Tom C. Karagiannis

γH2AX: a sensitive molecular marker of DNA damage and repair

Phosphorylation of the Ser-139 residue of the histone variant H2AX, forming γH2AX, is an early cellular response to the induction of DNA double-strand breaks. Detection of this phosphorylation event has emerged as a highly specific and sensitive molecular marker for monitoring DNA damage initiation and resolution. Further, analysis of γH2AX foci has numerous other applications including, but not limited to, cancer and aging research. Quantitation of γH2AX foci has also been applied as a useful tool for the evaluation of the efficacy of various developmental drugs, particularly, radiation modifying compounds. This review focuses on the current status of γH2AX as a marker of DNA damage and repair in the context of ionizing radiation. Although the emphasis is on γ-radiation-induced γH2AX foci, the effects of other genotoxic insults including exposure to ultraviolet rays, oxidative stress and chemical agents are also discussed.

Histone deacetylase inhibitors (HDACIs): multitargeted anticancer agents.

Histone deacetylase (HDAC) inhibitors are an emerging class of therapeutics with potential as anticancer drugs. The rationale for developing HDAC inhibitors (and other chromatin-modifying agents) as anticancer therapies arose from the understanding that in addition to genetic mutations, epigenetic changes such as dysregulation of HDAC enzymes can alter phenotype and gene expression, disturb homeostasis, and contribute to neoplastic growth. The family of HDAC inhibitors is large and diverse. It includes a range of naturally occurring and synthetic compounds that differ in terms of structure, function, and specificity. HDAC inhibitors have multiple cell type-specific effects in vitro and in vivo, such as growth arrest, cell differentiation, and apoptosis in malignant cells. HDAC inhibitors have the potential to be used as monotherapies or in combination with other anticancer therapies. Currently, there are two HDAC inhibitors that have received approval from the US FDA for the treatment of cutaneous T-cell lymphoma: vorinostat (suberoylanilide hydroxamic acid, Zolinza) and depsipeptide (romidepsin, Istodax). More recently, depsipeptide has also gained FDA approval for the treatment of peripheral T-cell lymphoma. Many more clinical trials assessing the effects of various HDAC inhibitors on hematological and solid malignancies are currently being conducted. Despite the proven anticancer effects of particular HDAC inhibitors against certain cancers, many aspects of HDAC enzymes and HDAC inhibitors are still not fully understood. Increasing our understanding of the effects of HDAC inhibitors, their targets and mechanisms of action will be critical for the advancement of these drugs, especially to facilitate the rational design of HDAC inhibitors that are effective as antineoplastic agents. This review will discuss the use of HDAC inhibitors as multitargeted therapies for malignancy. Further, we outline the pharmacology and mechanisms of action of HDAC inhibitors while discussing the safety and efficacy of these compounds in clinical studies to date.

Genome-wide analysis distinguishes hyperglycemia regulated epigenetic signatures of primary vascular cells

Emerging evidence suggests that poor glycemic control mediates post-translational modifications to the H3 histone tail. We are only beginning to understand the dynamic role of some of the diverse epigenetic changes mediated by hyperglycemia at single loci, yet elevated glucose levels are thought to regulate genome-wide changes, and this still remains poorly understood. In this article we describe genome-wide histone H3K9/K14 hyperacetylation and DNA methylation maps conferred by hyperglycemia in primary human vascular cells. Chromatin immunoprecipitation (ChIP) as well as CpG methylation (CpG) assays, followed by massive parallel sequencing (ChIP-seq and CpG-seq) identified unique hyperacetylation and CpG methylation signatures with proximal and distal patterns of regionalization associative with gene expression. Ingenuity knowledge-based pathway and gene ontology analyses indicate that hyperglycemia significantly affects human vascular chromatin with the transcriptional up-regulation of genes involved in metabolic and cardiovascular disease. We have generated the first installment of a reference collection of hyperglycemia-induced chromatin modifications using robust and reproducible platforms that allow parallel sequencing-by-synthesis of immunopurified content. We uncover that hyperglycemia-mediated induction of genes and pathways associated with endothelial dysfunction occur through modulation of acetylated H3K9/K14 inversely correlated with methyl-CpG content.

RNA interference and potential therapeutic applications of short interfering RNAs

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.

Transferrin Receptor-Mediated Endocytosis: A Useful Target for Cancer Therapy

Current cancer management strategies fail to adequately treat malignancies with multivariable dose-restricting factors such as systemic toxicity and multi-drug resistance limiting therapeutic benefit, quality of life and complete long-term remission rates. The targeted delivery of a therapeutic compound aims to enhance its circulation and cellular uptake, decrease systemic toxicity and improve therapeutic benefit with disease specificity. The transferrin peptide, its receptor and their biological significance, has been widely characterised and vastly relevant when applied to targeting strategies. Utilising knowledge about the physiological function of the transferrin–transferrin receptor complex and the efficiency of its receptor-mediated endocytosis provides rationale to continue the development of transferrin-targeted anticancer modalities. Furthermore, multiple studies report an upregulation in expression of the transferrin receptor on metastatic and drug resistant tumours, highlighting its selectivity to cancer. Due to the increased expression of the transferrin receptor in brain glioma, the successful delivery of anticancer compounds to the tumour site and the ability to cross the blood brain barrier has shown to be an important discovery. Its significance in the development of cancer-specific therapies is shown to be important by direct conjugation and immunotoxin studies which use transferrin and anti-transferrin receptor antibodies as the targeting moiety. Such conjugates have demonstrated enhanced cellular uptake via transferrin-mediated mechanisms and increased selective cytotoxicity in a number of cancer cell lines and tumour xenograft animal models. In addition, incubation of chemotherapy-insensitive cancer cells with transferrin-targeted conjugates in vitro has resulted in a reversal of their drug resistance. Transferrin immunotoxins have also shown similar promise, with a diphtheria toxin mutant covalently bound to transferrin (Tf-CRM107) currently involved in human clinical trials for the treatment of glioblastoma. Despite this, the inability to translate preliminary research into a clinical setting has compelled research into novel targeting strategies including the use of nanoparticulate theory in the design of drug delivery systems. The main objective of this review is to evaluate the importance of the transferrin–transferrin receptor complex as a target for cancer therapy through extensive knowledge of both the physiological and pathological interactions between the complex and different cell types. In addition, this review serves as a summary to date of direct conjugation and immunotoxin studies, with an emphasis on transferrin as an important targeting moiety in the directed delivery of anticancer therapeutic compounds.

Will broad-spectrum histone deacetylase inhibitors be superseded by more specific compounds?

Histone deacetylase (HDAC) inhibitors can induce differentiation, cell cycle and growth arrest or in certain cases apoptosis in cancer cells. In a remarkably short period of time, especially considering that their mechanism of action remains largely undefined, HDAC inhibitors have realized both success and failure as therapeutics for cancer in clinical trials. Notably, the pleiotropic HDAC inhibitors, suberoylanilide hydroxamic acid (SAHA) and depsipeptide, have shown efficacy in a wide range of cancers, in particular for cutaneous T-cell lymphoma (CTCL), and are progressing in phase II clinical studies. However, evidence is accumulating that specific HDAC enzymes are important with respect to clinical efficacy, calling the usefulness of the classical inhibitors into question. Class I enzymes are being heralded as the most clinically relevant, however, this is still controversial and much of the information is in the private domain. Nevertheless, the potential to alter the expression of a more focused, disease-related subset of genes and to limit adverse effects has prompted the development of isoform-specific HDAC inhibitors. Here, we consider the growing view that broad-spectrum HDAC inhibitors may be superseded by more specific compounds.

Modulation of cellular radiation responses by histone deacetylase inhibitors

Histone deacetylase (HDAC) inhibitors are emerging as a new class of targeted cancer chemotherapeutics. Several HDAC inhibitors are currently in clinical trials and promising anticancer effects at well-tolerated doses have been observed for both hematologic and solid cancers. HDAC inhibitors have been shown to induce cell-cycle and growth arrest, differentiation and in certain cases apoptosis in cell cultures and in vivo. However, it is known that these compounds induce varying responses in different cells and biological settings, and identifying their precise mechanisms of action is an area of great interest. Important findings are continually expanding our understanding of the cellular effects of HDAC inhibitors and recent studies will be briefly outlined in this review. In addition to their intrinsic anticancer properties, numerous studies have demonstrated that HDAC inhibitors can modulate cellular responses to other cytotoxic modalities including ionizing radiation, ultraviolet radiation and chemotherapeutic drugs. Hence, there is a growing interest in potential clinical use of HDAC inhibitors in combination with conventional cancer therapies. In this review, the interaction of HDAC inhibitors with other anticancer agents is discussed. The focus of the article is on the different mechanisms by which HDAC inhibitors enhance the sensitivity of cells to the effects of ionizing radiation.

Cancer metabolism and the Warburg effect: the role of HIF-1 and PI3K.

Cancer cells have been shown to have altered metabolism when compared to normal non-malignant cells. The Warburg effect describes a phenomenon in which cancer cells preferentially metabolize glucose by glycolysis, producing lactate as an end product, despite being the presence of oxygen. The phenomenon was first described by Otto Warburg in the 1920s, and has resurfaced as a controversial theory, with both supportive and opposing arguments. The biochemical aspects of the Warburg effect outline a strong explanation for the cause of cancer cell proliferation, by providing the biological requirements for a cell to grow. Studies have shown that pathways such as phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) as well as hypoxia inducible factor-1 (HIF-1) are central regulators of glycolysis, cancer metabolism and cancer cell proliferation. Studies have shown that PI3K signaling pathways have a role in many cellular processes such as metabolism, inflammation, cell survival, motility and cancer progression. Herein, the cellular aspects of the PI3K pathway are described, as well as the influence HIF has on cancer cell metabolism. HIF-1 activation has been related to angiogenesis, erythropoiesis and modulation of key enzymes involved in aerobic glycolysis, thereby modulating key processes required for the Warburg effect. In this review we discuss the molecular aspects of the Warburg effect with a particular emphasis on the role of the HIF-1 and the PI3K pathway.

γH2AX as a molecular marker of aging and disease

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 γH2AX is an early cellular response to DNA double-strand breaks. Visualization of discrete γH2AX 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 γH2AX as a molecular marker of aging and disease is analyzed.

Disparity of histone deacetylase inhibition on repair of radiation-induced DNA damage on euchromatin and constitutive heterochromatin compartments

Epigenetic regulation of chromatin structure is central to the process of DNA repair. A well-characterized epigenetic feature is the dynamic phosphorylation of the histone H2AX (γH2AX) and mobilization of double strand break (DSB) recognition and repair factors to the site. How chromatin structure is altered in response to DNA damage and how such alterations influence DSB repair mechanisms are currently relevant issues. Despite the clear link between histone deacetylases (HDACs) and radiosensitivity, how histone hyperacetylation influence DSB repair remains poorly understood. We have determined the structure of chromatin is a major factor determining radiosensitivity and repair in human cells. Trichostatin A (TSA) enhances radiosensitivity with dose modification factors of 1.2 and 1.9 at 0.2 and 1 μM, respectively. Cells treated with TSA causing hyperacetylation and remodelling on euchromatic alleles coexist with γH2AX accumulation in radiosensitized cells. Formation of γH2AX on heterochromatin was significantly reduced even when cells were treated with TSA, suggesting that chromatin structure and histone hyperacetylation are pronounced features of radiation sensitivity and repair in euchromatic regions.