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Histone deacetylase inhibitors are an emerging class of therapeutic agents that have gained FDA approval for clinical use for the treatment of cancer and inflammatory diseases since 2006. During pre-clinical, through to phase II trials this class of drugs demonstrated significant efficacy and safety, showing few side effect. Implementing there effect on target cells by promotion of cell cycle arrest, differentiation and/or apoptosis, these drugs may therefore be considered potential breakthrough drugs for many diseases. However, the molecular mechanisms involved in histone deacetylase inhibitors anti-cancer activity and in the signals triggered by cancer cells in response to these compounds in order to counteract cell death induction with regards to the immune system response remain poorly understood. The main proposed theory for many is Histone deacetylase inhibitors induce acetyl hyperactivation trough inhibition of the histone deacetylase enzymes, altering the compact state of DNA, and finally promoting gene transcription. To further complicate this theory we must consider other consequence of histone deacetylase inhibition, such as the repression of various genes, demonstrating supplementary functions beyond those previously considered. It is these Histone deacetylase inhibitors regulatory events concerning T-lymphocyte production that will be the primary focus of this thesis; specifically the effects of histone deacetylase 1 and/or 2 will be of interest.
The literature review will provide relevant knowledge into the Lymphatic system, T-lymphocyte differentiation and histone deacetylation.
The lymphatic system and T-lymphocytes
1.2.1 The Lymphatic system
The lymphoid system consists of several lymphoid organs including the spleen, lymph nodes, bone marrow, thymus and mucosa-associated lymphoid tissue (MALT). These concentration, interaction and deployment of lymphocytes. There are four main classes of lymphocytes involved in the monitoring and regulation of antigen (Ag) intrusions: lymphocytes, natural killer (NK) cells, macrophages and dendric cells. For the purpose of this study, we will focus on lymphocytes, and more specifically thymus derived lymphocytes (T-cells).
Lymphocytes are one of the five kinds of white blood cells or leukocytes that consist of two main types of immune cells, namely the bursa of Fabricius cells (B-cells) and T-cells. B-cells are derived from bone marrow stem cells and develop within the bone marrow. Final maturation occurs in the spleen whereby mature b-cells express surface immunoglobulin (Ig) that act as receptors for specific antigens, where they are primarily involved in the production of antibodies. T-cells also derive from the bone marrow, but unlike B-cells, these hematopoietic stem cells migrate to the richly innervated thymus, a gland with well-defined cortical and medullary regions, where they mature.
T-cells are required for full immunity expression and possess two forms of T-cell receptors (TCR): Alpha-Beta (Î±Î²) whose TCR is a heterodimer of an alpha chain with a beta chain, and Delta-gamma (Î³Î´) whose TCR is also a heterodimer of a gamma chain paired with a delta chain. Both classes of TCR do not occur autonomously.
1.2.2. T-lymphocyte subset differentiation
T-lymphocyte progenitors develop from multipotent hemopoietic foetal liver-derived stem cells in utero (Jotereau et al 1987) and bone marrow progenitors in adult life (Scollay et al 1986; Donskoy and Goldschneider 1992) where they migrate via the blood stream for maturation to the thymus and begin to express an array of surface glycoproteins. The prothymocyte enters the thymic cortex consisting of specialized stromal cells; in embryonic mice VÎ³3-bearing cells form via TCR-gene rearrangement that mature into Thy-1 + dendritic epidermal Î³Î´ cells found in the skin of adults (Havran and Allison 1990). During adult lymphoid development of early T and B lineage cells the enzyme terminal deoxynucleotidyl transferase (TdT), an enzyme that catalyzes the repetitive addition of dNTPs to the 3´-OH end of a DNA fragment, becomes expressed during TCR-gene rearrangement. TdT increases TCR and the immunoglobulin repertoire diversity during antigen-receptor gene rearrangement by insertion of nucleotides at the template-independent (N region), D and J junction sites (Alt and Baltimore 1982.; Komori el al 1993). Adult mice lacking TdT due to a gene mutation were found to possess an immature lymphocyte repertoire with only a small number of N nucleotides (Gilfillan et al 1993). IL-2 and IL-2R become expressed leading to autocrine proliferation of T-lymphocytes.
T-lymphocytes begin their cell cycle as Th0 type cells (Biedermann et al 2004). Rearrangement of the TCR-genes (Î±Î²/ Î³Î´) takes place where T-lymphocytes progeny progress to either
Cytotoxic T-lymphocytes that inflict direct damage to target cells as a result of various mechanisms such as CD95 or the perforin/granzyme system and also the production of effector cytokines such as TNF-a or IFN-Î³ (Russell and Ley 2002), characterised by the surface expression of CD8,
Helper T-lymphocytes, characterised by CD4 expression,
CD4+ cells recognize antigens presented in the context of Class II major histocompatibility complex (MHC), while CD8 positive cells recognize antigen presented in the context of Class I MHC.
Figure 1: MHC class II molecules present antigens to CD4+ T-cells and MHC class I molecules present antigens to CD8+ T-cells. CD4+ T-cells activate and direct other cells of the immune system e.g. B-cell antibody class switching and activation and growth of Cytotoxic T Cells. Their TCR has affinity for Class II MHC, but possess no cytotoxic or phagocytic activity. CD8+ T-cells inflict exocytosis of perforin and granzymes resulting in apoptosis of infected target cells. Their TCR has affinity for Class I MHC and possess cytotoxic activity, "Killer T-Cell".
At this stage, the pre-T-lymphocytes are CD3+CD4-CD8- or "double-negative" cells. Double-negative cells that productively rearrange gamma and delta chain gene segments develop into CD3+CD4-CD8- gamma/delta T-lymphocytes that become exported to the periphery in small numbers. Successful rearrangement of a set of TCR genes suppresses further rearrangement of TCR genes on the sister chromatid (allelic exclusion), thus each cell only expresses TCR with a single specificity. The majority of double-negative cells will go on to rearrange Î± and Î²-chain gene segments. The Î²-chain genes rearrange and are expressed first with a pre-T Î±-chain to form the pre-TCR. Once the pre-TCR recognizes an intrathymic ligand, a signal is generated and transmitted through CD3 which:  Halts further beta chain gene rearrangement (allelic exclusion)  Enhances alpha chain gene rearrangement  Causes CD4 and CD8 to be expressed. These immature double-positive T-lymphocytes express both CD4+ and CD8+ and later mature into single-positive T-lymphocytes, allowing them to become categorized based upon the loss of a cell surface co-receptors expression of either CD4+ CD8- or CD4- CD8+ .
In 1961, Miller discovered the importance of the thymus in T-lymphocyte development when carrying out studies of neonatal thymectomized mice (Miller J.F.A.P 1961). In 1986, Mosmann et al. published their findings of two identified subsets of activated CD4 positive T-lymphocytes, Th1 and Th2 cells, which differed from each other amongst mouse CD4+ T-lymphocytes clones, (Mosmann et al. 1986; Cherwinski et al. 1987), and later where identified amongst human T-lymphocytes (Del Prete et al. 1991) by their pattern in cytokine antigen-induced production of cytokines and there effector functions.
Epigenetics, Chromatin and Histone Modifying Enzymes
The term epigenetic refers to the change(s) in genetic expression and cellular phenotype without alterations in the DNA sequence itself, which can persist through one or more generations (Shilatifard, 2006). With the exception of controlled genomic rearrangements, such as those of the immunoglobulin and T-cell receptor genes in B and T cells, all other differentiation processes are initiated or maintained through epigenetic processes (Lund, A. H. & van Lohuizen, M. Epigenetics and cancer. Genes Dev. 18, 2315-2335 (2004)). Studies on the post-translational modifications of histones and DNA methylation (not discussed here) of conserved lysine residues on the amino-terminal domains has shown epigenetic programming to be crucial in mammalian development. Stable inheritance of epigenetic settings is essential for the maintenance of tissue- and cell-type-specific functions (Li, E. 2002)Chromatin modification and epigenetic reprogramming in mammalian development. Nat. Rev. Genet. 3: 662-673).
Histones can be defined as small, highly basic, acid soluble proteins (Bloch, D. P. 1963) that associate with nuclear DNA. In eukaryotes, DNA is wrapped around a histone octamer comprising of two copies of each nucleosomal core histones (H2A, H2B, H3, and H4), providing a nuclear scaffold of repeated units of chromatin called the nucleosome. Each of these core histone proteins poses globular domains, which allow for the mediation of specific histone interactions; namely histone-histone and histone-DNA (Luger, K. (2006). Dynamic nucleosomes. Chromosome Res. 14, 5-16). The mediation of an unstructured amino-terminal by these globular domains also takes place which in turn serves as a substrate for histone modifying enzymes that allow for the induction of post-transitional modifications, in the case of this discussion, acetylation (Cosgrove, M.S. and Wolberger, C. (2005). How does the histone code work? Biochem Cell Biol. 83, 468-476).
Although chromatin structure organisation is highly complex, two major forms exist; heterochromatin and euchromatin. The former holds a compact structure intrinsic to transcriptional inactive regions of DNA (Cosgrove, M.S. and Wolberger, C. (2005). How does the histone code work? Biochem. Cell Biol. 83, 468-476). The latter stands at the opposite end of the spectrum, comprising of uncondensed chromatin lacking histone HI and exists n gene rich environments (Benbow, R.M. (1992). Chromosome structures. Sci. Progress 76, 425-450). Two histone modifying enzymes involved in determining the state of acetylation of histones are histone acetyl-transferases and histone deacetylases, of which both play a major role in the regulation of gene expression (Grunstein, 1997), both which will be discussed in detail later. Many studies over almost two decades have reference to an altered are histone acetyl-transferase and histone deacetylase activity being present in several cancers (Muraoka et al. 1996; He et al. 1998; Lin et al.1998; Timmerman et al. 2001).
Another factor known to modulate the structure of chromatin is ATP-dependant chromatin remodelling complexes (ACRC). Switch 2 (SWI2) and Imitation switch (ISWI) are two of the most highly characterised ATPase subunits within this class of complexes (Eberharter, A. and Becker, P.B. (2004). ATP-dependent nucleosome remodelling: factors and functions. J. Cell Sci. 117, 3707-3711), both have been noted to regulate transcription in an opposing manor; SWI2 provides support for ACRC allowing nucleosomes to become disorganised and reorganised so as to allow for the DNA to become increasingly accessible. ISIW acts in the reverse of SWI2 causing the nucleosomes to organise in to precisely spaced arrays promoting transcriptional repression (Johnson, C.N., Adkins, N.L. and Georgel, P. (2005). Chromatin remodeling complexes: ATP-dependent machines in action. Biochem. Cell Biol. 83, 405-417; Saha, A., Wittmeyer, J. and Cairns, B.R. (2006). Chromatin remodelling: the industrial revolution of DNA around histones. Nat. Rev. Mol. Cell Biol. 7, 437-447).
Histone Modifying Enzymes
Including histone deacetylation and acetylation, roughly 150 different modifications have been identified on specific histone residues alterations, indicating epigenetic pathways clearly have an important role in tumorigenesis. The majority of modifications are instituted on the amino terminal tails, while fewer are located on the histone globular domains (Cosgrove and Wolberger, 2005). Later in this section two histone modifiers will be discussed; histone acetyltransferase and histone deacetylase enzymes, and the outcome of some of the effects by changes made to the chromatin organisation and gene regulation made known.
Histone acetylation has long been hypothesised in play an integral role in transcriptional regulation, DNA replication and repair by disrupting certain electrostatic interactions, with acetylation playing a critical role in influencing histone interactions with specific non-histone regulatory proteins (Brownell and Allis, 1996). A group of enzymes collectively known as histone acetyltransferase (HATs) catalyses the conversion of positively charged primary epsilon (Îµ)-amines to uncharged secondary amines in which acetate from the substrate acetyl-CoA is covalently added to specific lysine residues of histone (Pazin and Kadonaga 1997). Disruption of the negatively charged phosphodiester DNA backbone and positive amino-terminal histone tails allows the chromatin structure to weaken and become more relaxed. This relaxed structure then becomes open to transcription factor target site binding.
There are two classes of HATs; A-type and B-type, categorised based on their subcellular localisation (origin) and substrate specificity (function). A-type HATs carry out nuclear histone acetylation, related to transcription i.e. generating chromatin-based histone modifications (Brownell and Allis, 1996). B-type HATs catalyze acetylation of newly synthesized H4, transported from the cytoplasm to newly replicated DNA at the nucleus i.e. cytoplasmic modification in chromatin assembly (Ruiz-Carrillo et al. 1975; Allis et al. 1985). So far five HAT families have been classified: Gcn5-related acetyltransferases (GNATs); the MYST-related HATs; p300/CBP HATS; the TATA bining protein (TBP)-assosiated factors (TAFs) and the nuclear horomone-related HATs (SRC3) (Carrozza et al. 2003) all which display HAT activity in humans.
Although histones were the first recognized targets of HATs, non-histone proteins were soon acknowledged as being HAT substrates, leading to the new term 'acetylome' being adopted to describe all histone proteins collectively (Minucci and Pelicci, 2006 (Minucci, S. and Pelicci, P.G. (2006). Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer. Nat. Rev. Cancer 6, 38-51.). The transcription factor p53 was the first of the non-histone proteins to be identified (Gu and Roeder, 1997) which is activated following DNA damage. Activation occurs by kinase-mediated phosphorylation and the acetyltransferases p300/CBP at specific residues forming the p53-p300/CBP-DNA stable complex, thereby promoting transcription.
Interestingly HATs acetylate many targets beyond histones and are the catalytic core of various large protein co-activator complexes. As a result activation of gene expression HATs modulate various properties of target proteins regulating their stability, origin and function. HATs therefore regulate many processes beyond that of chromatin organisation.
188.8.131.52 Histone Deacetylase
HATs and HDACs together control the steady-state levels of histone acetylation. HATs catalyse histone acetylation and in opposition HDACs control gene transcription by regulating acetylation of DNA sequence-specific transcription factors by catalysing the removal of the acetyl groups on acetylomes (Gu and Roeder, 1997; Wilson et al., 2006). Through these mechanisms, HDACs are emerging as critical regulators of gene expression with studies showing several HDAC inhibitors (HDACi) having successful results in the treatment of malignant cells where HDACi shown to revert the malignant phenotype in studies by Minucci et al 2001.
Rpd3 was the first HDAC gene to be identified in the yeast S.cerevisiae (Vidal, M., A. M. Buckley, F. Hilger, and R. F. Gaber. 1990. Direct selection for mutants with increased K+ transport in Saccharomyces cerevisiae. Genetics 125:313-320; Vidal, M., R. Strich, R. E. Esposito, and R. F. Gaber. 1991. RPDI (SIN3) is required for maximal activation and repression of diverse yeast genes. Mol. Cell. Biol. 11:6306-6316). Since then several other HDAC genes have been identified from yeast to human through genetic and biochemical approaches, and in more recent years through the use of bioinformatics. At least 10 yeast genes have been identified possessing deacetylase activity and are grouped and divided into three classes of HDACs based their functional similarities and sequence: class 1 relate to reduced potassium dependency 3 (Rpd3); class 2 correlate histone deacetylase 1 (Hda1) and lastly class 3 associate to the silent information regulator 2 (Sir2). (Brachmann, C. B., Sherman, J. M., Devine, S. E., (1995). The SIR2 gene family, conserved from bacteria to humans, functions in silencing, cell cycle progression, and chromosome stability. Genes Dev. 9, 2888-2902; Kurdistani, S.K. and Grunstein M 2003. Histone acetylation and deacetylation in yeast. Nat. Rev. Mol. Cell. Biol. 4: 276-284). Eighteen HDACs identified from mouse and humans exist. These are grouped and divided into three classes of HDACs based their protein homology to yeast HDACs. Class 1 include HDAC 1, 2, 3, 8; class 2 contains of HDAC 4, 5, 6, 7 and 9; class 3 is comprised of SIRT1-7; and finally class contains HDAC 11 only. For the purpose of tis paper further referance will only be made to class 1 HDAC1 and 2.
Figure 2: The histone switch. HAT and HDAC activities regulate the acetylation status of chromatin. Acetylation establishes a structure that permits ATP-dependent chromatin remodelling factors to open promoters. Acetylated histone tails are shown as purple circles
Class 1 Deacetylases:
HDAC1 was the first of the histone deacetylase family to be identified in mammals and are closely related to the yeast Rpd3. HDAC1 and HDAC2 interact with one another and form the catalytic core of several multi-subunit complexes. Mammalian Sin3, nucleosome remodeling deacetylase (NuRD) and corepressor of REST (repressor element 1 silencing transcription factor (CoREST)) complexes are three protein complexes identified in mammals containing these HDAC1 and HDAC2 catalytic core enzymes (Yang and Seto, 2008 Yang, X.J., and Seto, E. (2008). The Rpd3/Hda1 family of lysine deacetylases: from bacteria and yeast to mice and men. Nat. Rev. Mol. Cell Biol. 9, 206218). These HDAC1/2 complexes lack the ability to bind directly with DNA and thus rely on verifying associated target genes of interaction partners via sequence specific DNA binding capability.
The tumour suppressor protein p53 promotes cell cycle arrest and/or apoptosis in response to DNA damage along with other forms of stress and is mutated in ~50% of all cancers; therefore its affiliation with acetyl modification has become one of the most highly studied and best characterised. Acetyl modification of p53 at six sites within the C-treminus of p53 increases transcription activity (Kawaguchi, Y., Ito, A., Appella, E. & Yao, T. P. Charge modification at multiple C-terminal lysine residues regulates p53 oligomerization and its nucleus-cytoplasm trafficking. J. Biol. Chem. 281, 1394-1400 2006). p21, an inhibitor of cyclin dependant kinases, expression is induced by affiliation, affecting the activity of cyclin D-, E- and A- dependant kinases through the G1 phase of the cell cycle causing cell cycle arrest and promotion of DNA repair pathways. Acetylation of p53 is also crucial in the ubiquitination of p53 degradation; MDM2 inhibits p53 acetylation by binding and reducing p300/CBP acetyltransferase activity (Kobet et al., 2000; Ito et al., 2001(Ito,A., Lai,C.H., Zhao,X., Saito,S., Hamilton,M.H., Appella,E., Yao,T.P. (2001) p300/CBP-mediated p53 acetylation is commonly induced by p53-activating agents and inhibited by MDM2. EMBO J., 20, 1331±1340. Kobet,E., Zeng,X., Zhu,Y., Keller,D. and Lu,H. (2000) MDM2 inhibits p300-mediated p53 acetylation and activation by forming a ternary complex with the two proteins. Proc. Natl Acad. Sci. USA, 97, 12547±12552.)). Studies have confirmed a direct association with the deacetlyation of several sites within p53 responsible for reduction of its activity and the HDAC1/2 complex, rendering its presence essential. Reduction of p53 activity via the HDAC1 and possibly HDAC1/2 complex, results in the repression of genes inhibiting cell cycle progression (Ito et al. 2002 Ito, A., Kawaguchi, Y., Lai, C. H., Kovacs, J. J., Higashimoto, Y., Appella, E. & Yao, T. P. (2002) EMBO J. 21 , 6236-6245.pmid:12426395 ; Zhang et al.,1998 Zhang,Y., LeRoy,G., Seelig,H.P., Lane,W.S. and Reinberg,D. (1998) The dermatomyositis-speci®c autoantigen Mi2 is a component of a complex containing histone deacetylase and nucleosome remodelling activities. Cell, 95, 279±289.)
HDAC catalytic domain Nuclear Localisation Signal
Figure 3: Schematic representation of the mammalian Class I HDACs. boxes HDAC 1, 2 and 3 are ubiquitously expressed in all tissue types, while HDACs 1, 2 and 8 are restricted to the nucleus. Homology between HDACs 1 and 2 is ~85% varying only at the C' terminus. Number of amino acids (aa) in each protein are shown.
Histone Deacetylase Inhibition:
As discussed previously in this thesis, it has become clear that HDACs modulate chromatin plasticity, facilitating protein-DNA interactions. Transcriptional control becomes disrupted leading to biological consequences of HDAC inhibition including: cell differentiation; cell cycle arrest; apoptosis; cytoskeletal alterations; and angiogenesis (Burgess et al., 2004 and Carey and La Thangue, 2006). Over-expression of various HDACs in many cancer types have lead to HDAC inhibitors (HDACis) being extensively investigated as potential anticancer agents in recent years. In a study carried out by Van Lint et al. where two different forms of HDACi were investigated for their effect on histone hyperacetylation on gene expression. Contrary to what we might be expect from the extensive presence of HDACs among chromatin, it was found that these forms of inhibitors are highly selective, exerting their affect on transcription on <2% of expressed genes in cultured cells. Although these drugs are becoming more rapidly approved for the treatment in cancer, the concern is now turning to the primary mechanism by which these agents trigger cell death specifically in tumour cells.
Many studies over the last decade have reported the over expression of both HDAC1 and HDAC2 in several forms of cancer such as gastric tumours, colon cancer and hormone refractory prostate carcinoma (HDAC1 only) (Halkidou et al., 2004; Song et al., 2005; Bolden et al., 2006 and Wilson et al., 2006). It is due to these studies and hundreds more observations like them, which have prudently lead to the intense study of HDACis as a therapeutic means of restoring the balance in tumour cells, and other various diseases, whereby HATs and HDAC activities have become imbalanced.
It was previously believed that HDACis offered anti-tumour activity by simply disrupting the balance of histones within chromatin allowing for hyperacetylation of the original histone state, ultimately allowing altered gene regulation. Although the mechanisms by which HDACis exert their effect at a molecular level have still yet to be fully characterised, a proposed mechanism has been introduced suggesting the deacetylation of histones is brought about by chelating of Zn2+ ion of class 1 and 2 Zn2+-dependant HDAC, via the binding of the HDACi at the active site pocket (Finnin et al., 1999; Somoza et al., 2004 and Vannini et al., 2004). This new hypothesis is highly supported by the work of Van Lint, Mariadason and Peart that found that approximately 10-22% of regulated by HDACis (Van Lint et al., 1996 Mariadason et al., 2000 and Peart et al., 2005). These investigations along with many other results (Nair et al., 2001 and Sasakawa et al., 2003) make available an argument that HDAC activity is not solely committed to the regulation of histone acetylation, but may be involved in a much broader range of gene regulation mechanisms.
p53-dependent activation of the p21WAF1 and Bax gene promoters are one of many in a series of complexes that has be reported to be disrupted by misregulation of histone acetylation. The induction of cell cycle arrest and apoptosis, respectively (Bolden et al., 2006 and Lin et al., 2006) have been reported to be induced by stabilisation of the non-histone protein levels of p53 via mediated acetylation by HDACis. This example stands amongst many other modifications to have been reported on HDACi activity. Other proposed machanisms include that of HDAC-protein phosphate 1 complexes (Brush et al., 2004; Canettieri et al., 2003 and Chen et al., 2005).
G2 check point
Figure 4: Proposed regulatory mechanisms of HDACis. HATs and HDACs have major roles in the control of cell fate and their misregulation is involved in the development of some human tumours. HDAC inhibitors allow for the correct regulation of cell by inducing antitumor activities in cancerous cells such as activation of differentiation programs, inhibition of the cell cycle, and induction of apoptosis.
HDAC inhibitors, although described here as monotherapies, are also being investigated to synergize with classic chemotherapeutic agents as well as newer signal transduction pathway modulators and angiogenesis inhibitors. HDAC inhibitors could span multiple cancers and be used alongside a broad range of therapeutics. Most HDACi currently in clinical development act by interfering unspecifically with the enzymatic activity of all class I HDACs, therefore the development of isoform-specific HDACi could lead to better therapeutic efficacy.
Knockout mouse models have demonstrated the importance of HDACs in cell differentiation. Genetic deletion of the class I genes HDAC1 (Lagger et al. 2002) or HDAC2 (Trivedi et al. 2007) results in lethality during the period around embryonic development or childbirth (from around week 28 of pregnancy to around one month after the birth), respectively. With the role of HDAC being widely studied, we have yet to determine its effects on immune function.
Histone acetylation is a particularly important modification of histone amino-termini. Hyperacetylation are associated with transcriptionally permissive chromatin, hypoacetylation are associated with repression of gene expression (Marks et al., 2003). The study of HDAC inhibitors is still in its infancy. While this class of agents holds great promise as anticancer therapy, we have yet to learn how best to administer these drugs. In this era of personalized medicine, we strive to individualize therapy so that maximal benefit may be achieved and unnecessary toxicity minimized. Our ability to do so depends on furthering our understanding of the various mechanisms by which HDAC inhibitors exert their effects, elucidating the optimal sequence and schedule of administration, and identifying individuals who are most likely to benefit from this particular therapy.