The Role of Histone Deacetylases in Human Cancer - Coursework Example

Topic:  The Role of Histone deacetylases in Human Cancer – an essay Histones are lysine rich basic proteins which are responsible for stabilization of the chromatin organization. Selective opening of the chromatin at specific sites makes the genes available for transcription and histones play an important role in this activity. Post-translational modifications of histone proteins has become the topic of interest in last two decades since the alteration in these modifications could result in changes in the gene expression or behavior of the cells to the advantage of the human race. The following essay summarizes the structure of chromatin with reference to the histones and discusses the post-translational modifications of these proteins. The enzymes involved in acetylation/deacetylation of histones and the inhibitors of these enzymes are described in view of their applications in controlling human cancer. Structure of chromatin The packaging unit of the chromatin is called nucleosome. Nucleosome is formed of a histone core of eight molecules of histones; two H2A, two H2B, two H3 and two H4. Each nucleosome consists of 1.75 superhelical turns (146 nucleotides length) of DNA wrapped around the core histone barrel-shaped octamers. The N-terminus of each octamer subunit is protruding free to be accessible for covalent modifications. A linker stretch of DNA (30 nucleotide in length) connects between the nucleosome cores and is covered by one molecule of histone H1 so as to close two turns of DNA (Figure 1). Histones are basic non-heat coagulable proteins that are very rich in lysine and arginine (20-30%). Thus, they are positively charged at the physiological pH to bind the negatively charged DNA molecules. There are five types of histones; H1, H2A, H2B, H3 and H4 (ranging from 22-11 kDa, respectively). Histone proteins in the nucleosome have been assigned several dynamic regulatory roles induced by the reversible covalent modifications of their N-terminal tail, e.g., acetylation, methylation, ADP-ribosylation, ubiquitinization and phosphorylation. The regulatory roles of histones include - control of rate of gene expression (Induction/repression and silencing); chromosomal condensation and assembly during replication; and DNA repair. During assembly of the nucleosome, the flexible tails entwine with the DNA superhelices and bring them closer to the other histone-DNA complexes to stabilize the beaded structures (Luger and Richmond, 1998). The nucleosome structure and position are not stable but they are modifiable by remodeling, cis-sliding or trans-positioning so as to increase/decrease access to transcription control sequences. The organization of the 2 nm DNA double strand in nucleosomes makes it to look like beads-in-a-string that is called the 10 nm fibers. This fiber is then coiled again around a linear hollow axis as a helix with 6 - 7 nucleosomes per turn to form the solenoid 30 nm fiber (like the telephone cord). In the extended interphase chromosome, the 30 nm fiber is organized into loops or domains through binding into the nuclear scaffold or matrix proteins leading to the 300 nm thickness fiber. Each loop contains 20 - 100 kb nucleotides in the euchromatin regions. The looping is brought about through attachment to the nuclear matrix proteins via a specific AT-rich DNA segments called matrix-associated regions or scaffold attachment regions. This level of packaging is the maximum condensation for the interphase chromosomes. With the help of other organizing proteins, the 300 nm fiber form of interphase chromosomes is further condensed into 700 nm thickness sister-chromatid to appear as discrete metaphase mitotic chromosomes. One metaphase chromosome with two sister chromatids is ~1400 nm thick and nearly totally transcriptionally inactive heterochromatin. Therefore, when the cell is about to enter mitosis, the linear chromosomal DNA is 50,000x condensed into the short metaphase chromosome (Figure 2). Chromatin that is transcriptionally active is called euchromatin which stains light and is DNase I hypersensitive. But densely packed dark stained chromatin is inactive and is called heterochromatin and is not DNase I hypersensitive due to inaccessibility of its DNA. Constitutive heterochromatin is always inactive and found at centromeres and telomeres. When there is heterochromatin DNA that becomes active during specific developmental phase and is inactive elsewhere, it is called facultative heterochromatin. Post-translation Modification of Histones The N-terminal tails of the core histone molecules hang out of the nucleosome and are, thus, exposed to post-translational modifications (Fig 3). These modifications include i) methylation at lysine and arginine residues; ii) acetylation at the lysine residues; and iii) phosphorylation, iv) ubiquitination and v) ADP-ribosylation of serine and threonine residues (Robin et al.., 2007). The modifications of a histone protein are not mutually exclusive; rather, initial covalent attachment(s) determine the course of the subsequent modifications. These observations led to the development of the ‘histone code’ hypothesis (Jenuwein and Allis, 2001). The histone code serves as a signature sequence for the recruitment of different regulatory proteins, which, by interaction with the DNA, triggers the expression or repression of the associated genes (Berger, 2002) as well as other physiological activities of the nucleosome such as remodeling of the chromatin. Methylation: Methylation of histone N-terminal tails has been studied extensively by a number of research workers. Initially reported in 1968 by Tidwell et al.., the importance of the methylation of histones has been elusive till Rice and Allis (2001) demonstrated its role in epigenetic regulation. The specific methylases that act on histone proteins are named as histone methyltransferases (HMT). Most of the regulatory methylations of histones have been found to be on N-terminal tails of H3 histones. Examples include ‘suv39H1’ that acts on H3 histone and methylates its lysine 9; ‘DOT1’ methylates H3 protein at lysine 79; ‘G9a’ at lysine 9 and lysine 27 of H3. Different types of histone methylations and their effect on gene expression is well studied by Robin et al. (2007). HMTs carry a conserved domain known as SET domain [SET = Su(var)3-9 Enhancer-ofzeste, Trithorax]. Methylation and demethylation can have variable effects on the gene expression, e.g. H3K4 (methylation of lysine 4 on H3 histone) is related with enhanced gene expression, whereas H3K9, H4K29 and H3K27 are associated with repression of the genes. Multiple methylations of a single histone are also reported and could have a role in the histone code. Trimethylation of H3K9 is more commonly seen in the heterochromatin regions and the enzyme responsible is ‘suv38h1’ – a pericentromeric enzyme; single or double methylation are commonly seen in transcriptionally active euchromatin regions and the enzyme involved is ‘G9a’. Lysine at position 4 (K4) in H3 histone is a very prominent methylation site and has been implicated in gene silencing. The methylation is essential for cellular growth and normal regulation of the gene expression (Strahl et al., 1999; Briggs et al., 2001). Very few methylation site have been detected in the globular regions of the histone proteins – Methylation of K79 on H3 is one on the first, catalyzed by ‘Dot1’, the modification has been implicated in telomeric silencing (Ng et al.., 2002). Emre and Berger (2006) reviewed various post-translational modifications of histone proteins. They discussed that the site specific methylation could itself subject of regulation, e.g., K4 methylation of H3 histone can be regulated by ubiquitination of the C-terminal H2B histones. The de-ubiquitination by ubiquitin proteases can regulate the co-activator dependent gene expression (Ubp8 protease) or silencing (Ubp10 protease). Acetylation: The positively charged lysine residues of the histone proteins interact with negatively charged DNA to form histone-DNA complexes and is very important for the stabilization of the nucleosome and thus the chromatin. Acetylation of these lysine residues results in neutralization of the positive charges and thus disruption of the ionic interaction between histone/DNA or between histone/histone proteins. Acetylation of histones was initially demonstrated by Allfrey (1964). Loosening of the chromatin structure exposes the genes to the transcription factors as well as the enzymes for replication and transcription. Hyper-acetylation and hypo-acetylation are catalyzed by two opposing enzyme systems, i.e., histone acetylases (HATs) and histone deacetylases (HDACs); balance between the two determines the acetylation status of the histone in a genome. Hyperacetylation of core histones is, therefore, involved in up-regulation of the gene expression as well as differentiation and cell proliferation. Hypo-acetylation, on the other hand, facilitates the electrostatic interactions and promotes chromosomal condensation and repression of the underlying genes (Zhang, 2003). Acetylation of lysine residues, viz., K9, K14, K18 and K23 on the core histone H3 and lysines located at K5, K8, K12 and K16 on the core histone H4 are particularly important since they are specifically involved in DNA-histone and histone-histone interactions (Roth et al., 2001; Strahl and Allis, 2000). Other Post-translational modifications: Apart from acetylation and methylation, other post-translational modifications of histone proteins include – ubiquitination and phosphorylation/dephosphorylation. The phosphorylation of histones, particularly H1, H2A and H3, have been indicated to be involved in DNA fragmentation and might motivate the cells to proceed towards apoptosis. Ubiquitination, on the other hand, has been shown to play a regulatory role in the methylation of specific lysine residues in histone proteins (Robin et al., 2007). Emre and Berger (2006) investigated the role of phosphorylation of histone H3 at residue serine 10. They demonstrated that the phosphorylation affects the recruitment of TATA-binding protein (TBP) during activation of transcription. The effect of phosphorylation could be modified by the acetylation of histone proteins, but some effect on activation could be acetylation independent as well. Histone Deacetylases and their Classification Histone deacetylases (HDACs) as mentioned above, are generating a lot of interest in the molecular scientific world with particular reference to the application of the inhibitors of these enzymes in oncogenesis and cell differentiation. Marks et al. (2001) have published an excellent review on the enzymes. The major work on the understanding the mechanism of action of these enzymes in regulating the gene expression has been based on the observations of types of histone deacetylases in yeast. The following classification (Table 1), therefore, compares the relationship between yeast and the human HDACs and their sensitivities to different inhibitors. Table 1: Classification and characteristics of histone deacetylases (Marks et al., 2001) HDAC Group Yeast HDAC Inhibitor sensitivity* Human HDAC Inhibitor Sensitivity* Class I Rpd3 S HDAC1 HDAC2 HDAC3 HDAC8 S S S S Class II Hda1 S HDAC4 HDAC5 HDAC6 HDAC7 HDAC9 HDAC10 S S S S S - Class III Sir2 Hst1 Hst2 Hst3 Hst4 NS - - - - SIRT1 SIRT2 SIRT3 SIRT4 SIRT5 SIRT6 SIRT7 - - - - - - - *Inhibitor sensitivity has been characterized with trichstatin (TSA) and suberoylanalide hydorxamic acid (SAHA) or related compounds. It is becoming more and more clear that the HDACs, through deacetylation of histone proteins are not only involved in the packaging of chromatins or gene expression. They also appear to participate in cell cycle regulation, differentiation and apoptosis. Deacetylation of p53, the central protein that regulates the cell cycle and apoptosis is one example, but they could also be acting on the transcription factors and some other proteins involved in down regulation of cell signals, e.g. E2F, α-tubulin and MyoD (Hubbert, 2002; Juan, 2000). Histone Deacetylase inhibitors and Cancer HDAC inhibitors are emerging as the most important group of potential anti-cancer agents. Although studies on the expression of HDACs in cancer tissues compared to the normal tissues have yielded negative results, their inhibitors (HDACIs) show the arrest of the tumor growth and stimulate apoptosis and differentiation. HDACs are known to associate with cellular oncogenes as well as with well characterized tumor suppressor genes like retinoblastoma (Rb) protein (de Ruijter et al., 2003). HDACs are recruited by the oncoproteins that are generated upon fusion of retinoic acid receptor with promyelocytic leukemia (PML) gene and HDACs are then able to suppress the differentiation pathway leading to unregulated proliferation of promyelocytic leukemia cells. Similar observations have been reported after the fusion of PLZF with retinoic receptor in causing promyelocytic leukeami, ETO with AML1 fusion to cause acute myelocytic leukemia and a number of solid tumors, e.g., breast cancer (Munster et al., 2001). The HDACs recruitment appears to be mediated by the so-called CpG islands through CpG binding proteins or the proteins containing methylated CpG binding domains and the enzymes involved in methylation of the CpG islands. Involvement of HDACs in oncogenesis triggered a search for the inhibitors which could curtail the activity of these enzymes and hence prevent oncogenesis. A number of these inhibitors have shown the potential to inhibit the partially purified HDACs in vitro, but a few have been tested in animal models and some have even progressed into clinical trials in human cancers. The most promising HDACIs which have been or are being tried in clinical trials include – butyrates, depsipeptide, CI-994 (N-acetyl dinaline), pyroxamide, suberoylanilide hydraxamic acid (SAHA) and valproic acid. X-ray christallographic images of the complexes of hydroxamic acids with HDACs have shown the inhibitors to bind at the catalytic site of the enzyme near the zinc ion thus blocking its access to the substrates. The latest HDACIs are related to SAHA known as CHAP compounds which are highly potent and show their inhibitory activity at very minute (nanomolar) concentrations. Sulfonamide hydroxamic acids have been tried and demonstrated anti-cancer potential in human colon cancer. Another class of HDACIs is cyclic tetrapeptides, e.g. apicidin and trapoxin, which are irreversible inhibitors of HDACs and bind at their active sites. Among the HDACIs, small chain fatty acids like phenylbutyrate were the first to go into clinical trial as anticancer drugs, but the tumor regression in leukemia or myelodysplastic anemia were not very significant (Gore and Garducci, 2000). The compound could be a very good anti-cancer agent particularly because it is well absorbed from GIT and the pharmacological levels are achievable through oral administration (Gilbert et al., 2001). Combination therapy of phenylbutyrate and all-trans retinoic acid did show remission in a leukemia patient but later resulted in development of resistance (Warrell et al., 1998). Novich et al. (1999) reported that the response to phenylbutyrate in the clinical trial was highly variable from patient to patient. The clinical trials of SAHA in Hodgkin’s lymphoma and a number of solid tumors undertaken by Kelli et al (2001) demonstrated regression in the size of the tumors as well as the clinical symptoms. At the doses testes, there were minimal or no toxic effects and the drug appeared to be well tolerated. Analysis of Anticancer Activity of SAHA on Squamous cell carcinoma cell lines As discussed above, suberoylanilide hydroxamic acid (SAHA) is one of the few highly promising anti-cancer agents that acts as inhibitor of histone deacetylases. The anti-tumor activities of any inhibitor can be tested by a number of in-vitro assays, viz., mitogen viability assays and cell-cycle arrest by flowcytometry. To test the in-vivo effectiveness of SAHA, the tumor cells can be grafted into nude mice or other animal model(s) and tested for tumor size. The potential of SAHA has been tested by Tatsuhito et al. (2009) on human oral squamous cell carcinoma (OSCC) cell lines. They have employed in vitro as well as in vivo experiments to demonstrate the antiproliferative activity of SAHA. Cell lines tested: Human OSCC cell lines SAS, HSC-2m HSC-4 and CA9-22 were used. a) Mitogenic assay – The crystal violet mitogenic assays with a number of tumor cell lines demonstrated that SAHA inhibited the tumor growth. Tumor cells were exposed to SAHA at concentrations ranging between 0.5 to 8 µM for 72 hours. The inhibitor reduced the cell viability in a dose as well as time dependent manner. The LD50 (the dose required for 50% kill) of SAHA was between 0.7 µMto 1.7 µM. The HDACI appeared to be most effective against CA9-22 cell line. b) Cell-cycle arrest – SAHA was also tested on SAS, HSC-2 and HSC-4 cell lines for its effect on the cell cycle. It was observed that the incubation with SAHA resulted in the increase inproportion of cells in G1 phase of cell cycle and decrease in the proportion of cells in S-phase, in all the cell lines tested. c) In-vivo assays – SAHA appeared to inhibit the tumor growth in vivo experiments as well. The size of SAS tumors xenografts was reduced in mice administered with 50 mg/kg BW of SAHA. They also studied the acetylation pattern of p53 in these tumors by using immunohistochemistry using anti-acetylated-p53 antibodies and reported that the SAHA treated tumors showed much higher levels of acetylated-p53 proteins. d) Mechanism of action – To study the mechanism of action of SAHA in controlling tumor cell proliferation, Tatsuhito et al. used western blot assays to look at the expression of different proteins involved in cell cycle regulation. A significant stimulation of acetylated-p53 and p21 proteins was demonstrated along with down regulation of cyclin D2. Induction of p21 protein along with mRNA The expression of p21 protein in the tumor cell lines can be tested by western blot assays using anti-p21 antibodies. The cell cultures exposed to SAHA can be harvested after a sufficient period of incubation and the cell digest split into two sets. One can be tested for the expression of mRNA for p21 by RT-PCR whereas the other can be used for western blot analysis. If the western blot positivity for p21 proteins corresponds with the positivity in PCR assays, it can be inferred that the transcription of p21 mRNA is accompanied with the translation into p21 protein. Alternatively, the cell cultures showing p21 mRNA expression, after fixation, can be subjected immunohistochemistry using labeled anti-p21 antibodies to demonstrate the presence of p21 protein. References Allfrey, V. G., Faulkner, R. M., Mirsky, A. E., 1964. Acetylation and methylation of histones and their possible role in the regulation of RNA synthesis. Proc. Natl. Acad. 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