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The Journal of Visualized Experiments (JoVE) is a peer reviewed, PubMed-indexed video journal. Our mission is to increase the productivity of scientific research.

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In JoVE (4)

Other Publications (35)

Articles by Tom C. Karagiannis in JoVE

 JoVE General

Quantification of γH2AX Foci in Response to Ionising Radiation


JoVE 1957 4/06/2010

1,2, 1,2,3, 1,3, 1, 2,3, 1,2

1Epigenomic Medicine, Baker IDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct, 2Department of Pathology, The University of Melbourne, 3Epigenetics in Human Health and Disease, Baker IDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct

Quantification of DNA double-strand streaks using γH2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of γH2AX foci after exposure of cells to radiation.

 JoVE General

Quantitation of γH2AX Foci in Tissue Samples


JoVE 2063 6/28/2010

1,2, 1,3, 1,2,3, 1, 2,3, 3,4,5, 1,3

1Epigenomic Medicine, Baker IDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct, 2Epigenetics in Human Health and Disease, Baker IDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct, 3Department of Pathology, The University of Melbourne, 4Department of Allergy and Immunology, Murdoch Children's Research Institute, Royal Children's Hospital, 5Department of Pediatrics, The University of Melbourne

Quantitation of DNA double-strand breaks on the basis of γH2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of γH2AX foci in tissue samples.

 JoVE General

Evaluation of the Spatial Distribution of γH2AX following Ionizing Radiation


JoVE 2203 8/07/2010

1,2,3, 1,2, 2,3, 2, 1,3, 2,3

1Epigenetics in Human Health and Disease, BakerIDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct, 2Epigenomic Medicine, BakerIDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct, 3Department of Pathology, University of Melbourne

Microscopic analysis of γH2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced γH2AX formation within the nucleus.

 JoVE General

Clonogenic Assay: Adherent Cells


JoVE 2573 3/13/2011

1,2, 1,2,3, 1, 1,4, 2,3, 1,2

1Epigenomic Medicine, BakerIDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct, 2Department of Pathology, The University of Melbourne, 3Epigenetics in Human Health and Disease, BakerIDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct, 4Department of Anatomy and Cellular Biology, The University of Melbourne

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

Other articles by Tom C. Karagiannis on PubMed

Epigenetic Changes Activate Widespread Signals in Response to Double-strand Breaks

Cancer Biology & Therapy. Jul, 2004  |  Pubmed ID: 15136760

Double-strand breaks are one the most severe types of DNA damage with respect to cell survival and the preservation of genomic integrity. Therefore, cells have evolved complex mechanisms including cell cycle regulation, activation of repair pathways and in certain cases induction of apoptosis in response to these lesions. The molecular details of many of the cellular responses to double-strand breaks have been well characterized. Our understanding of these responses in the context of chromatin has also progressed recently. In this review, we focus our discussion on the significance of DNA damage-induced chromatin modifications in double-strand break signaling and repair pathways. In particular, findings from recent studies suggest mechanisms by which highly localized double-strand breaks may activate widespread signals throughout the cell by inducing alterations in chromatin structure.

Plasmid DNA Breakage by Decay of DNA-associated Auger Electron Emitters: Approaches to Analysis of Experimental Data

Radiation Research. Jul, 2004  |  Pubmed ID: 15222798

Plasmid DNA is a popular substrate for the assay of DNA strand breakage by a variety of agents. The use of the plasmid assay relies on the assumption that individual damaging events occur at random, which allows the application of Poisson statistics. This assumption is not valid in the case of damage arising from decay of DNA-associated Auger electron emitters, since a single decay event can generate a few breaks in the same DNA strand, which is indistinguishable from a single break in the assay. The consequent analytical difficulties are overcome by considering relaxation events rather than single-strand breaks, and linearization events rather than double-strand breaks. A further consideration is that apart from damage at the site of DNA-associated decay, which is the principal interest of the analysis, some DNA damage also arises from the radiation field created by all decay events. These two components of damage are referred to as internal and external breakage, respectively, and they can be separated in the analysis since their contribution depends on the experimental conditions. The DNA-binding ligand Hoechst 33258 labeled with 125I was used in our experiments to study breakage in pBR322 plasmid DNA arising from the decay of this Auger electron emitter. The values obtained for the efficiency (per decay) of plasmid relaxation and linearization by the 125I-labeled ligand were 0.090 +/- 0.035 and 0.82 +/- 0.04, respectively. When dimethylsulfoxide was included as a radical scavenger, the efficiency values for relaxation and linearization were 0.15 +/- 0.02 and 0.65 +/- 0.05, respectively.

SiRNAs: Mechanism of RNA Interference, in Vivo and Potential Clinical Applications

Cancer Biology & Therapy. Nov, 2004  |  Pubmed ID: 15539939

Small interfering RNAs are currently the most widely used nucleic acid-based sequence-specific gene silencing molecules. These molecules mediate RNA interference--a natural post-transcriptional gene-silencing pathway. Given the high reliability and higher efficiency of small interfering RNA-mediated RNA interference, compared to earlier reverse genetic technologies, this is now the preferred technique in functional genomics. Furthermore, the exquisite specificity and exceptional gene-silencing potency of small interfering RNAs has resulted in intense research related to potential target-specific therapeutic applications of these molecules. This review will discuss the mechanism of RNA interference and applications of the pathway in molecular biology including functional genomics will be overviewed. The article will outline in vivo and potential clinical applications of small interfering RNA molecules.

Radio- and Chemo-sensitization of Human Erythroleukemic K562 Cells by the Histone Deacetylase Inhibitor Trichostatin A

Hellenic Journal of Nuclear Medicine. Sep-Dec, 2004  |  Pubmed ID: 15841297

Histone deacetylase inhibitors are emerging therapeutic agents for cancer. In addition to effecting hyperacetylation of core histones, they have been shown to induce biologic effects such as cell cycle redistribution, cytostasis and in certain cases apoptosis in a variety of cell lines. In this study, the purpose was to investigate the effects of Trichostatin A (TSA) - the most potent histone deacetylase inhibitor identified to date - in human erythroleukemic K562 cells. Further aims were to examine the effect of TSA pre-treatment on the chemosensitivity of the cells to the anthracycline, doxorubicin, and on radiosensitivity. In all experiments the cells were treated with 0.2, 0.5 and 2 mM TSA for 24 h prior to analysis for histone acetylation status, cell growth and survival. In parallel assays, TSA treated cells were exposed to doxorubicin or g-radiation and subsequently analyzed for clonogenic survival. The findings indicated that TSA exhibits potent histone deacetylase inhibitor activity in K562 cells, resulting in hyperacetylation of histones 3 and 4 at the concentrations tested. Furthermore, treatment of cells with TSA resulted in dose-dependent inhibition of proliferation, reduction in clonogenic survival and induction of apoptosis. Moreover, the findings of clonogenic survival assays indicated that pre-treatment of K562 cells with TSA augmented the cytotoxic potency of doxorubicin. The magnitude of sensitization to 10 mM doxorubicin-mediated cell death was approximately 2-fold in cells that were treated with 0.2 mM TSA and 5-fold in cells exposed to 0.5 and 2 mM TSA, compared to cells that had not been pre-treated with the histone deacetylase inhibitor. Similarly, exposure of K562 cells to TSA prior to irradiation resulted in dose-dependent radiosensitization. The dose modification factors at D(37) were calculated to be 1.3, 1.6 and 2.5 for cells treated with 0.2, 0.5 and 2 mM TSA, respectively. These findings provide additional evidence that histone deacetylase inhibitors can increase the cytotoxic efficiency of chemotherapeutic drugs, particularly those which target DNA, and can enhance the sensitivity of cells to g-radiation. More generally, the results support the development of histone deacetylase inhibitors as potential clinical chemo- and radio-sensitizers.

Radioimmunotherapy: Principles, Current Trends and Future Directions

Hellenic Journal of Nuclear Medicine. Jan-Apr, 2004  |  Pubmed ID: 16868640

Radioimmunotherapy has recently been introduced as a therapeutic modality for B-cell non-Hodgkin's lymphoma. The US Food and Drug Administration (FDA) has approved (February, 2002) 90Y-ibritumomab tiuxetan (Zevalin; IDEC Pharmaceuticals Corp., San Diego CA) and the FDA's Oncologic Drugs Advisory Committee has voted (December, 2002) in favor of 131I-tositumomab (Bexxar; Corixa Corp., Seattle WA), for use in radioimmunotherapy. It is anticipated that once various technical compliance issues are fulfilled Zevalin will be approved for use in Europe, where it will be marketed and distributed by Schering AG (Berlin, Germany). Similarly, approval for Bexxar is anticipated in Europe and it will be marketed by Amersham PLC (London, UK). The aim of this review is to discuss the principles of radioimmunotherapy. The scientific advances that have lead to the acceptance of radioimmunotherapy as a therapeutic modality in cancer are highlighted. Various limitations of radioimmunotherapy particularly for the treatment of solid tumors are considered and future directions for this treatment modality are examined.

RNA Interference and Potential Therapeutic Applications of Short Interfering RNAs

Cancer Gene Therapy. Oct, 2005  |  Pubmed ID: 15891770

RNA interference is an endogenous gene-silencing mechanism that involves double-stranded RNA-mediated sequence-specific mRNA degradation. The discovery of this pathway together with the elucidation of the structure and function of short interfering RNAs--the effector molecules of RNA interference--has had an enormous impact on experimental biology. RNA interference technologies are currently the most widely utilized techniques in functional genomic studies. Furthermore, there is an intense research effort aimed at developing short interfering RNAs for therapeutic purposes. A number of proof-of-principle experiments have demonstrated the clinical potential of appropriately designed short interfering RNAs in various diseases including viral infections, cancer and neurodegenerative disorders. Already, in such a short time from their discovery, Acuity Pharmaceuticals (August 2004) and Sirna Therapeutics (September 2004) have filed Investigational New Drug applications with the US FDA to begin clinical trials with modified siRNA molecules in patients with age-related macular degeneration. This review will give a brief overview of the mechanism of RNA interference and applications of the pathway in experimental biology will be discussed. The article will focus on recent developments related to the use of RNA interference technologies in mammalian systems and on potential clinical applications of short interfering RNA-mediated RNA interference.

The Histone Deacetylase Inhibitor, Trichostatin A, Enhances Radiation Sensitivity and Accumulation of GammaH2A.X

Cancer Biology & Therapy. Jul, 2005  |  Pubmed ID: 16082178

Histone deacetylase inhibitors have been shown to induce numerous biologic effects including, altering cell cycle distribution, cytostasis and in certain cases apoptosis. Given their ability to disrupt critical biological processes in cancer cells, these agents are emerging as potential therapeutics for cancer. Recently, it has been identified that histone deacetylase inhibitors can also efficiently enhance the radiation sensitivity of cells, both in vitro and in vivo. In this study, we investigated whether the potent histone deacetylase inhibitor, Trichostatin A, modulates the radiation sensitivity of human erythroleukemic K562 cells. The endpoints we used were clonogenic survival, apoptosis and gammaH2AX immunoprecipitations of soluble chromatin. The findings from clonogenic survival assays indicated that incubation with Trichostatin A 24 hours prior to irradiation enhances the radiation sensitivity of K562 cells. The dose modification factors ranged from 1.1 when cells were incubated with 0.1 microM Trichostatin A to 2.3 at 1 microM Trichostatin A. Similarly, caspase-3 and caspase-7 assays indicated that Trichostatin A potentiates radiation-induced apoptosis in K562 cells, in a concentration dependent manner. Our results suggest the modulation of radiation effects observed at the lower Trichostatin A concentrations was associated with histone hyperacetylation and changes in phosphorylated gammaH2A.X formation on euchromatin. In contrast, at the higher Trichostatin A concentrations mechanisms such as drug-mediated cytotoxicity and G1 cell cycle arrest, contributed to the sensitization effect. More generally, our findings are consistent with those from recent studies and support the development of histone deacetylase inhibitors for use as radiation sensitizers, particularly for targeting radioresistant cancers.

The Paradox of Histone Deacetylase Inhibitor-mediated Modulation of Cellular Responses to Radiation

Cell Cycle (Georgetown, Tex.). Feb, 2006  |  Pubmed ID: 16418577

Given the widespread use of radiotherapy in cancer, there has been a longstanding interest in the development of chemical compounds that can modify cellular responses to ionizing radiation. Additionally, recent terrorism threats suggesting attacks with 'dirty bombs' containing combinations of radioactive isotopes with conventional explosives, has increased the interest in compounds that can protect from radiation injury. Histone deacetylase inhibitors represent a new class of compounds that can modulate the effects of radiation. Research with histone deacetylase inhibitors has largely focussed on the consequences of their ability to alter gene transcription via histone acetylation and on their properties as anti-cancer agents. They have been shown to cause cell cycle and growth arrest, differentiation and in certain cases apoptosis in cell cultures and in vivo. In addition to their intrinsic anti-cancer properties, numerous studies have demonstrated that histone deacetylase inhibitors can modulate cellular responses to other toxicity-inducing modalities including ionizing radiation. The consensus is that histone deacetylase inhibitors markedly enhance the sensitivity of cells to radiation by altering numerous molecular pathways. Intriguingly, a report has also shown that histone deacetylase inhibitors can reduce radiation induced acute and late skin damage using a well-established animal model of cutaneous radiation syndrome. Hence, there is an emerging interest in potential use of histone deacetylase inhibitors as radiation sensitizers or protectors. This review focuses on the different mechanisms by which histone deacetylase inhibitors modify cellular responses to ionizing radiation.

DNA Targeted UVA Photosensitization: Characterization of an Extremely Photopotent Iodinated Minor Groove Binding DNA Ligand

Journal of Photochemistry and Photobiology. B, Biology. Jun, 2006  |  Pubmed ID: 16488619

Previous studies have described UVA-induced DNA strand breakage at the binding sites of iodinated DNA minor groove binding bisbenzimidazoles. The DNA breakage, presumably mediated by the carbon-centred ligand radical produced by photodehalogenation, was also shown to be cytotoxic. The earlier studies included a comparison of three ligand isomers, designated ortho-, meta- and para-iodoHoechst, and the efficiency of photo-induction of strand breaks in plasmid DNA proved to be much higher for the ortho-isomer. We have now extended the comparison of the three isomers with respect to photo-induced cytotoxicity in K562 cells. Although the relationship between the extent of nuclear uptake and the concentration of the ligand in the medium was similar for the three isomers, assay of in situ dehalogenation in drug-treated cells indicated that the apparent cross-section for dehalogenation of the ortho-isomer was greater than 5-fold higher than that for the meta- and para-isomers. Also, analysis of clonogenic survival data showed that the dehalogenation event associated with ortho-iodoHoechst was a more efficient mediator of UVA-induced cytotoxicity in K562 cells than that for meta- or para-iodoHoechst. The number of dehalogenation events associated with 50% cell-kill for ortho-iodoHoechst (1.23+/-0.04 x 10(4)) was less than that for the para- (3.92+/-0.29 x 10(4)) and meta- (11.6+/-0.90 x 10(4)) isomers. Thus it is concluded that the photopotency of ortho-iodoHoechst, which is an important feature in the context of its potential use in clinical phototherapy, is due not only to more efficient UVA-mediated dehalogenation of the ligand, but also to greater cytotoxic potency per dehalogenation event.

Clinical Potential of Histone Deacetylase Inhibitors As Stand Alone Therapeutics and in Combination with Other Chemotherapeutics or Radiotherapy for Cancer

Epigenetics : Official Journal of the DNA Methylation Society. Jul-Sep, 2006  |  Pubmed ID: 17965606

Histone deacetylase inhibitors are emerging as a new class of cancer chemotherapeutics and already are being heralded as the first anti-cancer drugs targeting the epigenome. Through histone hyperacetylation-mediated changes in chromatin conformation and gene expression, histone deacetylase inhibitors induce differentiation, cell cycle arrest, apoptosis, growth inhibition and cell death, which are more pronounced in transformed cell-lines than in normal cells. Additional anti-cancer effects of HDAC inhibitors include inhibition of migration, invasion and angiogenesis in vivo. Indeed, clinical anti-cancer activity has been observed using HDAC inhibitors as single agents or in combination with conventional chemotherapeutics, in phase I and II trials. Furthermore, numerous preclinical studies are suggesting a potential clinical role for HDAC inhibitors in radiotherapy either as radiation sensitizers or protectors. In this article the molecular basis for the clinical potential of HDAC inhibitors, either as stand alone cancer therapeutics or in combination with other chemotherapy agents or ionizing radiation will be overviewed.

The Epigenetic Modifier, Valproic Acid, Enhances Radiation Sensitivity

Epigenetics : Official Journal of the DNA Methylation Society. Jul-Sep, 2006  |  Pubmed ID: 17965607

Valproic acid is an established therapeutic for a variety of seizure disorders and in certain cases for depression and anxiety. In addition, valproic acid has been shown to possess histone deacetylase inhibition activity and is currently being investigated as an anti-cancer agent, either alone or in combination with other conventional cancer therapies such as ionizing radiation. In this study, we investigated whether valproic acid modulates cellular responses to radiation in human erythroleukemic, K562 cells. Hyperacetylation of nuclear histones 3 and 4 was used to correlate the effects of valproic acid to inhibition of histone deacetylase activity, clonogenic survival, apoptosis and apoptosis. The findings from the clonogenic survival and caspase induction assays indicated that pretreatment of cells with valproic acid for 24 hours, markedly enhanced radiation induced cell-death and apoptosis in K562 cells, respectively. Mechanisms involving drug-mediated cytotoxicity and changes in cell cycle distribution were associated with the radiation sensitizing properties of valproic acid, particularly at the higher concentrations. Overall, our findings are consistent with the general consensus that HDAC inhibitors efficiently sensitize cancer cells to the effects of ionizing radiation and support the idea of developing clinically relevant combinations of HDAC inhibitors and radiotherapy.

Receptor-mediated DNA-targeted Photoimmunotherapy

Cancer Research. Nov, 2006  |  Pubmed ID: 17079478

We show the efficacy of a therapeutic strategy that combines the potency of a DNA-binding photosensitizer, UV(A)Sens, with the tumor-targeting potential of receptor-mediated endocytosis. The photosensitizer is an iodinated bibenzimidazole, which, when bound in the minor groove of DNA and excited by UV(A) irradiation, induces cytotoxic lesions attributed to a radical species resulting from photodehalogenation. Although reminiscent of photochemotherapy using psoralens and UV(A) irradiation, an established treatment modality in dermatology particularly for the treatment of psoriasis and cutaneous T-cell lymphoma, a critical difference is the extreme photopotency of the iodinated bibenzimidazole, approximately 1,000-fold that of psoralens. This feature prompted consideration of combination with the specificity of receptor-mediated targeting. Using two in vitro model systems, we show the UV(A) cytotoxicity of iodo ligand/protein conjugates, implying binding of the conjugate to cell receptors, internalization, and degradation of the conjugate-receptor complex, with release and translocation of the ligand to nuclear DNA. For ligand-transferrin conjugates, phototoxicity was inhibited by coincubation with excess native transferrin. Receptor-mediated UV(A)-induced cytotoxicity was also shown with the iodo ligand conjugate of an anti-human epidermal growth factor receptor monoclonal antibody, exemplifying the potential application of the strategy to other cancer-specific targets to thus improve the specificity of phototherapy of superficial lesions and for extracorporeal treatments.

Comparison of Different Classes of Radionuclides for Potential Use in Radioimmunotherapy

Hellenic Journal of Nuclear Medicine. May-Aug, 2007  |  Pubmed ID: 17684582

Currently, beta-emitting radionuclides are used almost exclusively in the clinic and in clinical radioimmunotherapy studies. The main advantage of beta-emitters is the relatively long path length in biological tissue (in the mm range), which is sufficient to irradiate cancer cells that do not have bound radiolabelled antibody (cross-fire effect). This alleviates problems with inadequate uptake and heterogeneous distribution of radiolabelled antibodies in tumours. Hence, beta-emitters provide a relatively uniform radiation dose to the tumour and it is generally accepted that this class of radionuclides is more appropriate for radioimmunotherapy of solid tumours and large tumour burdens (> 0.5 cm). However, the shorter-range alpha-emitters (50-100 mm) and the ultra-short range Auger electron-emitting radionuclides (the majority of electrons traverse a few nm), have been shown to be more efficient than beta-emitters at inducing lethal lesions in single cells. It has been suggested that these classes of radionuclides may have the potential to provide a more favourable therapeutic index than beta-emitters for radioimmunotherapy of single tumour cells in the circulation, micrometastases and in certain cases, minimal residual disease. The aim of this article is to discuss the different classes of radionuclides with potential for clinical use in radioimmunotherapy.

Antibody-based Cancer Treatment with Ultra-short Range Auger Electron-emitting Radionuclides: Dual Receptor and DNA Targeting Strategies

Hellenic Journal of Nuclear Medicine. Sep-Dec, 2007  |  Pubmed ID: 18084655

The long-heralded potential of targeted cancer treatment using monoclonal antibodies is finally being realized. Several antibodies are already used in the oncology clinic and many others are undergoing preclinical evaluation. In addition to the development of unconjugated antibodies, there is intense interest in the potential clinical use of antibodies as vehicles for targeting cytotoxic agents specifically to cancer cells. For example, radioimmunotherapy which involves the use of antibodies to deliver radionuclides to target cells is an approved treatment modality for cancer. Our laboratory is involved in developing technologies for radioimmunotherapy using a unique class of radionuclides, known as Auger electron emitters. A key feature of the Auger electrons emitted by these radionuclides is that they traverse very small ranges (molecular dimensions) in biological tissues. The emission of Auger electrons results in a gradient of energy deposition with the majority of the radiochemical damage occurring in the immediate vicinity (within a few cubic nanometers) of the decaying radionuclide. Therefore, realizing the full potential of Auger electron emitting isotopes in radioimmunotherapy requires more sophisticated approaches than directly radiolabeling anticancer antibodies. Strategies which involve targeting the radionuclide not only to cancer cells but also to the DNA of those cells are necessary. In this paper potential dual, receptor and DNA, targeting systems for radioimmunotherapy with Auger electron-emitting radionuclides are discussed.

Effect of Valproic Acid on Radiation-induced DNA Damage in Euchromatic and Heterochromatic Compartments

Cell Cycle (Georgetown, Tex.). Feb, 2008  |  Pubmed ID: 18239454

The distinction between heterochromatin and euchromatin in the double-strand break (DSB) damage pathway is of interest, recent reports indicate that chromatin is not created equally nor is it acquiescent to DSBs. Using the classical histone deacetylase inhibitor, Trichostatin A, we have previously demonstrated that chromatin represents a heterogeneous substrate with respect to histone tail modification by histone deacetylase inhibitors and consequent responses to DNA damage and repair. Here, we extended the initial findings by investigating the radiation sensitizing properties of the widely used antiepileptic, valproic acid. Clonogenic survival assays confirm that valproic acid is an efficient sensitizer of radiation-induced cell death. The radiosensitizing effect is correlated with valproic acid-mediated histone hyperacetylation, chromatin decondensation and enhanced formation of radiation-induced gammaH2AX preferentially on euchromatic alleles. Heterochromatin was much more resistant to histone tail modification, changes in chromatin architecture and DNA damage. These findings are consolidated by studies with the structurally related analogue, valpromide, which does not inhibit histone deacetylase enzymes. At a relatively low concentration (1 mM) valpromide did not cause chromatin modifications and radiation sensitivity, providing further evidence that the radiation sensitizing properties of valproic acid are at least in part, due to histone modification-dependent effects on euchromatin. When higher concentrations (5 mM) were used, both compounds resulted in significant radiation sensitivity, albeit, with differing efficacy (dose modifying factors of 1.5 and 1.2 for valproic acid and valpromide, respectively). The findings imply that histone-modification independent mechanisms also contribute to the radiation sensitizing properties of valproic acid. Overall, our findings are consistent with the emerging interest in the use histone deacetylase inhibitors in combination with radiotherapy for the treatment of cancer.

Gamma-radiation-induced GammaH2AX Formation Occurs Preferentially in Actively Transcribing Euchromatic Loci

Cellular and Molecular Life Sciences : CMLS. Jan, 2010  |  Pubmed ID: 19859659

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 gamma-irradiation-induced gammaH2AX foci formation in euchromatin and heterochromatin. Here, we have extended those studies and provide evidence that gammaH2AX foci form preferentially in actively transcribing euchromatin following gamma-irradiation.

GammaH2AX As a Molecular Marker of Aging and Disease

Epigenetics : Official Journal of the DNA Methylation Society. Feb, 2010  |  Pubmed ID: 20150765

Double-strand breaks are one of the most critical DNA lesions with respect to cell-death and preservation of genomic integrity. Rapid phosphorylation of the histone variant H2AX at Ser-139 to form gammaH2AX is an early cellular response to DNA double-strand breaks. Visualization of discrete gammaH2AX foci using immunofluorescence-based assays has provided a sensitive and effective method for detecting DSBs which may be implicated in various pathologies including cancer, age-related diseases, chronic inflammatory diseases and ischemia-reperfusion injury. In this review, the potential utility and significance of gammaH2AX as a molecular marker of aging and disease is analysed.

Methylproamine Protects Against Ionizing Radiation by Preventing DNA Double-strand Breaks

Mutation Research. Oct, 2010  |  Pubmed ID: 20732333

The majority of cancer patients will receive radiotherapy (RT), therefore, investigations into advances of this modality are important. Conventional RT dose intensities are limited by adverse responses in normal tissues and a primary goal is to ameliorate adverse normal tissue effects. The aim of these experiments is to further our understanding regarding the mechanism of radioprotection by the DNA minor groove binder, methylproamine, in a cellular context at the DNA level.

Trichostatin A Accentuates Doxorubicin-induced Hypertrophy in Cardiac Myocytes

Aging. Oct, 2010  |  Pubmed ID: 20930262

Histone deacetylase inhibitors represent a new class of anticancer therapeutics and the expectation is that they will be most effective when used in combination with conventional cancer therapies, such as the anthracycline, doxorubicin. The dose-limiting side effect of doxorubicin is severe cardiotoxicity and evaluation of the effects of combinations of the anthracycline with histone deacetylase inhibitors in relevant models is important. We used a well-established in vitro model of doxorubicin-induced hypertrophy to examine the effects of the prototypical histone deacetylase inhibitor, Trichostatin A. Our findings indicate that doxorubicin modulates the expression of the hypertrophy-associated genes, ventricular myosin light chain-2, the alpha isoform of myosin heavy chain and atrial natriuretic peptide, an effect which is augmented by Trichostatin A. Furthermore, we show that Trichostatin A amplifies doxorubicin-induced DNA double strand breaks, as assessed by γH2AX formation. More generally, our findings highlight the importance of investigating potential side effects that may be associated with emerging combination therapies for cancer.

Epigenome Targeting by Probiotic Metabolites

Gut Pathogens. 2010  |  Pubmed ID: 21172038

The intestinal microbiota plays an important role in immune development and homeostasis. A disturbed microbiota during early infancy is associated with an increased risk of developing inflammatory and allergic diseases later in life. The mechanisms underlying these effects are poorly understood but are likely to involve alterations in microbial production of fermentation-derived metabolites, which have potent immune modulating properties and are required for maintenance of healthy mucosal immune responses. Probiotics are beneficial bacteria that have the capacity to alter the composition of bacterial species in the intestine that can in turn influence the production of fermentation-derived metabolites. Principal among these metabolites are the short-chain fatty acids butyrate and acetate that have potent anti-inflammatory activities important in regulating immune function at the intestinal mucosal surface. Therefore strategies aimed at restoring the microbiota profile may be effective in the prevention or treatment of allergic and inflammatory diseases.

Double-strand Breaks and the Concept of Short- and Long-term Epigenetic Memory

Chromosoma. Apr, 2011  |  Pubmed ID: 21174214

Double-strand breaks represent an extremely cytolethal form of DNA damage and thus pose a serious threat to the preservation of genetic and epigenetic information. Though it is well-known that double-strand breaks such as those generated by ionising radiation are among the principal causative factors behind mutations, chromosomal aberrations, genetic instability and carcinogenesis, significantly less is known about the epigenetic consequences of double-strand break formation and repair for carcinogenesis. Double-strand break repair is a highly coordinated process that requires the unravelling of the compacted chromatin structure to facilitate repair machinery access and then restoration of the original undamaged chromatin state. Recent experimental findings have pointed to a potential mechanism for double-strand break-induced epigenetic silencing. This review will discuss some of the key epigenetic regulatory processes involved in double-strand break (DSB) repair and how incomplete or incorrect restoration of chromatin structure can leave a DSB-induced epigenetic memory of damage with potentially pathological repercussions.

Evaluation of the Efficacy of Radiation-modifying Compounds Using γH2AX As a Molecular Marker of DNA Double-strand Breaks

Genome Integrity. 2011  |  Pubmed ID: 21261999

Radiation therapy is a widely used therapeutic approach for cancer. To improve the efficacy of radiotherapy there is an intense interest in combining this modality with two broad classes of compounds, radiosensitizers and radioprotectors. These either enhance tumour-killing efficacy or mitigate damage to surrounding non-malignant tissue, respectively. Radiation exposure often results in the formation of DNA double-strand breaks, which are marked by the induction of H2AX phosphorylation to generate γH2AX. In addition to its essential role in DDR signalling and coordination of double-strand break repair, the ability to visualize and quantitate γH2AX foci using immunofluorescence microscopy techniques enables it to be exploited as an indicator of therapeutic efficacy in a range of cell types and tissues. This review will explore the emerging applicability of γH2AX as a marker for monitoring the effectiveness of radiation-modifying compounds.

Effects of Oral Intake of Water in Patients with Oropharyngeal Dysphagia

BMC Geriatrics. 2011  |  Pubmed ID: 21356121

Dysphagia is associated with numerous medical conditions and the major intervention to avoid aspiration in people with dysphagia involves modifying the diet to thickened fluids. This is associated with issues related to patient quality of life and in many cases non-compliance leading to dehydration. Given these concerns and in the absence of conclusive scientific evidence, we designed a study, to further investigate the effects of oral intake of water in people with dysphagia.

Photosensitization by Iodinated DNA Minor Groove Binding Ligands: Evaluation of DNA Double-strand Break Induction and Repair

Journal of Photochemistry and Photobiology. B, Biology. May, 2011  |  Pubmed ID: 21440453

Iodinated DNA minor groove binding bibenzimidazoles represent a unique class of UVA photosensitizer and their extreme photopotency has been previously characterized. Earlier studies have included a comparison of three isomers, referred to as ortho-, meta- and para-iodoHoechst, which differ only in the location of the iodine substituent in the phenyl ring of the bibenzimidazole. DNA breakage and clonogenic survival studies in human erythroleukemic K562 cells have highlighted the higher photo-efficiency of the ortho-isomer (subsequently designated UV(A)Sens) compared to the meta- and para-isomers. In this study, the aim was to compare the induction and repair of DNA double-strand breaks induced by the three isomers in K562 cells. Further, we examined the effects of the prototypical broad-spectrum histone deacetylase inhibitor, Trichostatin A, on ortho-iodoHoechst/UVA-induced double-strand breaks in K562 cells. Using γH2AX as a molecular marker of the DNA lesions, our findings indicate a disparity in the induction and particularly, in the repair kinetics of double-strand breaks for the three isomers. The accumulation of γH2AX foci induced by the meta- and para-isomers returned to background levels within 24 and 48 h, respectively; the number of γH2AX foci induced by ortho-iodoHoechst remained elevated even after incubation for 96 h post-irradiation. These findings provide further evidence that the extreme photopotency of ortho-iodoHoechst is due to not only to the high quantum yield of dehalogenation, but also to the severity of the DNA lesions which are not readily repaired. Finally, our findings which indicate that Trichostatin A has a remarkable potentiating effect on ortho-iodoHoechst/UVA-induced DNA lesions are encouraging, particularly in the context of cutaneous T-cell lymphoma, for which a histone deacetylase inhibitor is already approved for therapy. This finding prompts further evaluation of the potential of combination therapies.

Histone Deacetylase Inhibitors Augment Doxorubicin-induced DNA Damage in Cardiomyocytes

Cellular and Molecular Life Sciences : CMLS. Dec, 2011  |  Pubmed ID: 21584806

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.

Chromatin Modifying Agents - the Cutting Edge of Anticancer Therapy

Drug Discovery Today. Jul, 2011  |  Pubmed ID: 21664485

Chromatin modifying compounds are emerging as the next generation of anticancer therapies. By altering gene expression they could be able to correct uncontrolled proliferation and, in certain cases, aberrant apoptotic pathways, which are hallmarks of malignant cells. The modulation of gene expression is regulated via chromatin remodelling processes that include DNA methylation and chromatin modifications. The identification of aberrant methylation of genes and dysregulated histone acetylation status in cancer cells provides a basis for novel epigenetic therapies. Currently available chromatin modifying agents, a group that includes DNA methyltransferase and histone deacetylase inhibitors, exert anticancer effects by reactivating tumour suppressor genes, inhibiting proliferation and inducing apoptosis. It is anticipated that massive parallel sequencing will identify new epigenetic targets for drug development.

Investigation into the Biological Properties of the Olive Polyphenol, Hydroxytyrosol: Mechanistic Insights by Genome-wide MRNA-Seq Analysis

Genes & Nutrition. Sep, 2011  |  Pubmed ID: 21953375

The medicinal properties of the leaves and fruit of Olea Europaea (olive tree) have been known since antiquity. Numerous contemporary studies have linked the Mediterranean diet with increased health. In particular, consumption of olive oil has been associated with a decreased risk of cardiovascular disease and certain cancers. Increasingly, there has been an interest in the biological properties of polyphenols, which are minor constituents of olive oil. For example, hydroxytyrosol has been shown to be a potent antioxidant and has anti-atherogenic and anti-cancer properties. The overall aim of this study was to provide insights into the molecular mechanisms of action of hydroxytyrosol using genome-wide mRNA-Seq. Initial experiments were aimed at assessing cytotoxicity, apoptosis and cell cycle effects of hydroxytyrosol in various cell lines. The findings indicated a dose-dependent reduction in cell viability in human erythroleukemic K562 and human keratinocytes. When comparing the viability in parental CEM-CCRF and R100 cells (which overexpress the P-glycoprotein pump), it was determined that the R100 cells were more resistant to effects of hydroxytyrosol suggesting efflux by the multi-drug resistance pump. By comparing the uptake of Hoechst 33342 in the two cell lines that had been pretreated with hydroxytyrosol, it was determined that the polyphenol may have P-glycoprotein-modulating activity. Further, initial studies indicated modest radioprotective effects of relatively low doses of hydroxytyrosol in human keratinocytes. Analysis of mRNA sequencing data identified that treatment of keratinocytes with 20 μM hydroxytyrosol results in the upregulation of numerous antioxidant proteins and enzymes, including heme oxygenase-1 (15.46-fold upregulation), glutaredoxin (1.65) and glutathione peroxidase (1.53). This may account for the radioprotective activity of the compound, and reduction in oxidative stress suggests a mechanism for chemoprevention of cancer by hydroxytyrosol. Alteration in the expression of transcription factors may also contribute to the anti-cancer effects described in numerous studies. These include changes in the expression of STAT3, STAT6, SMAD7 and ETS-1. The telomerase subunit TERT was also found to be downregulated in K562 cells. Overall, our findings provide insights into the mechanisms of action of hydroxytyrosol, and more generally, we identify potential gene candidates for further exploration.

Potential Non-oncological Applications of Histone Deacetylase Inhibitors

American Journal of Translational Research. 2011  |  Pubmed ID: 22046487

Histone deacetylase inhibitors have emerged as a new class of anticancer therapeutic drugs. Their clinical utility in oncology stems from their intrinsic cytotoxic properties and combinatorial effects with other conventional cancer therapies. To date, the histone deacetylase inhibitors suberoylanilide hydroxamic acid (Vorinostat, Zolinza®) and depsipeptide (Romidepsin, Istodax®) have been approved by the US Food and Drug Administration for the treatment of refractory cutaneous T-cell lymphoma. Further, there are currently over 100 clinical trials involving the use of histone deacetylase inhibitors in a wide range of solid and hematological malignancies. The therapeutic potential of histone deacetylase inhibitors has also been investigated for numerous other diseases. For example, the cytotoxic properties of histone deacetylase inhibitors are currently being harnessed as a potential treatment for malaria, whereas the efficacy of these compounds for HIV relies on de-silencing latent virus. The anti-inflammatory properties of histone deacetylase inhibitors are the predominant mechanisms for other diseases, such as hepatitis, systemic lupus erythematosus and a wide range of neurodegenerative conditions. Additionally, histone deacetylase inhibitors have been shown to be efficacious in animal models of cardiac hypertrophy and asthma. Broad-spectrum histone deacetylase inhibitors are clinically available and have been used almost exclusively in preclinical systems to date. However, it is emerging that class- or isoform-specific compounds, which are becoming more readily available, may be more efficacious particularly for non-oncological applications. The aim of this review is to provide an overview of the effects and clinical potential of histone deacetylase inhibitors in various diseases. Apart from applications in oncology, the discussion is focused on the potential efficacy of histone deacetylase inhibitors for the treatment of neurodegenerative diseases, cardiac hypertrophy and asthma.

Protective Effects of Valproic Acid Against Airway Hyperresponsiveness and Airway Remodeling in a Mouse Model of Allergic Airways Disease

Epigenetics : Official Journal of the DNA Methylation Society. Dec, 2011  |  Pubmed ID: 22139576

Airway remodeling and airway hyperresponsiveness are major aspects of asthma pathology that are not targeted optimally by existing anti-inflammatory drugs. Histone deacetylase inhibitors have a wide range of effects that may potentially abrogate aspects of remodeling. One such histone deacetylase inhibitor is valproic acid (2-propylvaleric acid). Valproic acid is used clinically as an anti-epileptic drug and is a potent inhibitor of class I histone deacetylases but also inhibits class II histone deacetylases. We used valproic acid as a molecular model of histone deacetylase inhibition in vivo in chronic allergic airways disease mice with airway remodeling and airway hyperresponsiveness. Wild-type Balb/c mice with allergic airways disease were treated with valproic acid or vehicle control. Airway inflammation was assessed by bronchoalveolar lavage fluid cell counts and examination of lung tissue sections. Remodeling was assessed by morphometric analysis of histochemically stained slides and lung function was assessed by invasive plethysmography measurement of airway resistance. Valproic acid treatment did not affect inflammation parameters; however, valproic acid treatment resulted in reduced epithelial thickness as compared to vehicle treated mice (p < 0.01), reduced subepithelial collagen deposition (p < 0.05) and attenuated airway hyperresponsiveness (p < 0.05 and p < 0.01 for the two highest doses of methacholine, respectively). These findings show that treatment with valproic acid can reduce structural airway remodeling changes and hyperresponsiveness, providing further evidence for the potential use of histone deacetylase inhibitors for the treatment of asthma.

Utility of γH2AX As a Molecular Marker of DNA Double-Strand Breaks in Nuclear Medicine: Applications to Radionuclide Therapy Employing Auger Electron-Emitting Isotopes

Current Radiopharmaceuticals. Jan, 2011  |  Pubmed ID: 22191615

There is an intense interest in the development of radiopharmaceuticals for cancer therapy. In particular, radiopharmaceuticals which involve targeting radionuclides specifically to cancer cells with the use of monoclonal antibodies (radioimmunotherapy) or peptides (targeted radiotherapy) are being widely investigated. For example, the ultra-short range Auger electron-emitting isotopes, which are discussed in this review, are being considered in the context of DNAtargeted radiotherapy. The efficient quantitative evaluation of the levels of damage caused by such potential radiopharmaceuticals is required for assessment of therapeutic efficacy and determination of relevant doses for successful treatment. The DNA double-strand break surrogate marker, γH2AX, has emerged as a useful biomonitor of damage and thus effectiveness of treatment, offering a highly specific and sensitive means of assessment. This review will cover the potential applications of γH2AX in nuclear medicine, in particular radionuclide therapy.

Protection by Methylproamine of Irradiated Human Keratinocytes Correlates with Reduction of DNA Damage

International Journal of Radiation Biology. Mar, 2011  |  Pubmed ID: 21087168

The therapeutic ratio for ionising radiation treatment of tumour is a trade-off between normal tissue side-effects and tumour control. Application of a radioprotector to normal tissue can reduce side-effects. Here we study the effects of a new radioprotector on the cellular response to radiation. Methylproamine is a DNA-binding radioprotector which, on the basis of published pulse radiolysis studies, acts by repair of transient radiation-induced oxidative species on DNA. To substantiate this hypothesis, we studied protection by methylproamine at both clonogenic survival and radiation-induced DNA damage, assessed by γH2AX (histone 2AX phosphorylation at serine 139) focus formation endpoints.

Genetic and Epigenetic Events in Diabetic Wound Healing

International Wound Journal. Feb, 2011  |  Pubmed ID: 21159125

The prevalence of the chronic metabolic disorder, diabetes mellitus, is expected to increase in the coming years and worldwide pandemic levels are predicted. Inevitably, this will be accompanied by an increase in the prevalence of diabetic complications, including diabetic foot ulcers. At present, treatment options for diabetic foot ulcers are in many cases insufficient, and progression of the condition results in the requirement for limb amputation in a proportion of patients. To improve therapy, an increase in our understanding of the pathobiology of diabetic complications such as impaired wound healing is necessary. In this review, recent advances in molecular aspects of normal and impaired diabetic wound healing are discussed. Furthermore, investigations of the role of epigenetic processes in the pathogenesis of impaired diabetic wound healing are now emerging. Indeed, epigenetic changes have already been identified as key factors in diabetes and related complications and these are overviewed in this review.

Influence of Natural and Synthetic Histone Deacetylase Inhibitors on Chromatin

Antioxidants & Redox Signaling. Feb, 2012  |  Pubmed ID: 22229817

Abstract Significance: Histone deacetylase inhibitors (HDACIs) have emerged as a new class of anticancer therapeutics. The hydroxamic acid, suberoylanilide hydroxamic acid (Vorinostat, Zolinza™), and the cyclic peptide, depsipeptide (Romidepsin, Istodax™), were approved by the U.S. Food and Drug Administration (FDA) for the treatment of cutaneous T-cell lymphoma in 2006 and 2009, respectively. At least 15 HDACIs are currently undergoing clinical trials either alone or in combination with other therapeutic modalities for the treatment of numerous hematological and solid malignancies. Recent Advances: The potential utility of HDACIs has been extended to nononcologic applications, including autoimmune disorders, inflammation, diseases of the central nervous system, and malaria. Critical Issues: Given the promise of HDACIs, there is growing interest in the potential of dietary compounds that possess HDAC inhibition activity. This review is focused on the identification of and recent findings with HDACIs from dietary, medicinal plant, and microbial sources. We discuss the mechanisms of action and clinical potential of natural HDACIs. Future Directions: Apart from identification of further HDACI compounds from dietary sources, further research will be aimed at understanding the effects on gene regulation on lifetime exposure to these compounds. Another important issue that requires clarification. Antioxid. Redox Signal. 00, 000-000.

Histone Deacetylases and Their Role in Asthma

The Journal of Asthma : Official Journal of the Association for the Care of Asthma. Mar, 2012  |  Pubmed ID: 22233225

Objective. The aim of this article is to provide an overview of the classical histone deacetylase (HDAC) enzymes and HDAC inhibitors. The discussion is focused on the potential anti-asthmatic effects of this group of compounds. Methods. Medline was used with the search terms, "asthma and HDAC," "asthma and Trichostatin A," "asthma and valproic acid," "allergic airways disease and HDAC," "allergic airways disease and Trichostatin A," and "allergic airways disease and valproic acid." Manuscripts from the past decade were accessed. Historical literature dating from the 1960s was accessed for the use of anti-epileptics in the treatment of asthma. Results. Preliminary clinical trials with anti-epileptic drugs including the well-known HDAC inhibitor, valproic acid, have shown long-lasting anti-asthmatic effects providing the basis for the evaluation of this class of compounds in asthma. Studies using the prototypical HDAC inhibitor, Trichostatin A, in well-established murine models of allergic airways disease have also indicated beneficial effects. Conclusion. Although the precise mechanisms are still controversial, inhibition of airway hyperresponsiveness and agonist-induced contraction as well as anti-inflammatory effects have been described for HDAC inhibitors in asthma.

Molecular Model of Naphthalene-induced DNA Damage in the Murine Lung

Human & Experimental Toxicology. Jan, 2012  |  Pubmed ID: 21508073

Airway epithelial damage and repair represents a novel therapeutic target in asthma and chronic obstructive pulmonary disease. An established mouse model of airway epithelial damage involves the Clara cell cytotoxicity of parenterally administered naphthalene, an important environmental toxicant with genotoxic and carcinogenic potential. The objective of the current study was to investigate naphthalene-induced toxicity and to identify and quantify DNA double-strand breaks in a murine naphthalene model of airway epithelial damage. Male C57/BL6 mice were injected with 200 mg/kg naphthalene and culled at 12-, 24-, 48- and 72-h time points. Lung function and bronchoalveolar lavage was performed and the lungs were dissected for histological analysis and for quantitation of DNA double-strand breaks using γH2AX as a molecular marker. Mice injected with naphthalene had increased epithelial denudation, bronchoalveolar lavage fluid cellularity and reactivity to nebulized methacholine chloride as compared to corn oil vehicle controls. Histological changes were most pronounced at the 12- and 24-h time points. DNA double-strand breaks, quantitated as the number of γH2AX foci per cell, were highest at the 24- and 48-h time points. All parameters had decreased at the 72-h time point, consistent with airway re-epithelization and cellular repair. Our findings indicate a time-dependent accumulation of γH2AX foci in mouse airway epithelial cells following administration of naphthalene. Naphthalene airway epithelial injury constitutes a model of DNA double-strand breaks in mice, which can be adapted as a suitable model for further investigation of genotoxic damage for evaluating the efficacy of potential therapeutics.

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