactivation of one of the three Xas. Here, we showed that the Xi of somatic cells is reprogrammed to the Xa state and that cellular differentiation occurs randomly, i.e., regardless of the Xa or Xi state, indicating that the memory of the Xi of somatic cells has been erased and reset to the ground state (i.e., inner cell mass-like state), indicating that random X-chromosome inactivation occurs upon differentiation.