- freely available
Int. J. Mol. Sci. 2010, 11(7), 2715-2745; doi:10.3390/ijms11072715
Published: 16 July 2010
Abstract: The free radical nitric oxide (NO•) is known to play a dual role in human physiology and pathophysiology. At low levels, NO• can protect cells; however, at higher levels, NO• is a known cytotoxin, having been implicated in tumor angiogenesis and progression. While the majority of research devoted to understanding the role of NO• in cancer has to date been tissue-specific, we herein review underlying commonalities of NO• which may well exist among tumors arising from a variety of different sites. We also discuss the role of NO• in human physiology and pathophysiology, including the very important relationship between NO• and the glutathione-transferases, a class of protective enzymes involved in cellular protection. The emerging role of NO• in three main areas of epigenetics—DNA methylation, microRNAs, and histone modifications—is then discussed. Finally, we describe the recent development of a model cell line system in which human tumor cell lines were adapted to high NO• (HNO) levels. We anticipate that these HNO cell lines will serve as a useful tool in the ongoing efforts to better understand the role of NO• in cancer.
NO• is a free radical which was discovered in 1980 as a ubiquitous diffusible second messenger. While some authors use the term “nitric oxide” to refer to any of the nitric oxide reactive species (NO•, NO−, and NO+), in biological settings, nitric oxide usually refers to the free radical. NO• was determined to be a prominent constituent of what was then called endothelium-derived relaxing factor (EDRF) . Later in 1985, Eschericia coli lipopolysacchride (LPS) was found to initiate production of NO• by LPS-stimulated mouse macrophages . We now know the molecule plays a significant role in both normal and abnormal physiology of human beings, as well as plants and invertebrates [3–5]. NO• has a well characterized two-step synthetic metabolic pathway in which L-arginine is first converted to NG-hydroxy-L-arginine and then to L-citrulline and NO•. This reaction is catalyzed by the enzymatic family of nitric oxide synthases (NOSs). Three NOS isoforms exist: 1) nNOS/NOS1, a calcium-dependent enzyme discovered in neurons that is involved in neural transmission; 2) iNOS/NOS2, a calcium-independent enzyme that releases large amounts of NO• in response to macrophage activation with endotoxin and cytokines, and is involved in cytotoxicity; and 3) eNOS/NOS3, also a calcium-dependent enzyme that is constitutively expressed, isolated from endothelial cells, and is found in normal vascular endothelium [6,7]. Once NO• is produced, it can react with various molecules, resulting in more stable compounds such as S-nitrosothiols, metal adducts, peroxynitrites (while in the presence of oxygen), and tetrahydrobiopterin (THB) . THB is recognized as a necessary prerequisite for the biosynthesis of key aromatic amino acid hydroxylase enzyme precursors necessary for synthesis of neurotransmitters such as serotonin, melatonin, epinephrine and dopamine.
It is believed NO• regulates the physiology and pathophysiology of the body through one of three biomolecular mechanisms: 1) redox interactions with thiols, 2) coordinating interactions with metal functional centers, and 3) through protein kinase activity . The role and impact of NO• is believed to be widespread because of its ability to cross cell membranes in an unaltered chemical form, diffuse rapidly, interact with key generative and target cell-response proteins, and quickly interact with key transition metal containing proteins .
The various biomolecular mechanisms of NO• result in numerous biological functions, including: 1) antitumor and microbial immunity, including against gram positive organisms , 2) immuno-modulation and allo-antigenicity [9,10], and 3) a signaling pathway . Nearly every cell throughout the body has the ability to express calcium-independent iNOS . Within the central nervous system, learning, sleep, feeding, male and female reproductive behavior are all impacted by NO• . It also influences the neurotransmitters in synapses between peripheral organs  and regulates angiogenesis and neurogenesis after stroke activity . NO• also delays the aging of oocytes , controls resting potential in skeletal muscle , regulates contraction-excitation coupling , and modulates chondrocyte development during endochondral ossification . Significantly, NO• depravation is a critical underlying cause for endothelial dysfunction, which in turn is a key common contributor to diabetes-related cardiovascular disease, myocardial infarction, and atherosclerosis [16,17].
NO• in low concentrations is now known to have benign, modulating, and regulatory effects on normal mammalian and human biology and physiology. Higher concentration levels of NO• are now shown to be both damaging and pathologic to physiologic processes . What is defined as a high or low level can vary enormously depending upon which physiologic system the free radical is found and the apparent contradictory functional impact of NO• presence on a particular molecular mechanism and biochemical microenvironment . For instance, at NO• concentrations of less than 100 nM, cyclic guanosine monophosphate (cGMP), cGMP-dependent protein kinase (PKG) and extracellular signal regulated kinase (ERK) activation can occur. As concentration levels increase Akt is phosphorylated, and in ranges between 300 to 800 nM hypoxia inducible factor-1 alpha and p53 are stabilized . As concentrations increase further, nitrosation and oxidative processes become prominent and initiate stressful cellular events . However, the role of NO• can be either protective or toxic, depending upon the unique biochemical content of the microenvironment in which it exists .
It is also now well established that NO• plays a multifaceted and contradictory role in the biology and growth of tumors . Over-expression of NOS has been shown to be responsible for tumor angiogenesis and maintaining vascular tone within tumor blood vessel systems [22–25], as well as the facilitation of neoplastic transformation [22,26,27]. Studies have shown that in cancer patients, NO• regulates blood flow to tumors, and by down-regulating NO• synthesis, a distinct vasoconstricting event results . This has been demonstrated through the use of N-nitro-L-arginine (L-NNA) to reduce blood flow to tumors in BD9 rats with P22 carcinosarcoma [22,28]. In humans cancer patients reducing NOS results in an increase in blood pressure [22,29,30]. At higher concentrations in the proper microenvironment, for an extended period of time, NO• exposure initiates inflammation, can stimulate tumor growth and/or metastatic behavior [6,7], and can lead to mutations and the clinical presentation of cancer [9,31,32]. Exogenous sources of NO•, such as cigarette smoke, contribute to subcellular damage through the formation of N-nitrosoamines and N-nitrosamides, contributing to elevated expression of head and neck cancers [7,33–36].
It is also well recognized that reactive oxygen species (ROSs) play an important function in either the protective or pathologic expression of NO• reactions with oxygen [6,7]. ROSs provide beneficial impacts through killing of microorganisms and malignant cells, or a pathologic effect, again depending upon the microenvironment. Higher concentrations of ROS generate oxidative and nitrosative environmental stressors leading to: 1) DNA damage, 2) down-regulated antioxidants, and 3) an impact on transcription/translation activities, thereby generally impairing normal cellular function [7,37,38]. Should DNA become damaged from either endogenous or exogenous sources of NO•, a number of defensive apoptotic systems are initiated to protect against unwanted cellular transformation [6,7]. Amongst the most important defensive apoptotic systems is the up regulation of DNA damage sensing proteins, such as p53. Damage not repaired typically results in cellular death through apoptosis. NO• is also known to inhibit caspase activation, which in turn is known to induce normal apoptosis. Studies have also shown NO• prohibits apoptosis in a variety of cell types, as well as in some tumor cells [39,40].
Thiols are a group of biological molecules which act as intracellular antioxidants. Among the most studied forms is glutathione (GSH), known to react with and neutralize electrophilic centers of a number of environmental and oxidative cellular stressors. The enzyme which catalyzes these reactions is glutathione S-transferase-pi (GST-pi), one of a family of Phase II detoxifying glutathione S-transferases (GSTs) responsible not only for detoxification, but also activation of significant biochemical pathways essential to normal physiology . These oxidative stressors include elevated levels of NO•. The GST isoenzymes and their behavior are essential to providing yet another tool toward protection of DNA from a variety of endogenous and exogenous pathogenic sources [42–44]. Equally important, the GSTs catalyze the conjugation of GSH to an array of xenobiotic or toxic compounds rendering them non-toxic . Due to these behaviors GSTs are an important area of research in molecular biology.
While GST isoenzymes relieve the source of toxicity, the catalytic group is also strongly implicated in the development of cellular resistance to anti-cancer drug therapy [42,45]. It provides a mechanism to explain cancer patients’ observed resistance to anticancer drug therapies . Tumor cell lines which over-express GST-pi have heightened detoxification responses and acquire increased resistance to compounds perceived by the body as being toxic, including chemotherapeutic drugs [42,45,46]. GST isoenzymes are categorized into three primary groups: 1) cytosolic, 2) membrane-bound microsomal, and 3) mitochondrial . The cytosolic type is further divided into seven classes: 1) Alpha, 2) Mu, 3) Omega, 4) Pi, 5) Sigma, 6) Theta, and 7) Zeta . GST-pi is now recognized to be the predominant isoform subclass [42,44].
The GST-pi gene has four functional polymorphisms (GSTP1*A, GSTP1*B, GSTP1*C, and GSTP1*D), with each allelic genotype having different treatment response outcomes among individual cancer patients [45,47]. Examples include: 1) GSTP1*A is responsible for acquired resistance to cisplatin treatment due to creation of platinum-GSH conjugates , 2) GSTP1*B, which in certain circumstances, is associated with an impaired ability to detoxify platinum based therapeutic treatment , and 3) patients testing positive for GSTP1*C appear to experience breast cancer with less frequency . All polymorphisms have been shown to impact, to varying degrees: 1) anticancer therapy treatment, 2) chemotherapeutic response, and 3) susceptibility to cancer. Most importantly, GSTP1 has been reported to be over-expressed in a number of different tumor types including: colon, lymphoma, pancreas, breast, NSCLC and ovarian [42,51]. One effort analyzed GST enzymes, GST composition, and GSH concentration levels in normal and squamous cell carcinoma tissues among 25 patients (14 with oropharyngeal or oral tumors, 11 with laryngeal tumors) . GST-pi levels increased in 11 of the 14 oral cavity tumors, and elevated expression of GST-pi was found in all laryngeal tumors . Another report provides evidence for heighten risk of relapse for laryngeal cancer associated with GST-pi over-expression . Others propose the possibility that up-regulated GST-pi, GST-mu, and GST-alpha can be predictors of a second primary tumor in head and neck cancers . Additional studies demonstrate over-expression of GST-pi within normal mucosa adjacent to tumors, dysplastic mucosal lesions, and head & neck squamous cell carcinoma (HNSCC) [54,55]. GST-pi expression increases through a step-wise progression, correlating with up-regulated NOS and molecular markers of oxidative injury [54,55]. It has been hypothesized that GST-pi is over-expressed in mucosal cells in response to oxidative injury by toxins such as NO• and known nitrosative carcinogens resulting from smoking cigarettes [54,55].
Presented herein is a study in which we investigated GST-pi expression in laryngeal tumors (Table 1). Patient charts were reviewed for TNM stage and course of treatment. Tissue sections were reviewed, and the intensity of tumor staining was graded on an immunohistochemical scale (0–4).
In ten patients, seven had previously undergone radiation therapy; five of the seven patients were concurrently treated with chemotherapy and radiation therapy. All patients failed treatment or had recurrence or persistent disease. The seven patients who received radiation exhibited higher levels of GST-pi expression than the three patients who were not treated with radiation. Figure 1 shows examples of GST-pi immunostaining observed in human this study.
To investigate the commonality among squamous cell carcinomas arising in different sites, we also investigated the NOS and GST-pi expression of cervical squamous cell carcinomas (CSCC). Presented herein are results showing expression of eNOS, iNOS, and GST-pi in a series of patients with CSCC. Patient charts were reviewed for TNM stage, tumor grade, and course of treatment. All samples were obtained prior to treatment. Tissue sections were reviewed; the intensity of tumor staining was graded on an immunohistochemical scale (0–3). Table 2 summarizes the results.
Both iNOS and GST-pi were highly expressed in CSCC, whereas eNOS showed only limited expression. Examples of the observed staining are shown in Figure 2. The eNOS expression in CSCC was in contrast to previously reported HNSCC work which showed highly expressed eNOS [56,57].
Another reported study confirms our findings that NO• is a significant contributor to cervical cancer, and suggests a link between NO• and a number of prominent risk factors associated with the onset of cervical cancer. These factors include: 1) chronic inflammation, 2) HPV infections, 3) extended use of oral contraceptives, 4) sexually transmitted diseases, and 5) smoking tobacco . All of these factors cause increases in NO• levels [58–61] and markers of NO•-mediated mutagenesis in patients with cervical intraepithelial neoplasia [58,62,63].
3. Reactive Nitrogen Species
The role of reactive nitrogen species (RNSs) has been well documented for many decades. The impact of RNSs originates in inflammatory tissues and can result in mutations in tumor suppressor genes, leading to subsequent tumor neoplastic growth . RNSs also cause post-translational modifications of proteins involved in fundamental cellular functions such as apoptosis, cell cycle check point, and DNA repair . RNSs are known to cause both oxidation and nitration reactions resulting in DNA strand breaks, mutations in DNA base pairs, and helix modifications . What has also become increasingly evident over time is how important the molecular composition of the microenvironment is relative to the degree of DNA alterations. The molecular composition of the microenvironment is influenced by a number of RNS factors, including: 1) biomolecular profile, 2) type, 3) concentration level, 4) accessibility, 5) bioavailability, and 6) half life . RNSs can evolve further into a variety of related molecules including 4-hydroxynoneal (4-HNE) and reactive aldehydes-malondialdehydes (MDA), both of which are associated with increased cancer risk in chronic inflammatory diseases [65–67]. Both 4-HNE and MDA are also known to cause point mutations within tumor suppressor genes [65–67]. Further, RNSs play a critical role as a facilitator between signal transduction receptors such as the MAPK signaling cascade. This can lead to the expression of proto-oncogenes such as c-Jun, c-Fos, and AP-1. These proto-oncogenes impact differentiation, proliferation, cellular death, and transformation [65,68,69]. Free radical exposure is also well recognized to cause post-translational modifications which affect the functionality of key cellular proteins. For example, exposure to NO•, an abundant RNS, leads to post-translational modifications of both p53 and Rb tumor suppressor genes at critical concentration levels [65,70,71]. Exposure to NO• also activates DNA repair and signal transduction species including DNA protein kinases [65,72,73].
4. Epigenetics and NO•
In the early 1940’s C.H. Waddington first used “epigenetics” to describe the mechanisms responsible for the developmental pathway from fertilized egg to an adult [74–76]. Epigenetics is known to regulate primary biological functions, including, but not limited to: 1) memory function, 2) development and aging, 3) mobile elements activity, 4) genomic imprinting, 5) viral infections, 6) somatic gene therapy, 7) cloning, 8) X-inactivation, and 9) the biology of cancer [77–81]. The list of diseases associated with epigenetic dysregulation continues to grow as research efforts progress [77–81]. Over time and with the expanded knowledge base created by the efforts of many, the term has evolved to reference the heritable modifications to chromatin, which regulate gene expression, but do not change the underlying DNA sequence [75,78,82]. The impact on chromatin composition can be rapid and reversible, originating from endogenous and exogenous sources and which may well modulate gene expression behavior [47,74,75,82].
Gene expression and silencing can be carried out via a number of interrelated epigenetic mechanisms that may be modulated by NO•. These mechanisms include, but are not limited to: 1) DNA methylation [75,82–84], 2) microRNA (miRNA) [82,85,86], and 3) histone modifications [75,78,82,83,85,87–89]. The three mechanisms combine synergistically to regulate and affect epigenetic programming and reprogramming behavior [82,85,90–97]. Herein we discuss these three mechanisms and the emerging research to date as to how these may be affected by NO•. Interestingly, a recent study involving Duchenne muscular dystrophy indicated that a diminution of NO• results in global epigenetic changes, thereby implicating NO• as an “epigenetic molecule” .
4.1. DNA Methylation
Chromatin is made up of nucleosomes. The nucleosomes are comprised of DNA (146–147 base pairs in length, depending upon the literature cited [82,84]) and histones. The DNA is wrapped in a left-handed super-helix 1.7 times surrounding a core complex of eight histones , two each of H2A, H2B, H3, and H4 [82,86]. Each histone within the core has two active functional regions: 1) a “histone-fold” area to facilitate histone-to-histone and histone-to-DNA interactions in nucleosomes, and 2) a NH2-terminal with COOH-terminal “tails,” which are the sites for post-translational modifications that include methylation, phosphorylation, ubiquitination, and acetylation . The tails also appear to facilitate linkage between other nucleosomes and/or DNA . Chromatin also allows DNA molecules, comprised of millions of nucleotides, hundreds of millions of base pairs in length, to be housed highly compressed within the cell nucleus [82,98]. Less tightly bound chromatin usually has more reactive sites available for histone alterations, which in turn reversibly modify chromatin structure .
The DNA methylation and chromatin reconfiguration processes have equally prominent, yet reversible roles in mediating the genome into transcriptionally expressed or unexpressed segments [77,78]. Some patterns of DNA methylation remain constant throughout adulthood, while others are reversible. The on-going and mutable role of histones is reversible and also facilitates the silencing or unsilencing of gene expression. Tumorigenesis is a key example of pathological dysregulation in chromatin remodeling, or a lack of normalcy in DNA methylation processing behavior [77,78]. DNA methylation involves the addition of a methyl group at the carbon 5 position of the cytosine ring. The event is reversible and is a significant factor in gene expression [77,78]. It takes place primarily within the 5’CG3’ (also known as the CpG dinucleotide or CpG loci or sites), which are usually depleted and irregularly positioned throughout the genome with weakly concentrated locations. However, more dense areas known as CpG islands also exist [89,90,93,99]. CpG sites are usually methylated whereas the CpG islands are unmethylated. As we age, this mechanism reverses with intermittent methylation of the CpG islands taking place with a corresponding loss of overall methylation patterns throughout the genome; this is prominent with oncogenic events [82,90,99]. Abnormal or DNA hypermethylation patterns are known to impact promoter regions, which in turn, silence genes and are strongly evident in most cancers. Methylation anomalies also fail concurrently to express many tumor suppressor genes, further contributing to oncogenesis [82,90,91,99]. The specific relationship among CpG island hypermethylation activity, genetic alteration, and epigenetic inactivation of tumor suppressor genes is currently being studied in colorectal cancers (CRCs) . CpG islands exist in about 50% of all human genes within promoter regions, and when hypermethylated, result in transcriptional silencing and tumor suppressor gene activity being down-regulated . A smaller group of CRCs demonstrate extensive methylation behavior referred to as CpG island methylator phenotype (CIMP). There are three principle mechanisms driving genomic instability in CRCs: 1) CIMP, 2) microsatellite instability (MSI), a unique phenotype within CRC, and 3) chromosomal instability (CIN). All three mechanisms all contribute to epigenetically alter gene expression in CRCs [101,102].
DNA methylation is facilitated by DNA methyltransferases (DNMTs), including DNMT 1, DNMT 3A, and DNMT 3B [92,99]. Collectively, all three enzymes ensure proper DNA methylation patterns [95,99]. DNA methylation (see Table 3 below) is being studied because cancer cells display elevated levels of altered DNA methylation patterns when compared to normal cells [77,78]. There are a variety of tumor types with associated hypermethylation of at least one gene, including: lung cancer, breast cancer, leukemia, and hematologic diseases [77,78,94,96,97]. Certain patterns of hypomethylation can also contribute to the formation of other cancer types as well, including but not limited to: metastatic hepatocellular cancer, cervical cancer, prostate and B-cell chronic lymphocytic leukemia [77,78,103–106]. It has been reported that transcription is impeded when methylcytosine binding domain proteins (MBDs) bind to methylated DNA. This binding process interferes with the interdependent relationship between the DNA methylation and chromatin reconfiguration processes, thereby precluding further gene transcription [78,90].
An additional study demonstrated that NO• regulates chromatin and gene expression through inactivation of nuclear and cytoplasmic proteins by tyrosine-(Tyr) nitration and/or S-nitrosylation/nitrosation . In S-nitrosylation, primary, tertiary and quaternary protein architecture is affected ; in Tyr-nitration, the impact is more widespread across a variety of proteins . Although NO• is diffusible, it must also exist physically within the proper microenvironment and limited macroenvironment with proteins/substrates in order to react. Evidence of this includes the presence of iNOS in the caveolae of endothelial cells, in neurons, and within the nucleus [5,134]. Additionally, it has been suggested that NO•, through S-nitrosylation, impacts a number of targets: 1) a variety of transcription factors, including tissue specific transcription factors, 2) some oncoproteins, 3) DNA binding, and 4) transactivation of nuclear receptors . NO•-mediated changes on transcription factors through Tyr-nitration have also been reported; these changes primarily through impacting normal protein-to-protein interactions and limiting nuclear localization . Table 4 lists the impact of NO• on various epigenetic modulators.
It has further been suggested that DNA is susceptible to transition metal-mediated reductive/oxidative modifications which can affect double helix strength [5,135]. Others have hypothesized NO• may be impacting a “genome-wide oscillation” and expression/suppression of hundreds of genes, through reactions with thiols/cystine residues in partnership with Fe2+ ions (both of which are believed to reside in chromatin) [5,136,137]. This “genome-wide oscillation” could remain in place with oscillating cycles, constructing and deconstructing—of protein complexes, resulting in a corresponding impact on transcription processing [5,138]. Finally, there is evidence to suggest synthesis of NO• within the nucleus due to the presence of THB enzymes and two isoforms of NOS (eNOS and iNOS) [5,139].
MicroRNAs are relatively small, non-coding RNAs, typically 20–23 nucleotides in size. miRNAs originate from 60–110 nucleotide fold-back RNA precursors  and have an enormous impact on the control of gene expression [99,140,141]. The biosynthetic pathway originates from proteins of the Argonaute family. These proteins are transcribed first via RNA polymerase II, and then by RNases III Drosha and DGCR8. Finally, in the cytoplasm, RNA III Dicer transforms the proteins into the fully functioning miRNA [99,140]. Typically miRNAs act as post-transcriptional regulators by impeding protein production of specific messenger RNA (mRNA) molecular species and apparently interacting through base-pairing between the 5’-end tails of miRNA (nucleotides 2–8) and the anti-parallel sequences of the 3’-untranslated (3’-UTRs) areas within selected mRNAs [141–144]. Originally believed to be relatively small in number, there are now over 460 human miRNAs identified , with the possibility of greater than 1,000 in existence . They have been linked to aberrant cell growth patterns and appear to play a dual role in both oncogenesis and tumor suppression, depending upon which portion of the genome is being affected. For example, the miR-17-92 cluster has a role in tumor neovascularization, when c-myc activates transcription [145,146]. This gene is found to be over-expressed in the miR17-92 cluster in B-cell lymphomas and lung cancers. c-Myc also contributes to tumor angiogenesis, as well as tumor growth in mouse B-cell lymphoma [145–148]. It has also been demonstrated that human cancer types can be classified using expression patterns of miRNA . Furthermore, at least one significant relational link has been identified between DNA methylation patterns and miRNA; the strength of this relationship is possibly affected by changing the amounts of DNMT1, DNMT 3A, and/or DNMT 3B present [98,150,151].
As is the case with DNA hypermethylation, regulating the abnormal activity levels of certain miRNAs could be important in initiating and controlling tumorigenesis. It may be possible to target these species through interventions or blocking drugs, such that the miRNAs serve as therapeutic molecular markers. Increasing efforts are also being directed towards providing evidence to support the development of miRNAs as diagnostic and prognostic products . A few examples include: 1) stimulating apoptosis in cultured glioblastoma cells by deprogramming miR-21 (an oncogenic miRNA) [152,153], 2) up-regulating miR-372 and 373 in testicular germ cell tumors [99,154], and 3) over-expressing miR-155 in both breast cancers and B-cell lymphomas [99,155–157]. Initial clinical results using epigenetic drugs, such as 4-phenylbutyric acid (PBA) and 5-Aza-2’-deoxycytidine (5-Aza-CdR) exhibit the capability to up-regulate miR-127, which in turn down-regulates Bcl6. This finding provides hope that additional therapeutic options will become available by developing drugs that act via epigenetic mechanisms [99,158]. It also suggests that epigenetic drugs may be able to up-regulate tumor-suppressor genes abnormally de-programmed epigenetically, while also causing miRNAs to turn off oncogenic mRNAs [99,159].
As research on miRNAs continues to emerge, a number of studies have found a link between NO• expression and a number of different miRNAs. In one study, human umbilical vein endothelial cells (HUVECs) were exposed to prolonged unidirectional shear stress, which resulted in the significant up-regulation of 13 miRNAs . Among the 13 miRNAs identified, miR-21 exhibited the greatest level of up-regulation. miR-21 serves as a regulator of smooth muscle apoptosis  and has been found to be regulated in both cardiac hypertrophy  and human tumors . Notably, HUVECs which over-expressed miR-21 exhibited increased eNOS phosphorylation and NO• production, as well as decreased apoptosis. Similarly, another study found that decreasing the levels of miR-145—another smooth muscle miRNA regulator—resulted in decreased NO• expression .
Two other miRNAs have been shown to indirectly modulate iNOS expression: miR-155 and miR-661. Mice transfected with miR-155 exhibited reduced expression of Suppressor of Cytokine Signal-1, and in turn, enhanced iNOS expression . In a different study, human liver cancer cells expressing the hepatitis B virus transactivator protein HBx were studied . When the miRNA miR-661 was depleted in these HBx-expressing cells, HBx activity was impaired, leading to enhanced iNOS and nitrite production.
4.3. Histone Modifications
Histones are yet another fundamental epigenetic pathway mechanism and are influential in both transcriptional and post-translational modifications [5,82]. These proteins are positively attracted to the more negatively charged DNA molecules present, making them particularly susceptible to post-transcriptional changes in DNA binding through: 1) acetylation, 2) methylation, 3) phosphorylation, 4) ubiquitination, 5) SUMOylation (small ubiquinine-like modifier), and 6) isomerization [5,78]. More specifically, they also include: 1) lysine acetylation, 2) lysine and arginine methylation, 3) serine and threoine phosphorylation, 4) lysine ubiquitylation, and 5) lysine SUMOylation, with over 60 modification sites currently known . Histone post-translational modifications occur in the globular domains and the amino-terminal tails [82,167,168], and along with ATP–dependent chromatin remodeling, are among the most significant influencers of gene expression . Coupled with DNA methylation activities, these histone mechanisms collectively create an adaptive epigenetic environment [82,169]. This is of particular importance towards understanding the enormous impact the histone post-translation changes can have on chromatin steric formation. By altering the molecular landscape, it transforms transcriptional regulators to interact with cis-DNA binding elements . This pattern has been studied and verified in lysine acetylation [82,170].
Histone modification activities take place primarily through two groups of enzymes. The first, histone acetyltranferases (HATs) is comprised of three classes: GNAT, MYST, and CBP/p300. They are characterized by the ability to transfer acetyl groups from acetyl-CoA to amino-ɛ groups for lysines within H3 and H4 and are principally responsible for the opening up of chromatin structure, thereby permitting access for transcription processes to take place . The second group, histone deacetylases (HADCs) reverse the process, resulting in a tightening or constriction of chromatin, making the epigenome less accessible to reactions . There are four classes of HDAC enzymes . Class I consists of HDAC1, HDAC2, HDAC3, and HDAC8. Class II is further divided into two subgroups: IIa, which includes HDAC4, HDAC5, HDAC6, and HDAC7; and IIb, which includes HDAC9 and HDAC10. Class III consists of the sirtuins (SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, and SIRT7) [173,174]. Class IV is comprised of one enzyme, HDAC11 [172,174]. All play significant roles in human physiology and processes, ranging from embryogenesis and cellular differentiation to tumorigenesis, by facilitating deacetylating enzymatic reactions [175–177]. Significantly, HATs are drawn to promoter locations to become part of protein complexes, and many transcriptional co-activators demonstrate HAT enzymatic characteristics . Co-activators have emerged as a significant participant in chemical signaling between systemic and cellular metabolism, including regulating both mitochondrial oxidative metabolism and the balance between lipid, glucose, and energy homeostatic functionality . There are a number of other histone modification pathways which we will not explore within the limitations of this review including, but not limited to: 1) histone methyltransferases, 2) lysine and arginine methyltransferases, and 3) non-SET K-methyltransferases. There is synergy and transience between the HATs and HDACs which creates an oscillating-feedback loop and reversible gene transcription control mechanism environment. Table 5 indicates some of the cancers believed attributable to dysregulated histone protein behavior within the epigenome.
The most recent advances in epigenetic research have been aided by a concurrent evolution of many laboratory techniques, including: 1) cDNA microarray, 2) restriction landmark genomic scanning, 3) CpG island microarrays, and 4) sodium bisulfate conversion [77,78,179]. Sodium bisulfate is particularly useful in differentiating areas of normal and abnormal methylation activity by converting unmethylated cytosines to uracil, while leaving methylated cytosines intact. There are a number of methods useful in exploring CpG island methylation patterns, including: 1) combined bisulfate restriction analyses, 2) methylation-sensitive single nucleotide extension, 3) methylation-sensitive single-strand conformational polymorphism, and 4) methylation-specific polymerase chain reaction assays [77,78,179,180]. Research efforts have also indicated that preemptive assessment of methylation patterns can predict possible malignancies and aid in more timely detection and diagnosis of tumors [77,78].
While much progress has already been made, it is clear that the analytical techniques used to study epigenetics are still evolving. For example, researchers have attempted to analytically differentiate between DNA cytosine methylation (5mC), and hydroxymethylcytosine (hmC). 5mC is involved with transcriptional repression, while the functionality of hmC is still unknown. A recent report concludes the two compounds are “experimentally indistinguishable” from one another using established 5mC mapping criteria and suggest existing 5mC data-bases should be re-examined to ensure that no hmC data has been included erroneously .
To date, only limited data exists regarding the role of NO• in histone modification. A recent study has shown that eNOS gene expression relies upon underlying epigenetic causal mechanisms . It was found that when the human eNOS gene in vascular endothelial cells is expressed, the promoter region is free of DNA methylation, and histone complexes initiate post-transcriptional changes. H3 lysine 4 methylation, H3 lysine 9 acetylation, and H4 lysine 12 acetylation all impacted chromatin by inducing an open steric formation. These reactions thereby permit access by appropriate transcription factors and mechanisms, most important among them, RNA polymerase II at the eNOS promoter location . In contrast, the iNOS gene was found to be silent in cultured endothelial cells containing hypermethylated CpG dinucleotides within the promoter while they are complexed with the methyl-binding protein, MeCP2. These reactions result in silencing of post-translational histones H3 lysine 9 methylation and are suggested to be prevalent in transcriptionally unexpressed heterochromatin. Sterically, the chromatin structure is tightly configured, and RNA polymerase II is not present . It is also postulated that this and other studies provide evidence that non-expressing cells have the necessary transcriptional mechanisms to directly affect eNOS expression, and more significantly, a chromatin-linked down-regulating system which prevents eNOS from being expressed in non-endothelial cells [82,182–184].
A different study found that the hyporesponsiveness of the iNOS promoter in humans is at least partially due to epigenetic silencing in direct response to the hypermethylation of CpG dinucleotides and histone H3 lysine 9 methylation . More specifically, the study found that the iNOS promoter was highly methylated at CpG dinucleotides in various human endothelial cells and vascular smooth muscle cells, two cell types in which iNOS induction is known to be difficult. Furthermore, a human pulmonary adenocarcinoma cell line (A549), a colon adenocarcinoma cell line (DLD-1), and primary hepatocyte cell cultures are all capable of iNOS induction . The iNOS promoter is hypomethylated in DLD-1 cells that have been treated with a DNA methyltransferase. This stimulates both global and iNOS promoter DNA hypomethylation. Use of a chromatin immunoprecipitation assay showed significant presence of methyl-CpG-binding transcriptional repressor MeCP2 within the iNOS promoter location in these endothelial cells. In its entirety the study provided a definition of chromatin-based epigenetic mechanisms controlling human iNOS gene expression .
5. Biological Model System
As indicated earlier in this review, our prior work has focused on the role of NO• in both squamous cell carcinomas (head & neck, cervix) and adenocarcinomas (lung, breast). We and others have reported a spectrum of NOS expression in patient populations of these tumors, as well as other tumor types. It has also been observed that patients who present with and/or progress to high levels of NOS expression portend to have poorer clinical outcomes than those with low level expression. It has been hypothesized that immune system cells are being killed by the comparatively high free radical NO• environment encountered in the tumor bed . Since there is no practical way to study this in human patients, we sought to produce a unique, in vitro tissue culture model system of free radical stressed tumor cells to determine if in fact they could adapt to increasing levels of NO•. The resulting model system would mimic the spectrum of NO• expression found clinically .
Our model system was developed by “adapting” low NO• expressing cell lines to increasing levels of NO• donor. These “parent” cells were gradually exposed to high NO• (HNO) levels, resulting in a new set of HNO cell lines. DETA-NONOate was selected as the NO• donor for the adaptation process due to: a) its high level of free radical donation (two moles of NO• per mole of DETA-NONOate), and relatively long half-life (approximately 24 h. at 37 °C and pH 7.4). During the adaptation process, the cell lines successfully withstood incremental increases of 25 μM DETA-NONOate. For each cell line, the adaptation endpoint was selected as the concentration in which the exogenous NO• introduced to the cells was lethal to the parent cell lines. At this endpoint concentration, the HNO cells still grow robustly and are not morphologically altered from the original (untreated) parent cells. Six different parent/HNO cell line pairs have already been developed: one human lung adenocarcinoma cell line (A549) , one mouse lung adenocarcinoma (LP07) , and four human breast adenocarcinomas (T-47D, Hs578t, BT-20, and MCF-7) . Ongoing work is focusing on extending this model to human head & neck, colon, prostate, and liver tumor cell lines.
While the A549 cells were adapted to DETA-NONOate (see Figure 3 below), the A549-HNO cell lines were also found to be resistant to other nitrogen-based free radical donors [186,187]. This suggests the A549-HNO cell line could have been generated by using any appropriate NO• donor, and that the cells were adapted to the NO• free radical, and not the donor per se [186,187].
Additionally, the lung and breast tumor HNO cell lines were exposed to various concentrations of hydrogen peroxide (H2O2), an oxygen-based free radical donor [186,187]. The HNO cell lines were more resistant to exposure than the corresponding parent cell lines (see Figure 4 as an example). These results show that the HNO cells are similarly resistant to oxygen-based free radicals.
The reported adaptation process resulted in major biological changes, between the parent and HNO cells despite the identical morphology between the two. HNO cancer cell lines exhibited more aggressive growth than did their corresponding parent cell lines under both normal and low-nutrient growth conditions [186,187].
The HNO adapted cell lines are comparable to aggressive, fast growing tumors growing in high NO• environments, while the parent cell lines represent less aggressive, slower growing tumors existing in relatively lower NO• environments. Furthermore, our adaptation process demonstrated that long-term NO• exposure can alter slow growing, less resistant tumors, into faster growing and more resistant cancer cells [186,187]. The molecular mechanism for this parent-to-HNO transformation remains to be elucidated; however, high concentration levels of NO• (above 1 μM) are known to increase nitrosative cellular stress, which interferes with DNA repair and inhibits zinc finger complexes [187–189]. Our model system has proven that tumor cells are able to adapt to comparatively high NO• concentrations, regardless of tumor origin or their histological type. Understanding the role of NO• in tumor cells may in part lie with NO•-mediated epigenetics.
As was discussed above, epigenetic alterations that involve aberrant DNA methylation of CpG sequences in genes is increasingly being recognized as a key mechanism involved in transcriptional silencing of genes in both disease states and, healthy ageing populations [65,82,190]. Our HNO-adapted cell line system provides a robust, in vitro model for the identification of novel genetic targets that are associated with antioxidant stress. We also have evidence that, relative to the MCF-7 breast cancer parent cells, the HNO adapted MCF-7 cells demonstrate a significant increase in hypermethylation of both HPP1 (70-fold increase) and APC (22-fold increase) tumor suppressor genes (see Figure 5 below).
Research of NO• has evolved greatly over time. The protective/cytotoxic duality of NO•, once in question, is now generally accepted. As such, current studies are now more intently focused on understanding the role of NO• in cellular toxicity, particularly as it relates to tumor development and progression. The association between NOS and GST may be a key component of this story, given that over-expression of NOS (regardless of isotype) is often observed in parallel with the over-expression of GST (particularly GST-pi). As discussed above, these results are consistent across a number of different tumor types, suggesting that NO• behavior may be more consistent across different tumor types than originally thought. Whether this commonality in NO• behavior exists among different tumor types will become more apparent as research is further pursued in the field. The HNO cell line model system described above may prove to be a valuable tool for such studies. Similarly, work will continue in the area of NO• and epigenetics. While epigenetic research is still in its infancy, it is already clear that NO• may play an important role in a number of epigenetic functions, including DNA methylation, microRNAs, and histone modifications. Thus, while much is already known about the biological role of NO•, even more has yet to be discovered.
References and Notes
- Furchgott, RF; Zawadzki, JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 1980, 288, 373–376, doi:10.1038/288373a0. 6253831
- Stuehr, DJ; Marletta, MA. Mammalian nitrate biosynthesis: Mouse macrophages produce nitrite and nitrate in response to escherichia coli lipopolysaccharide. Proc. Natl. Acad. Sci. USA 1985, 82, 7738–7742, doi:10.1073/pnas.82.22.7738. 3906650
- Jacklet, JW. Nitric oxide signaling in invertebrates. Invert. Neurosci 1997, 3, 1–14, doi:10.1007/BF02481710. 9706698
- Wilson, ID; Neill, SJ; Hancock, JT. Nitric oxide synthesis and signalling in plants. Plant Cell Environ 2008, 31, 622–631, doi:10.1111/j.1365-3040.2007.01761.x. 18034772
- Illi, B; Colussi, C; Grasselli, A; Farsetti, A; Capogrossi, MC; Gaetano, C. No sparks off chromatin: Tales of a multifaceted epigenetic regulator. Pharmacol. Ther 2009, 123, 344–352, doi:10.1016/j.pharmthera.2009.05.003. 19464317
- Bentz, BG; Simmons, RL; Haines, GK, III; Radosevich, JA. The yin and yang of nitric oxide: Reflections on the physiology and pathophysiology of no. Head Neck 2000, 22, 71–83, doi:10.1002/(SICI)1097-0347(200001)22:1<71::AID-HED11>3.0.CO;2-G. 10585608
- Mocellin, S; Bronte, V; Nitti, D. Nitric oxide, a double edged sword in cancer biology: Searching for therapeutic opportunities. Med. Res. Rev 2007, 27, 317–352, doi:10.1002/med.20092. 16991100
- Stamler, JS. Redox signaling: Nitrosylation and related target interactions of nitric oxide. Cell 1994, 78, 931–936, doi:10.1016/0092-8674(94)90269-0. 7923362
- Hibbs, JB; Vavrin, A; Taintor, RR. L-arginine is required for expression of the activated macrophage effector mechanism causing selective metabolic inhibition in target cells. J. Immunol 1987, 138, 550–565. 2432129
- Langrehr, JM; Hoffman, RA; Lancaster, JR, Jr; Simmons, RL. Nitric oxide--a new endogenous immunomodulator. Transplantation 1993, 55, 1205–1212, doi:10.1097/00007890-199306000-00001. 8516803
- Garthwaite, J. Concepts of neural nitric oxide-mediated transmission. Eur. J. Neurosci 2008, 27, 2783–2802, doi:10.1111/j.1460-9568.2008.06285.x. 18588525
- Chen, J; Zacharek, A; Zhang, C; Jiang, H; Li, Y; Roberts, C; Lu, M; Kapke, A; Chopp, M. Endothelial nitric oxide synthase regulates brain-derived neurotrophic factor expression and neurogenesis after stroke in mice. J. Neurosci 2005, 25, 2366–2375, doi:10.1523/JNEUROSCI.5071-04.2005. 15745963
- Goud, AP; Goud, PT; Diamond, MP; Abu-Soud, HM. Nitric oxide delays oocyte aging. Biochemistry 2005, 44, 11361–11368, doi:10.1021/bi050711f. 16114873
- Stamler, JS; Meissner, G. Physiology of nitric oxide in skeletal muscle. Physiol. Rev 2001, 81, 209–237. 11152758
- Teixeira, CC; Agoston, H; Beier, F. Nitric oxide, c-type natriuretic peptide and cgmp as regulators of endochondral ossification. Dev. Biol 2008, 319, 171–178, doi:10.1016/j.ydbio.2008.04.031. 18514181
- Hayashi, T; Yano, K; Matsui-Hirai, H; Yokoo, H; Hattori, Y; Iguchi, A. Nitric oxide and endothelial cellular senescence. Pharmacol. Ther 2008, 120, 333–339, doi:10.1016/j.pharmthera.2008.09.002. 18930078
- Spinetti, G; Kraenkel, N; Emanueli, C; Madeddu, P. Diabetes and vessel wall remodelling: From mechanistic insights to regenerative therapies. Cardiovasc. Res 2008, 78, 265–273, doi:10.1093/cvr/cvn039. 18281374
- Thomas, DD; Espey, MG; Ridnour, LA; Hofseth, LJ; Mancardi, D; Harris, CC; Wink, DA. Hypoxic inducible factor 1alpha, extracellular signal-regulated kinase, and p53 are regulated by distinct threshold concentrations of nitric oxide. Proc. Natl. Acad. Sci. USA 2004, 101, 8894–8899, doi:10.1073/pnas.0400453101. 15178764
- Ridnour, LA; Thomas, DD; Mancardi, D; Espey, MG; Miranda, KM; Paolocci, N; Feelisch, M; Fukuto, J; Wink, DA. The chemistry of nitrosative stress induced by nitric oxide and reactive nitrogen oxide species. Putting perspective on stressful biological situations. Biol. Chem 2004, 385, 1–10, doi:10.1515/BC.2004.001. 14977040
- Yu, Z; Kuncewicz, T; Dubinsky, WP; Kone, BC. Nitric oxide-dependent negative feedback of parp-1 trans-activation of the inducible nitric-oxide synthase gene. J. Biol. Chem 2006, 281, 9101–9109. 16464859
- Mocellin, S. Nitric oxide: Cancer target or anticancer agent? Curr. Cancer Drug Targets 2009, 9, 214–236, doi:10.2174/156800909787581015. 19275761
- Ng, QS; Goh, V; Milner, J; Stratford, MR; Folkes, LK; Tozer, GM; Saunders, MI; Hoskin, PJ. Effect of nitric-oxide synthesis on tumour blood volume and vascular activity: A phase I study. Lancet Oncol 2007, 8, 111–118, doi:10.1016/S1470-2045(07)70001-3. 17267325
- Gratton, JP; Lin, MI; Yu, J; Weiss, ED; Jiang, ZL; Fairchild, TA; Iwakiri, Y; Groszmann, R; Claffey, KP; Cheng, YC; et al. Selective inhibition of tumor microvascular permeability by cavtratin blocks tumor progression in mice. Cancer Cell 2003, 4, 31–39, doi:10.1016/S1535-6108(03)00168-5. 12892711
- Murohara, T; Asahara, T; Silver, M; Bauters, C; Masuda, H; Kalka, C; Kearney, M; Chen, D; Symes, JF; Fishman, MC; et al. Nitric oxide synthase modulates angiogenesis in response to tissue ischemia. J. Clin. Invest 1998, 101, 2567–2578, doi:10.1172/JCI1560. 9616228
- Papapetropoulos, A; Garcia-Cardena, G; Madri, JA; Sessa, WC. Nitric oxide production contributes to the angiogenic properties of vascular endothelial growth factor in human endothelial cells. J. Clin. Invest 1997, 100, 3131–3139, doi:10.1172/JCI119868. 9399960
- Crowell, JA; Steele, VE; Sigman, CC; Fay, JR. Is inducible nitric oxide synthase a target for chemoprevention? Mol. Cancer Ther 2003, 2, 815–823. 12939472
- Hofseth, LJ; Hussain, SP; Wogan, GN; Harris, CC. Nitric oxide in cancer and chemoprevention. Free Radic. Biol. Med 2003, 34, 955–968, doi:10.1016/S0891-5849(02)01363-1. 12684081
- Tozer, GM; Prise, VE; Chaplin, DJ. Inhibition of nitric oxide synthase induces a selective reduction in tumor blood flow that is reversible with l-arginine. Cancer Res 1997, 57, 948–955. 9041200
- Haynes, WG; Noon, JP; Walker, BR; Webb, DJ. Inhibition of nitric oxide synthesis increases blood pressure in healthy humans. J. Hypertens 1993, 11, 1375–1380, doi:10.1097/00004872-199312000-00009. 7510736
- Lorente, JA; Landin, L; De Pablo, R; Renes, E; Liste, D. L-arginine pathway in the sepsis syndrome. Crit. Care Med 1993, 21, 1287–1295, doi:10.1097/00003246-199309000-00010. 8370291
- Bentz, BG; Haines, GK, III; Radosevich, JA. Increased protein nitrosylation in head and neck squamous cell carcinoma. Head Neck 2000, 22, 64–70, doi:10.1002/(SICI)1097-0347(200001)22:1<64::AID-HED10>3.0.CO;2-J. 10585607
- Jenkins, DC; Charles, IG; Thomsen, LL; Moss, DW; Holmes, LS; Baylis, SA; Rhodes, P; Westmore, K; Emson, PC; Moncada, S. Roles of nitric oxide in tumor growth. Proc. Natl. Acad. Sci. USA 1995, 92, 4392–4396, doi:10.1073/pnas.92.10.4392. 7538668
- Epperlein, MM; Nourooz-Zadeh, J; Noronha-Dutra, AA; Woolf, N. Nitric oxide in cigarette smoke as a mediator of oxidative damage. Int. J. Exp. Pathol 1996, 77, 197–200. 8977370
- Gaston, B; Drazen, JM; Loscalzo, J; Stamler, JS. The biology of nitrogen oxides in the airways. Am. J. Respir. Crit. Care Med 1994, 149, 538–551. 7508323
- Mirvish, SS. Role of n-nitroso compounds (noc) and n-nitrosation in etiology of gastric, esophageal, nasopharyngeal and bladder cancer and contribution to cancer of known exposures to noc. Cancer Lett 1995, 93, 17–48, doi:10.1016/0304-3835(95)03786-V. 7600541
- Gibson, QH; Roughton, FJ. The determination of the velocity constants of four successive reactions of carbon monoxide with sheep haemoglobin. Proc Soc Ser B: Biol Sci Lond 1957, 206–224.
- Davis, KL; Martin, E; Turko, IV; Murad, F. Novel effects of nitric oxide. Annu. Rev. Pharmacol. Toxicol 2001, 41, 203–236, doi:10.1146/annurev.pharmtox.41.1.203. 11264456
- Souza, JM; Peluffo, G; Radi, R. Protein tyrosine nitration--functional alteration or just a biomarker? Free Radic. Biol. Med 2008, 45, 357–366, doi:10.1016/j.freeradbiomed.2008.04.010. 18460345
- Lane, AP; Prazma, J; Baggett, HC; Rose, AS; Pillsbury, HC. Nitric oxide is a mediator of neurogenic vascular exudation in the nose. Otolaryngol. Head Neck. Surg 1997, 116, 294–300, doi:10.1016/S0194-5998(97)70263-8. 9121780
- Michel, O; Bloch, W; Rocker, J. Nos-mapping of human nasal mucosa under physiologic and pathophysiological conditions. In Proceedings of the Fourth International Meeting on the Biology of Nitric Oxide; Moncada, S, Stamler, J, Gross, S, Higgs, EA, Eds.; Portland Press: London, UK, 1996.
- Eaton, DL; Bammler, TK. Concise review of the glutathione s-transferases and their significance to toxicology. Toxicol. Sci 1999, 49, 156–164, doi:10.1093/toxsci/49.2.156. 10416260
- Hanna, E; MacLeod, S; Vural, E; Lang, N. Genetic deletions of glutathione-s-transferase as a risk factor in squamous cell carcinoma of the larynx: A preliminary report. Am. J. Otolaryngol 2001, 22, 121–123, doi:10.1053/ajot.2001.22571. 11283827
- Ketterer, B. Protective role of glutathione and glutathione transferases in mutagenesis and carcinogenesis. Mutat. Res 1988, 202, 343–361, doi:10.1016/0027-5107(88)90197-2. 3057366
- Mulder, TP; Manni, JJ; Roelofs, HM; Peters, WH; Wiersma, A. Glutatione s-transferases and glutathione in human head and neck cancer. Carcinogenesis 1995, 16, 619–624, doi:10.1093/carcin/16.3.619. 7697822
- McIlwain, CC; Townsend, DM; Tew, KD. Glutathione s-transferase polymorphisms: Cancer incidence and therapy. Oncogene 2006, 25, 1639–1648, doi:10.1038/sj.onc.1209373. 16550164
- Schumaker, L; Nikitakis, N; Goloubeva, O; Tan, M; Taylor, R; Cullen, KJ. Elevated expression of glutathione s-transferase pi and p53 confers poor prognosis in head and neck cancer patients treated with chemoradiotherapy but not radiotherapy alone. Clin. Cancer Res 2008, 14, 5877–5883, doi:10.1158/1078-0432.CCR-08-0998. 18794100
- Ali-Osman, F; Akande, O; Antoun, G; Mao, JX; Buolamwini, J. Molecular cloning, characterization, and expression in escherichia coli of full-length cdnas of three human glutathione s-transferase pi gene variants. Evidence for differential catalytic activity of the encoded proteins. J. Biol. Chem 1997, 272, 10004–10012, doi:10.1074/jbc.272.15.10004. 9092542
- Goto, S; Iida, T; Cho, S; Oka, M; Kohno, S; Kondo, T. Overexpression of glutathione s-transferase pi enhances the adduct formation of cisplatin with glutathione in human cancer cells. Free Radic. Res 1999, 31, 549–558, doi:10.1080/10715769900301121. 10630679
- Stoehlmacher, J; Park, DJ; Zhang, W; Groshen, S; Tsao-Wei, DD; Yu, MC; Lenz, HJ. Association between glutathione s-transferase p1, t1, and m1 genetic polymorphism and survival of patients with metastatic colorectal cancer. J. Natl. Cancer Inst 2002, 94, 936–942, doi:10.1093/jnci/94.12.936. 12072547
- Maugard, CM; Charrier, J; Pitard, A; Campion, L; Akande, O; Pleasants, L; Ali-Osman, F. Genetic polymorphism at the glutathione s-transferase (gst) p1 locus is a breast cancer risk modifier. Int. J. Cancer 2001, 91, 334–339, doi:10.1002/1097-0215(200002)9999:9999<::AID-IJC1057>3.0.CO;2-H. 11169956
- Tew, KD. Glutathione-associated enzymes in anticancer drug resistance. Cancer Res 1994, 54, 4313–4320. 8044778
- Miura, K; Suzuki, S; Tanita, J; Shinkawa, H; Satoh, K; Tsuchida, S. Correlated expression of glutathione s-transferase-pi and c-jun or other oncogene products in human squamous cell carcinomas of the head and neck: Relevance to relapse after radiation therapy. Jpn. J. Cancer Res 1997, 88, 143–151, doi:10.1111/j.1349-7006.1997.tb00359.x. 9119742
- Bongers, V; Snow, GB; de Vries, N; Cattan, AR; Hall, AG; van der Waal, I; Braakhuis, BJ. Second primary head and neck squamous cell carcinoma predicted by the glutathione s-transferase expression in healthy tissue in the direct vicinity of the first tumor. Lab. Invest 1995, 73, 503–510. 7474922
- Bentz, BG; Haines, GK, III; Lingen, MW; Pelzer, HJ; Hanson, DG; Radosevich, JA. Nitric oxide synthase type 3 is increased in squamous hyperplasia, dysplasia, and squamous cell carcinoma of the head and neck. Ann. Otol. Rhinol. Laryngol 1999, 108, 781–787. 10453787
- Bentz, BG; Haines, GK, III; Radosevich, JA. Glutathione s-transferase pi in squamous cell carcinoma of the head and neck. Laryngoscope 2000, 110, 1642–1647, doi:10.1097/00005537-200010000-00013. 11037818
- Bentz, BG; Haines, GK, III; Lingen, MW; Pelzer, HJ; Hanson, DG; Radosevich, JA. Nitric oxide synthase type 3 is increased in squamous hyperplasia, dysplasia, and squamous cell carcinoma of the head and neck. Ann. Otol. Rhinol. Laryngol 1999, 108, 781–787. 10453787
- Bentz, BG; Haines, GK, III; Radosevich, JA. Glutathione s-transferase pi in squamous cell carcinoma of the head and neck. Laryngoscope 2000, 110, 1642–1647, doi:10.1097/00005537-200010000-00013. 11037818
- Wei, L; Gravitt, PE; Song, H; Maldonado, AM; Ozbun, MA. Nitric oxide induces early viral transcription coincident with increased DNA damage and mutation rates in human papillomavirus-infected cells. Cancer Res 2009, 69, 4878–4884, doi:10.1158/0008-5472.CAN-08-4695. 19487298
- Benencia, F; Gamba, G; Cavalieri, H; Courreges, MC; Benedetti, R; Villamil, SM; Massouh, EJ. Nitric oxide and hsv vaginal infection in balb/c mice. Virology 2003, 309, 75–84, doi:10.1016/S0042-6822(02)00057-0. 12726728
- Carratelli, CR; Rizzo, A; Paolillo, R; Catania, MR; Catalanotti, P; Rossano, F. Effect of nitric oxide on the growth of chlamydophila pneumoniae. Can. J. Microbiol 2005, 51, 941–947, doi:10.1139/w05-080. 16333333
- Chang, K; Lubo, Z. Review article: Steroid hormones and uterine vascular adaptation to pregnancy. Reprod. Sci 2008, 15, 336–348, doi:10.1177/1933719108317975. 18497342
- Hiraku, Y; Tabata, T; Ma, N; Murata, M; Ding, X; Kawanishi, S. Nitrative and oxidative DNA damage in cervical intraepithelial neoplasia associated with human papilloma virus infection. Cancer Sci 2007, 98, 964–972, doi:10.1111/j.1349-7006.2007.00497.x. 17488337
- Tavares-Murta, BM; de Resende, AD; Cunha, FQ; Murta, EF. Local profile of cytokines and nitric oxide in patients with bacterial vaginosis and cervical intraepithelial neoplasia. Eur. J. Obstet. Gynecol. Reprod. Biol 2008, 138, 93–99, doi:10.1016/j.ejogrb.2007.06.015. 17683845
- Cerutti, PA. Prooxidant states and tumor promotion. Science 1985, 227, 375–381, doi:10.1126/science.2981433. 2981433
- Hussain, SP; Harris, CC. Inflammation and cancer: An ancient link with novel potentials. Int. J. Cancer 2007, 121, 2373–2380, doi:10.1002/ijc.23173. 17893866
- Hussain, SP; Raja, K; Amstad, PA; Sawyer, M; Trudel, LJ; Wogan, GN; Hofseth, LJ; Shields, PG; Billiar, TR; Trautwein, C; et al. Increased p53 mutation load in nontumorous human liver of wilson disease and hemochromatosis: Oxyradical overload diseases. Proc. Natl. Acad. Sci. USA 2000, 97, 12770–12775, doi:10.1073/pnas.220416097. 11050162
- Nair, J; Gansauge, F; Beger, H; Dolara, P; Winde, G; Bartsch, H. Increased etheno-DNA adducts in affected tissues of patients suffering from crohn's disease, ulcerative colitis, and chronic pancreatitis. Antioxid. Redox. Signal 2006, 8, 1003–1010, doi:10.1089/ars.2006.8.1003. 16771690
- Cerutti, PA; Trump, BF. Inflammation and oxidative stress in carcinogenesis. Cancer Cells 1991, 3, 1–7. 2025490
- Shaulian, E; Karin, M. Ap-1 as a regulator of cell life and death. Nat. Cell Biol 2002, 4, E131–E136, doi:10.1038/ncb0502-e131. 11988758
- Hofseth, LJ; Saito, S; Hussain, SP; Espey, MG; Miranda, KM; Araki, Y; Jhappan, C; Higashimoto, Y; He, P; Linke, SP; et al. Nitric oxide-induced cellular stress and p53 activation in chronic inflammation. Proc. Natl. Acad. Sci. USA 2003, 100, 143–148, doi:10.1073/pnas.0237083100. 12518062
- Ying, L; Marino, J; Hussain, SP; Khan, MA; You, S; Hofseth, AB; Trivers, GE; Dixon, DA; Harris, CC; Hofseth, LJ. Chronic inflammation promotes retinoblastoma protein hyperphosphorylation and e2f1 activation. Cancer Res 2005, 65, 9132–9136, doi:10.1158/0008-5472.CAN-05-1358. 16230367
- Wink, DA; Hanbauer, I; Grisham, MB; Laval, F; Nims, RW; Laval, J; Cook, J; Pacelli, R; Liebmann, J; Krishna, M; et al. Chemical biology of nitric oxide: Regulation and protective and toxic mechanisms. Curr. Top Cell Regul 1996, 34, 159–187. 8646847
- Xu, W; Liu, L; Smith, GC; Charles, G. Nitric oxide upregulates expression of DNA-pkcs to protect cells from DNA-damaging anti-tumour agents. Nat. Cell Biol 2000, 2, 339–345, doi:10.1038/35014028. 10854324
- Jablonka, E; Lamb, MJ. The changing concept of epigenetics. Ann. NY Acad. Sci 2002, 981, 82–96. 12547675
- Peters, J. Overview of mammalian genome special issue on epigenetics. Mamm. Genome 2009, 20, 529–531, doi:10.1007/s00335-009-9231-4. 19862571
- Waddington, CH. The epigenotype. Endeavour 1942, 18–20.
- Das, PM; Singal, R. DNA methylation and cancer. J. Clin. Oncol 2004, 22, 4632–4642, doi:10.1200/JCO.2004.07.151. 15542813
- DeAngelis, JT; Farrington, WJ; Tollefsbol, TO. An overview of epigenetic assays. Mol. Biotechnol 2008, 38, 179–183, doi:10.1007/s12033-007-9010-y. 17943463
- Jacob, S; Moley, KH. Gametes and embryo epigenetic reprogramming affect developmental outcome: Implication for assisted reproductive technologies. Pediatr. Res 2005, 58, 437–446, doi:10.1203/01.PDR.0000179401.17161.D3. 16148054
- Tang, WY; Ho, SM. Epigenetic reprogramming and imprinting in origins of disease. Rev. Endocr. Metab. Disord 2007, 8, 173–182, doi:10.1007/s11154-007-9042-4. 17638084
- Miller, CA; Sweatt, JD. Covalent modification of DNA regulates memory formation. Neuron 2007, 53, 857–869, doi:10.1016/j.neuron.2007.02.022. 17359920
- Matouk, CC; Marsden, PA. Epigenetic regulation of vascular endothelial gene expression. Circ. Res 2008, 102, 873–887, doi:10.1161/CIRCRESAHA.107.171025. 18436802
- Prokhortchouk, E; Defossez, PA. The cell biology of DNA methylation in mammals. Biochim. Biophys. Acta 2008, 1783, 2167–2173, doi:10.1016/j.bbamcr.2008.07.015. 18706939
- Horn, PJ; Peterson, CL. Molecular biology. Chromatin higher order folding--wrapping up transcription. Science 2002, 297, 1824–1827, doi:10.1126/science.1074200. 12228709
- Carthew, RW; Sontheimer, EJ. Origins and mechanisms of mirnas and sirnas. Cell 2009, 136, 642–655, doi:10.1016/j.cell.2009.01.035. 19239886
- Luger, K; Mader, AW; Richmond, RK; Sargent, DF; Richmond, TJ. Crystal structure of the nucleosome core particle at 2.8 a resolution. Nature 1997, 389, 251–260, doi:10.1038/38444. 9305837
- Hansen, JC. Conformational dynamics of the chromatin fiber in solution: Determinants, mechanisms, and functions. Ann. Rev. Biophys. Biomol. Struct 2002, 31, 361–392, doi:10.1146/annurev.biophys.31.101101.140858.
- Jenuwein, T; Allis, CD. Translating the histone code. Science 2001, 293, 1074–1080, doi:10.1126/science.1063127. 11498575
- Takai, D; Jones, PA. The cpg island searcher: A new www resource. In Silico Biol 2003, 3, 235–240. 12954087
- Esteller, M. Cancer epigenomics: DNA methylomes and histone-modification maps. Nat. Rev. Genet 2007, 8, 286–298, doi:10.1038/nrg2005. 17339880
- Jones, PA; Baylin, SB. The fundamental role of epigenetic events in cancer. Nat. Rev. Genet 2002, 3, 415–428. 12042769
- Klose, RJ; Bird, AP. Genomic DNA methylation: The mark and its mediators. Trends Biochem. Sci 2006, 31, 89–97, doi:10.1016/j.tibs.2005.12.008. 16403636
- Takai, D; Jones, PA. Comprehensive analysis of cpg islands in human chromosomes 21 and 22. Proc. Natl. Acad. Sci. USA 2002, 99, 3740–3745, doi:10.1073/pnas.052410099. 11891299
- Catteau, A; Morris, JR. Brca1 methylation: A significant role in tumour development? Semin. Cancer Biol 2002, 12, 359–371, doi:10.1016/S1044-579X(02)00056-1. 12191635
- Egger, G; Liang, G; Aparicio, A; Jones, PA. Epigenetics in human disease and prospects for epigenetic therapy. Nature 2004, 429, 457–463, doi:10.1038/nature02625. 15164071
- Leone, G; Teofili, L; Voso, MT; Lubbert, M. DNA methylation and demethylating drugs in myelodysplastic syndromes and secondary leukemias. Haematologica 2002, 87, 1324–1341. 12495905
- Tsou, JA; Hagen, JA; Carpenter, CL; Laird-Offringa, IA. DNA methylation analysis: A powerful new tool for lung cancer diagnosis. Oncogene 2002, 21, 5450–5461, doi:10.1038/sj.onc.1205605. 12154407
- Liu, J; Casaccia, P. Epigenetic regulation of oligodendrocyte identity. Trends Neurosci 2010, 33, 193–201, doi:10.1016/j.tins.2010.01.007. 20227775
- Chuang, JC; Jones, PA. Epigenetics and micrornas. Pediatr. Res 2007, 61, 24R–29R, doi:10.1203/pdr.0b013e3180457684. 17413852
- Goel, A. Cpg island methylator phenotype in colrectal cancer: A current perspective. Curr Colorect Cancer Rep 2008, 77–83.
- Boland, R. Promoter methylation in the genesis of gastrointestinal cancer. Yonsei Med. J 2009, 50, 309–321, doi:10.3349/ymj.2009.50.3.309. 19568590
- Vogelstein, B; Kinzler, KW. Cancer genes and the pathways they control. Nat. Med 2004, 10, 789–799, doi:10.1038/nm1087. 15286780
- Bedford, MT; van Helden, PD. Hypomethylation of DNA in pathological conditions of the human prostate. Cancer Res 1987, 47, 5274–5276. 2443238
- Ehrlich, M. DNA methylation in cancer: Too much, but also too little. Oncogene 2002, 21, 5400–5413, doi:10.1038/sj.onc.1205651. 12154403
- Kim, YI; Giuliano, A; Hatch, KD; Schneider, A; Nour, MA; Dallal, GE; Selhub, J; Mason, JB. Global DNA hypomethylation increases progressively in cervical dysplasia and carcinoma. Cancer 1994, 74, 893–899, doi:10.1002/1097-0142(19940801)74:3<893::AID-CNCR2820740316>3.0.CO;2-B. 8039116
- Lin, CH; Hsieh, SY; Sheen, IS; Lee, WC; Chen, TC; Shyu, WC; Liaw, YF. Genome-wide hypomethylation in hepatocellular carcinogenesis. Cancer Res 2001, 61, 4238–4243. 11358850
- Kawakami, K; Brabender, J; Lord, RV; Groshen, S; Greenwald, BD; Krasna, MJ; Yin, J; Fleisher, AS; Abraham, JM; Beer, DG; et al. Hypermethylated apc DNA in plasma and prognosis of patients with esophageal adenocarcinoma. J. Natl. Cancer Inst 2000, 92, 1805–1811, doi:10.1093/jnci/92.22.1805. 11078757
- Harden, SV; Tokumaru, Y; Westra, WH; Goodman, S; Ahrendt, SA; Yang, SC; Sidransky, D. Gene promoter hypermethylation in tumors and lymph nodes of stage i lung cancer patients. Clin. Cancer Res 2003, 9, 1370–1375. 12684406
- Virmani, AK; Rathi, A; Sathyanarayana, UG; Padar, A; Huang, CX; Cunnigham, HT; Farinas, AJ; Milchgrub, S; Euhus, DM; Gilcrease, M; et al. Aberrant methylation of the adenomatous polyposis coli (apc) gene promoter 1a in breast and lung carcinomas. Clin. Cancer Res 2001, 7, 1998–2004. 11448917
- Chan, KY; Ozcelik, H; Cheung, AN; Ngan, HY; Khoo, US. Epigenetic factors controlling the brca1 and brca2 genes in sporadic ovarian cancer. Cancer Res 2002, 62, 4151–4156. 12124354
- Dobrovic, A; Simpfendorfer, D. Methylation of the brca1 gene in sporadic breast cancer. Cancer Res 1997, 57, 3347–3350. 9269993
- Sanchez-Cespedes, M; Esteller, M; Wu, L; Nawroz-Danish, H; Yoo, GH; Koch, WM; Jen, J; Herman, JG; Sidransky, D. Gene promoter hypermethylation in tumors and serum of head and neck cancer patients. Cancer Res 2000, 60, 892–895. 10706101
- Villuendas, R. Loss of p16/ink4a protein expression in non-hodgkin's lymphomas is a frequent finding associated with tumor progression. Am J Pathol 1998, 887–897.
- Graff, JR; Herman, JG; Lapidus, RG; Chopra, H; Xu, R; Jarrard, DF; Isaacs, WB; Pitha, PM; Davidson, NE; Baylin, SB. E-cadherin expression is silenced by DNA hypermethylation in human breast and prostate carcinomas. Cancer Res 1995, 55, 5195–5199. 7585573
- Graff, JR; Greenberg, VE; Herman, JG; Westra, WH; Boghaert, ER; Ain, KB; Saji, M; Zeiger, MA; Zimmer, SG; Baylin, SB. Distinct patterns of e-cadherin cpg island methylation in papillary, follicular, hurthle's cell, and poorly differentiated human thyroid carcinoma. Cancer Res 1998, 58, 2063–2066. 9605742
- Waki, T; Tamura, G; Tsuchiya, T; Sato, K; Nishizuka, S; Motoyama, T. Promoter methylation status of e-cadherin, hmlh1, and p16 genes in nonneoplastic gastric epithelia. Am. J. Pathol 2002, 161, 399–403, doi:10.1016/S0002-9440(10)64195-8. 12163364
- Yang, X; Yan, L; Davidson, NE. DNA methylation in breast cancer. Endocr. Relat. Cancer 2001, 8, 115–127, doi:10.1677/erc.0.0080115. 11446343
- Li, LC; Chui, R; Nakajima, K; Oh, BR; Au, HC; Dahiya, R. Frequent methylation of estrogen receptor in prostate cancer: Correlation with tumor progression. Cancer Res 2000, 60, 702–706. 10676656
- Lee, WH; Morton, RA; Epstein, JI; Brooks, JD; Campbell, PA; Bova, GS; Hsieh, WS; Isaacs, WB; Nelson, WG. Cytidine methylation of regulatory sequences near the pi-class glutathione s-transferase gene accompanies human prostatic carcinogenesis. Proc. Natl. Acad. Sci. USA 1994, 91, 11733–11737, doi:10.1073/pnas.91.24.11733. 7972132
- Veigl, ML; Kasturi, L; Olechnowicz, J; Ma, AH; Lutterbaugh, JD; Periyasamy, S; Li, GM; Drummond, J; Modrich, PL; Sedwick, WD; et al. Biallelic inactivation of hmlh1 by epigenetic gene silencing, a novel mechanism causing human msi cancers. Proc. Natl. Acad. Sci. USA 1998, 95, 8698–8702, doi:10.1073/pnas.95.15.8698. 9671741
- Kondo, E. Not hmsh2 but hmlh1 is frequently silenced by hypermethylation in endometrial cancer but rarely silenced in pancreatic cancer with microsatellite instability. Int J Oncol 2000, 535–541.
- Strathdee, G; MacKean, MJ; Illand, M; Brown, R. A role for methylation of the hmlh1 promoter in loss of hmlh1 expression and drug resistance in ovarian cancer. Oncogene 1999, 18, 2335–2341, doi:10.1038/sj.onc.1202540. 10327053
- Harden, SV; Tokumaru, Y; Westra, WH; Goodman, S; Ahrendt, SA; Yang, SC; Sidransky, D. Gene promoter hypermethylation in tumors and lymph nodes of stage I lung cancer patients. Clin. Cancer Res 2003, 9, 1370–1375. 12684406
- Esteller, M; Garcia-Foncillas, J; Andion, E; Goodman, SN; Hidalgo, OF; Vanaclocha, V; Baylin, SB; Herman, JG. Inactivation of the DNA-repair gene mgmt and the clinical response of gliomas to alkylating agents. N. Engl. J. Med 2000, 343, 1350–1354, doi:10.1056/NEJM200011093431901. 11070098
- Melki, JR; Vincent, PC; Clark, SJ. Concurrent DNA hypermethylation of multiple genes in acute myeloid leukemia. Cancer Res 1999, 59, 3730–3740. 10446989
- Garcia, MJ; Martinez-Delgado, B; Cebrian, A; Martinez, A; Benitez, J; Rivas, C. Different incidence and pattern of p15ink4b and p16ink4a promoter region hypermethylation in hodgkin's and cd30-positive non-hodgkin's lymphomas. Am. J. Pathol 2002, 161, 1007–1013, doi:10.1016/S0002-9440(10)64261-7. 12213729
- Herman, JG; Jen, J; Merlo, A; Baylin, SB. Hypermethylation-associated inactivation indicates a tumor suppressor role for p15ink4b. Cancer Res 1996, 56, 722–727. 8631003
- Agathanggelou, A. Methylation associated inactivation of rassf1a from region 3p21.3 in lung, breast and ovarian tumours. Oncogene 2001, 1509–1518.
- Morrissey, C; Martinez, A; Zatyka, M; Agathanggelou, A; Honorio, S; Astuti, D; Morgan, NV; Moch, H; Richards, FM; Kishida, T; et al. Epigenetic inactivation of the rassf1a 3p21.3 tumor suppressor gene in both clear cell and papillary renal cell carcinoma. Cancer Res 2001, 61, 7277–7281. 11585766
- Kwong, J; Lo, KW; To, KF; Teo, PM; Johnson, PJ; Huang, DP. Promoter hypermethylation of multiple genes in nasopharyngeal carcinoma. Clin. Cancer Res 2002, 8, 131–137. 11801549
- Stirzaker, C; Millar, DS; Paul, CL; Warnecke, PM; Harrison, J; Vincent, PC; Frommer, M; Clark, SJ. Extensive DNA methylation spanning the rb promoter in retinoblastoma tumors. Cancer Res 1997, 57, 2229–2237. 9187126
- Gonzalez-Gomez, P; Bello, MJ; Alonso, ME; Arjona, D; Lomas, J; de Campos, JM; Isla, A; Rey, JA. Cpg island methylation status and mutation analysis of the rb1 gene essential promoter region and protein-binding pocket domain in nervous system tumours. Br. J. Cancer 2003, 88, 109–114, doi:10.1038/sj.bjc.6600737. 12556968
- Tannenbaum, SR; White, FM. Regulation and specificity of s-nitrosylation and denitrosylation. ACS Chem. Biol 2006, 1, 615–618, doi:10.1021/cb600439h. 17168564
- Sbaa, E; Frerart, F; Feron, O. The double regulation of endothelial nitric oxide synthase by caveolae and caveolin: A paradox solved through the study of angiogenesis. Trends Cardiovasc. Med 2005, 15, 157–162, doi:10.1016/j.tcm.2005.05.006. 16165011
- Minchenkova, LE; Ivanov, VI. Influence of reductants upon optical characteristics of the DNA-Cu2+ complex. Biopolymers 1967, 5, 615–625, doi:10.1002/bip.1967.360050703. 6074259
- Robbins, E; Fant, J; Norton, W. Intracellular iron-binding macromolecules in hela cells. Proc. Natl. Acad. Sci. USA 1972, 69, 3708–3712, doi:10.1073/pnas.69.12.3708. 4509333
- Bitny-Szlachto, S; Ochalska-Czepulis, M. Effects of disulphides and alpha-oxoglutarate on nuclear thiol formation and thiol content of chromatin in lysed rat spleen nuclei. Int. J. Biochem 1978, 9, 179–183, doi:10.1016/0020-711X(78)90147-7. 648701
- Vanin, AF; Ivanov, VI. Interaction of iron ions with oxygen or nitrogen monoxide in chromosomes triggers synchronous expression/suppression oscillations of compact gene groups (“Genomewide oscillation”): Hypothesis. Nitric Oxide 2008, 18, 147–152, doi:10.1016/j.niox.2008.01.007. 18291123
- Elzaouk, L; Laufs, S; Heerklotz, D; Leimbacher, W; Blau, N; Resibois, A; Thony, B. Nuclear localization of tetrahydrobiopterin biosynthetic enzymes. Biochim. Biophys. Acta 2004, 1670, 56–68, doi:10.1016/j.bbagen.2003.10.015. 14729142
- Calin, GA; Croce, CM. Microrna signatures in human cancers. Nat. Rev. Cancer 2006, 6, 857–866, doi:10.1038/nrc1997. 17060945
- Nilsen, TW. Mechanisms of microrna-mediated gene regulation in animal cells. Trends Genet 2007, 23, 243–249, doi:10.1016/j.tig.2007.02.011. 17368621
- Farh, KK; Grimson, A; Jan, C; Lewis, BP; Johnston, WK; Lim, LP; Burge, CB; Bartel, DP. The widespread impact of mammalian micrornas on mrna repression and evolution. Science 2005, 310, 1817–1821, doi:10.1126/science.1121158. 16308420
- Lewis, BP; Shih, IH; Jones-Rhoades, MW; Bartel, DP; Burge, CB. Prediction of mammalian microrna targets. Cell 2003, 115, 787–798, doi:10.1016/S0092-8674(03)01018-3. 14697198
- Stark, A; Brennecke, J; Bushati, N; Russell, RB; Cohen, SM. Animal micrornas confer robustness to gene expression and have a significant impact on 3'utr evolution. Cell 2005, 123, 1133–1146, doi:10.1016/j.cell.2005.11.023. 16337999
- Dews, M; Homayouni, A; Yu, D; Murphy, D; Sevignani, C; Wentzel, E; Furth, EE; Lee, WM; Enders, GH; Mendell, JT; et al. Augmentation of tumor angiogenesis by a myc-activated microrna cluster. Nat. Genet 2006, 38, 1060–1065, doi:10.1038/ng1855. 16878133
- O'Donnell, KA; Wentzel, EA; Zeller, KI; Dang, CV; Mendell, JT. C-myc-regulated micrornas modulate e2f1 expression. Nature 2005, 435, 839–843, doi:10.1038/nature03677. 15944709
- Hayashita, Y; Osada, H; Tatematsu, Y; Yamada, H; Yanagisawa, K; Tomida, S; Yatabe, Y; Kawahara, K; Sekido, Y; Takahashi, T. A polycistronic microrna cluster, mir-17-92, is overexpressed in human lung cancers and enhances cell proliferation. Cancer Res 2005, 65, 9628–9632, doi:10.1158/0008-5472.CAN-05-2352. 16266980
- He, L; Thomson, JM; Hemann, MT; Hernando-Monge, E; Mu, D; Goodson, S; Powers, S; Cordon-Cardo, C; Lowe, SW; Hannon, GJ; et al. A microrna polycistron as a potential human oncogene. Nature 2005, 435, 828–833, doi:10.1038/nature03552. 15944707
- Lu, J; Getz, G; Miska, EA; Alvarez-Saavedra, E; Lamb, J; Peck, D; Sweet-Cordero, A; Ebert, BL; Mak, RH; Ferrando, AA; et al. Microrna expression profiles classify human cancers. Nature 2005, 435, 834–838, doi:10.1038/nature03702. 15944708
- Benetti, R; Gonzalo, S; Jaco, I; Munoz, P; Gonzalez, S; Schoeftner, S; Murchison, E; Andl, T; Chen, T; Klatt, P; et al. A mammalian microrna cluster controls DNA methylation and telomere recombination via rbl2-dependent regulation of DNA methyltransferases. Nat. Struct. Mol. Biol 2008, 15, 268–279, doi:10.1038/nsmb.1399. 18311151
- Sinkkonen, L; Hugenschmidt, T; Berninger, P; Gaidatzis, D; Mohn, F; Artus-Revel, CG; Zavolan, M; Svoboda, P; Filipowicz, W. Micrornas control de novo DNA methylation through regulation of transcriptional repressors in mouse embryonic stem cells. Nat. Struct. Mol. Biol 2008, 15, 259–267, doi:10.1038/nsmb.1391. 18311153
- Chan, JA; Krichevsky, AM; Kosik, KS. Microrna-21 is an antiapoptotic factor in human glioblastoma cells. Cancer Res 2005, 65, 6029–6033, doi:10.1158/0008-5472.CAN-05-0137. 16024602
- Chandra, RK; Bentz, BG; Haines, GK, III; Robinson, AM; Radosevich, JA. Expression of glutathione s-transferase pi in benign mucosa, barrett's metaplasia, and adenocarcinoma of the esophagus. Head Neck 2002, 24, 575–581, doi:10.1002/hed.10093. 12112555
- Voorhoeve, PM; le Sage, C; Schrier, M; Gillis, AJ; Stoop, H; Nagel, R; Liu, YP; van Duijse, J; Drost, J; Griekspoor, A; et al. A genetic screen implicates mirna-372 and mirna-373 as oncogenes in testicular germ cell tumors. Cell 2006, 124, 1169–1181, doi:10.1016/j.cell.2006.02.037. 16564011
- Eis, PS; Tam, W; Sun, L; Chadburn, A; Li, Z; Gomez, MF; Lund, E; Dahlberg, JE. Accumulation of mir-155 and bic rna in human b cell lymphomas. Proc. Natl. Acad. Sci. USA 2005, 102, 3627–3632, doi:10.1073/pnas.0500613102. 15738415
- Iorio, MV; Ferracin, M; Liu, CG; Veronese, A; Spizzo, R; Sabbioni, S; Magri, E; Pedriali, M; Fabbri, M; Campiglio, M; et al. Microrna gene expression deregulation in human breast cancer. Cancer Res 2005, 65, 7065–7070, doi:10.1158/0008-5472.CAN-05-1783. 16103053
- Tam, W; Dahlberg, JE. Mir-155/bic as an oncogenic microrna. Genes Chromos. Cancer 2006, 45, 211–212, doi:10.1002/gcc.20282. 16252262
- Saito, Y; Liang, G; Egger, G; Friedman, JM; Chuang, JC; Coetzee, GA; Jones, PA. Specific activation of microrna-127 with downregulation of the proto-oncogene bcl6 by chromatin-modifying drugs in human cancer cells. Cancer Cell 2006, 9, 435–443, doi:10.1016/j.ccr.2006.04.020. 16766263
- Yoo, CB; Jones, PA. Epigenetic therapy of cancer: Past, present and future. Nat. Rev. Drug Discov 2006, 5, 37–50, doi:10.1038/nrd1930. 16485345
- Weber, M; Baker, MB; Moore, JP; Searles, CD. Mir-21 is induced in endothelial cells by shear stress and modulates apoptosis and enos activity. Biochem. Biophys. Res. Commun 2010, 393, 643–648, doi:10.1016/j.bbrc.2010.02.045. 20153722
- Ji, R; Cheng, Y; Yue, J; Yang, J; Liu, X; Chen, H; Dean, DB; Zhang, C. Microrna expression signature and antisense-mediated depletion reveal an essential role of microrna in vascular neointimal lesion formation. Circ. Res 2007, 100, 1579–1588, doi:10.1161/CIRCRESAHA.106.141986. 17478730
- Tatsuguchi, M; Seok, HY; Callis, TE; Thomson, JM; Chen, JF; Newman, M; Rojas, M; Hammond, SM; Wang, DZ. Expression of micrornas is dynamically regulated during cardiomyocyte hypertrophy. J. Mol. Cell Cardiol 2007, 42, 1137–1141, doi:10.1016/j.yjmcc.2007.04.004. 17498736
- Wang, Y; Lee, CG. Microrna and cancer--focus on apoptosis. J. Cell Mol. Med 2009, 13, 12–23. 19175697
- Zeng, L; Carter, AD; Childs, SJ. Mir-145 directs intestinal maturation in zebrafish. Proc. Natl. Acad. Sci. USA 2009, 106, 17793–17798, doi:10.1073/pnas.0903693106. 19805048
- Wang, X; Zhao, Q; Matta, R; Meng, X; Liu, X; Liu, CG; Nelin, LD; Liu, Y. Inducible nitric-oxide synthase expression is regulated by mitogen-activated protein kinase phosphatase-1. J. Biol. Chem 2009, 284, 27123–27134, doi:10.1074/jbc.M109.051235. 19651781
- Bui-Nguyen, TM; Pakala, SB; Sirigiri, DR; Martin, E; Murad, F; Kumar, R. Stimulation of inducible nitric oxide by hepatitis b virus transactivator protein hbx requires mta1 coregulator. J. Biol. Chem 2010, 285, 6980–6986, doi:10.1074/jbc.M109.065987. 20022949
- Bernstein, BE; Meissner, A; Lander, ES. The mammalian epigenome. Cell 2007, 128, 669–681, doi:10.1016/j.cell.2007.01.033. 17320505
- Chan, GC; Fish, JE; Mawji, IA; Leung, DD; Rachlis, AC; Marsden, PA. Epigenetic basis for the transcriptional hyporesponsiveness of the human inducible nitric oxide synthase gene in vascular endothelial cells. J. Immunol 2005, 175, 3846–3861. 16148131
- Goldberg, AD; Allis, CD; Bernstein, E. Epigenetics: A landscape takes shape. Cell 2007, 128, 635–638, doi:10.1016/j.cell.2007.02.006. 17320500
- Kouzarides, T. Chromatin modifications and their function. Cell 2007, 128, 693–705, doi:10.1016/j.cell.2007.02.005. 17320507
- Allis, CD; Berger, SL; Cote, J; Dent, S; Jenuwien, T; Kouzarides, T; Pillus, L; Reinberg, D; Shi, Y; Shiekhattar, R; et al. New nomenclature for chromatin-modifying enzymes. Cell 2007, 131, 633–636, doi:10.1016/j.cell.2007.10.039. 18022353
- Yang, XJ; Seto, E. The rpd3/hda1 family of lysine deacetylases: From bacteria and yeast to mice and men. Nat. Rev. Mol. Cell Biol 2008, 9, 206–218, doi:10.1038/nrm2346. 18292778
- Smith, BC; Hallows, WC; Denu, JM. Mechanisms and molecular probes of sirtuins. Chem. Biol 2008, 15, 1002–1013, doi:10.1016/j.chembiol.2008.09.009. 18940661
- Wang, GG; Allis, CD; Chi, P. Chromatin remodeling and cancer, part I: Covalent histone modifications. Trends Mol. Med 2007, 13, 363–372, doi:10.1016/j.molmed.2007.07.003. 17822958
- Sengupta, N; Seto, E. Regulation of histone deacetylase activities. J. Cell Biochem 2004, 93, 57–67, doi:10.1002/jcb.20179. 15352162
- Stamler, JS; Singel, DJ; Loscalzo, J. Biochemistry of nitric oxide and its redox-activated forms. Science 1992, 258, 1898–1902, doi:10.1126/science.1281928. 1281928
- Yang, XJ; Gregoire, S. Class ii histone deacetylases: From sequence to function, regulation, and clinical implication. Mol. Cell Biol 2005, 25, 2873–2884, doi:10.1128/MCB.25.8.2873-2884.2005. 15798178
- Lin, J; Handschin, C; Spiegelman, BM. Metabolic control through the pgc-1 family of transcription coactivators. Cell Metab 2005, 1, 361–370, doi:10.1016/j.cmet.2005.05.004. 16054085
- Fraga, MF; Esteller, M. DNA methylation: A profile of methods and applications. BioTechniques 2002, 33.
- Singal, R; Ginder, GD. DNA methylation. Blood 1999, 93, 4059–4070. 10361102
- Nestor, C. Enzymatic approaches and bisulfite sequencing cannot distinguish between 5-methylcytosine and 5-hydroxymethylcytosine in DNA. BioTechniques 2010, 48, 317–319, doi:10.2144/000113403. 20569209
- Chan, Y; Fish, JE; D'Abreo, C; Lin, S; Robb, GB; Teichert, AM; Karantzoulis-Fegaras, F; Keightley, A; Steer, BM; Marsden, PA. The cell-specific expression of endothelial nitric-oxide synthase: A role for DNA methylation. J. Biol. Chem 2004, 279, 35087–35100, doi:10.1074/jbc.M405063200. 15180995
- Guillot, PV; Liu, L; Kuivenhoven, JA; Guan, J; Rosenberg, RD; Aird, WC. Targeting of human enos promoter to the hprt locus of mice leads to tissue-restricted transgene expression. Physiol. Genomics 2000, 2, 77–83. 11015585
- Teichert, AM; Miller, TL; Tai, SC; Wang, Y; Bei, X; Robb, GB; Phillips, MJ; Marsden, PA. In vivo expression profile of an endothelial nitric oxide synthase promoter-reporter transgene. Am. J. Physiol. Heart Circ. Physiol 2000, 278, H1352–1361. 10749733
- Bentz, BG; Hammer, ND; Radosevich, JA; Haines, GK, III. Nitrosative stress induces DNA strand breaks but not caspase mediated apoptosis in a lung cancer cell line. J. Carcinog 2004, 3, 16, doi:10.1186/1477-3163-3-16. 15617570
- Radosevich, JA; Elseth, KM; Vesper, BJ; Tarjan, G; Haines, GK, III. Long-term adaptation of lung tumor cell lines with increasing concentrations of nitric oxide donor. Open Lung Cancer J 2009, 2, 35–44, doi:10.2174/1876819900902010035.
- Vesper, BJ; Elseth, KM; Tarjan, G; Haines, GK, III; Radosevich, JA. Long-term adaptation of breast tumor cell lines to high concentrations of nitric oxide. Tumor. Biol 2010, 31, 267–275, doi:10.1007/s13277-010-0028-6.
- Ridnour, LA; Thomas, DD; Donzelli, S; Espey, MG; Roberts, DD; Wink, DA; Isenberg, JS. The biphasic nature of nitric oxide responses in tumor biology. Antioxid. Redox. Signal 2006, 8, 1329–1337, doi:10.1089/ars.2006.8.1329. 16910780
- Wink, DA; Mitchell, JB. Chemical biology of nitric oxide: Insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide. Free Radic. Biol. Med 1998, 25, 434–456, doi:10.1016/S0891-5849(98)00092-6. 9741580
- Schetter, AJ; Heegaard, NH; Harris, CC. Inflammation and cancer: Interweaving microrna, free radical, cytokine and p53 pathways. Carcinogenesis 2010, 31, 37–49, doi:10.1093/carcin/bgp272. 19955394
|Table 1. GST-pi immunohistochemical staining in human laryngeal tumors.|
|Patient||Age/Sex||Tumor Location||Tumor Stage||Surgery Performed||Previous Treatment||GST-pi Intensity||GST-pi Pattern|
|1||74/M||Larynx||T3N0M0 (recurrent)||Total laryngectomy||Chemo/XRT||3+||diffuse|
|5||73/M||Supraglottis||T2N0M0 (recurrent)||Supraglottic laryngectomy||XRT||4+||diffuse|
|6||63/M||Supraglottis||T4N2M0 (recurrent)||Completion laryngectomy||Supraglottic laryngectomy, Chemo/XRT||2+||diffuse|
|7||61/M||Supraglottis||T2N2M0 (recurrent)||Completion laryngectomy||Supraglottic laryngectomy, Chemo/XRT||4+||diffuse|
|10||81/M||Larynx||T3N0M0 (recurrent)||Completion laryngectomy||Supraglottic laryngectomy||2+||focal|
Chemo: chemotherapy, XRT: radiation therapy. Study was carried out with IRB approval.
|Table 2. Cervical cancer patient summary and immunohistochemistry data.|
|Patient||Age||Stage||Grade||Treatment||Recurrence/Persistence||DFS (mos.)||iNOS Intensity||eNOS Intensity||GST-pi Intensity|
Staging by AJCC 2002 criteria. Treatment methods, C: chemotherapy, R: radiation therapy, S: surgery. DFS: Disease free survival. iNOS: inducible nitric oxide synthase; eNOS: endothelial constitutive nitric oxide synthase; GST-pi: glutathione S-transferase pi. Study was carried out with IRB approval.
|Table 3. The epigenetic impact of dysregulated DNA methylation on gene expression and human cancers.|
|APC||Inhibitor of β-catenin||Aerodigestive tract, lung, breast||Activation β –catenin route||[90,107–109]|
|AR||Androgen receptor||Prostate||Hormone insensitivity|||
|BRCA1||DNA repair, transcription||Breast, ovarian||Double strand breaks||[90,110,111]|
|CDH1||E cadherin, cell adhesion||Breast, stomach, Leukemia||Dissemination|||
|CDH13||H cadherin, cell division||Breast, lung||Dissemination|||
|CDKN2A/p16||Cyclin-dependent kinase inhibitor||Head, neck, gastrointestinal tract, lung, NHL||Entrance in cell cycle||[78,90,108,112,113]|
|COX2||Cycloxyenase-2||Colon, stomach||Anti-inflammatory resistance1|||
|CPBP1||Retinol-binding protein||Colon, stomach, lymphoma||Vitamin insensitivity|||
|DAPK1||Pro-apoptotic||Lymphoma, lung, colon||Resistance to apoptosis||[90,108]|
|DKK1||Extracellular Wnt inhibitor||Colon||Activation Wnt signaling|||
|E-cadherin||Increasing proliferation, invasion and/or metastasis||Breast, Thyroid, Gastric||[114–116]|
|ER||Oestrogen receptor||Breast, prostate||Hormone insensitivity||[117,118]|
|EXT1||Heparan intermediate filament||Leukemia, skin||Cellular detachment|||
|FAT||Cadherin, tumor suppressor||Colon||Dissemination|||
|GATA4||Transcription factor||Colon, stomach||Silencing of target genes|||
|GATA5||Transcription factor||Colon, stomach||Silencing of target genes|||
|GSTP1||Conjugation to glutathione||Prostate, breast, kidney||Adduct accumulation||[90,108,119]|
|HIC1||Transcription factor||Various forms||Currently unknown|||
|HOXA9||Homeobox protein||Neuroblastoma||Currently unknown|||
|hMLH1||Defective DNA mismatch repair, gene mutations||Colon, Renal, Gastric, Endometrim, Ovarian||[116,120–122]|
|ID4||Transcription factor||Leukemia, stomach||Currently unknown|||
|IGFBP3||Growth factor binding protein||Lung, skin||Resistance to apoptosis|||
|Lamin A/C||Nuclear intermediate filament||Lymphoma, leukemia||Currently unknown|||
|LKB1/STK11||Serine-theronine kinase||Colon, breast, lung||Currently unknown|||
|MGMT||DNA repair of 06-alkyl-guanine, p53||Lung, brain, various||Mutations, chemosensitivity||[90,123,124]|
|MLH1||DNA mismatch repair||Colon, endometrium, stomach, ovarian||Frameshift mutations, gene mutations|||
|NORE1A||Ras effector homologue||Lung||Currently unknown|||
|p14ARF||MDM2 inhibitor||Colon, stomach. kidney||Degradation of p53|||
|p15||Leukemia, Lymphoma||Entrance in cell cycle||[125–127]|
|p15INK4b||Cyclin-dependent kinase inhibitor||Leukemia, lymphoma, lung, SCC||Entrance in cell cycle|||
|p16INK4a||Cyclin-dependent kinase inhibitor||Various||Entrance in cell cycle||[90,108]|
|p73||P53 homologue||Lymphoma||Currently unknown|||
|PR||Progestrogen receptor||Breast||Hormone insensitivity|||
|PRLR||Prolactin receptor||Breast||Hormone insensitivity|||
|RARβ2||Retinoic acid receptor –β2||Colon, lung, head and neck||Vitamin insensitivity|||
|RASSF1A||Ras effector homologue||Lung, breast, ovarian, kidney, nasopharyngeal||Currently unknown||[128–130]|
|Rb||Cell-cycle inhibitor||Retinoblastoma, oligodenodroglioma||Entrance to cell||[90,131,132]|
|RIZ1||Histone/protein methyltransferase||Breast, liver||Abnormal gene expression|||
|SFRP1||Secreted frizzled-related protein 1||Colon||Activation Wnt signaling|||
|SLC5A8||Sodium transporter||Glioma, colon||Currently unknown|||
|SOC1||Inhibitor of JAK-STAT pathway||Liver, mieloma||JAK2 activation|||
|SOC3||Inhibitor of JAK-STAT pathway||Lung||JAK2 activation|||
|SRBC||BRCA1-binding protein||Breast, lung||Currently unknown|||
|SYK||Tyrosine kinase||Breast||Currently unknown|||
|TMS1||Pro-apoptotic||Breast||Resistance to apoptosis|||
|TPEF/HPP1||Transmembrane protein||Colon, bladder||Currently unknown|||
|TSHR||Thyroid-stimulating hormone receptor||Thyroid||Hormone insensitivity|||
|VHL||Ubiquitin ligase component||Kidney, haemangioblastoma||Loss of hypoxic response||[90,129]|
|WIF1||Wnt inhibitor factor||Colon, lung||Activation Wnt signaling|||
|WRN||DNA repair||Colon, stomach, sarcoma||DNA breakage, chemosensitivity|||
Abbreviations: NHL= Non-Hodgkin’s lymphoma, SCC= Squamous Cell Carcinoma, hMLH1= mutant homologue 1.
|Table 4. The epigenetic impact of NO•.|
|Substrate||Modification||Effect on Nucleosome/Chromatin||Transcription|
|Class II HDACs||Dephosphorylation||Indirect||_|
|OxyR and SoxR||S-N||Indirect||+|
Abbreviations: S-N= S-Nitrosylation, T-N= Tyr-Nitration. Adapted from reference .
|Table 5. The epigenetic impact of histone modification on gene expression and human cancers.|
|CBP1||Colon, stomach, endometrium, lung, leukemia||Mutations, translocations, deletions|
|EZH23||Various types||Gene amplification, over-expression|
|GASC14||Squamous cell carcinoma||Gene amplification|
|HDAC12||Various types||Aberrant expression|
|HDAC22||Various types||Aberrant expression, mutations in MSI+|
|MLL23||Glioma, pancreas||Gene amplification|
|MORF1||Haematological malignancies, leiomyomata||Translocations|
|p3001||Colon, stomach, endometrium||Mutations in MSI+|
|RIZ13||Various types||CpG-island hypermethylation|
© 2010 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland. This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).