Role of Histone Deacetylase in Cancer - Term Paper Example

Part A “Role of Histone Deacetylase in Cancer” (Molecular Biology Images http www.molecularstation.com/molecular-biology-images/502-dna-pictures/60-nucleosome.html) Introduction An initial thought that cancer is the outcome of modifications in genetic material either by their amplification or translocations, deletions or as a result of mutation was later added by the triggering of oncogenes or suppressing the expression of tumor-suppressor genes. In the present era, another concept has been given focus that epigenetic changes especially the methylation of DNA and posttranslational histone modifications namely, acetylation, methylation, hypermethylation and phosphorylation are responsible for causing cancer (Esteller, 2002). Chromatin (condensed heterochromatin and extended euchromatin) is a composite amalgamation of DNA, RNA and proteins that formulate chromosomes. It provides strength to the DNA (Dame, 2005) Amongst its three components, DNA and histone proteins (alkaline in nature and characteristic feature of eukaryotic nucleus) form nuclear matter and are responsible for packing DNA in small structural units called nucleosomes, a comportment to fit them inside the cell. Chromatin, directs all cellular tasks including gene expression, DNA replication, cell division and differentiation. Histones play an imperative role in chromosome vitality, stability and gene expression (Alberts, 2001). Histones are of various types but the most prominent ones reported are H1, H2A, H2B, H3 and H4. Except H1 rest form chromatin sub-units called nucleosomes. Each nucleosome is an octamer with 2 units each of H2A, H2B, H3, H4. Each nucleosome can accommodate 165 base pairs of DNA strands (2.85 turns of DNA) (Kornberg, 1974). A linker DNA with H1 histone links two nucleosomes and hence responsible for super-coiling (Alberts, 2001). Histone proteins present in chromatin undergoes chemical alterations. Acetylation of lysine residues of H3 and H4 is a resultant of equilibrium between histone acetyltransferases (HAT) and histone deacetylases (HDAC). Levels of these modifications are imperative for gene regulation, its expression and chromatin remodeling (Goll, Bestor, 2002). It is observed that acetylated lysine present in the tails of histone is related to euchromatin state while its deacetylation is related to heterochromatin state, responsible for transcriptional gene silencing (Johnstone, 2002; Iizuka and Smith, 2003). X-ray diffraction pattern discloses conservation of histone proteins in eukaryotes (Lunger 1997) with variation in H1 amino acids (Alberts, 2001). Deacetylation of histones causes greater ionic interactions between positive histones and negative DNA, forming compressed chromatin structure, suppressing gene transcription. On the contrary, acetylation of histones induce various genome functions, assembly of chromatin, repair of damaged DNA and recombination of genetic material (Polo, 2005; Vidanes et al., 2005). Modified histones link with centromeres (site of spindle connection in cell division), telomeres and inactive X-chromosomes (Barr body). In mammals Barr body encompass histone variant, macroH2A, which is three times of normal H2A (Chadwick, 2002). Another variant of H3 is observed in Drosophila chromosomes, CENP-A, it is said to be an evolutionary alteration and has a role in cell cycle in combination with CENP-B and CENP-C (Redon, 2002). Reports say that H2A and H3 also possess variants as H2AZ, H2AX and H3.3 respectively. H2AZ is responsible for reduced nucleosome stability, on the other hand H2AX is the most profuse variant of histone, play vital role in the breakage and repair mechanism of DNA and also in V(D)J recombination which aids in producing diversified immunoglobulin molecules (Redon, 2002). In a similar manner various histone variants are responsible for the gene expression and DNA stabilization in eukaryotes. Recent researches reveal that H2AX is essential for cell-cycle checkpoint arbitration and is responsible for cancer induction as a result of DNA damage (Kastan, Bartek, 2004). Additionally, variant H3.3 is associated with the chromosomes present in non-dividing cells and helps in the transcription of DNA in active genes (Ahmad, 2002). It is also observed that H1 is replaced by H5 histones in RBCs of birds and is shown to possess role in silencing of chromosomal DNA in their condensed nuclei (Aviles, 1978). Nucleosomal histones undergo the process of acetylation and deacetylation. These modifications are important for inflection of chromatin configuration, their purpose for existence and also for expression of genes. (HATs) and (HDACs) balance directs stability of histone acetylation while imbalance generates cancer (Mei, 2004). Acetylation and deacetylation of histone play an imperative part in the gene expression and its regulation. It is evident that HDAC cause hypoacetylation and gene silencing. If genes of HDACs are altered due to mutation or expression is misrepresented, vital proteins for cell proliferation, cell-cycle regulation and apoptosis are altered leading to cancer. Therefore, HDACs are emerging as potential therapeutic agents for cancer therapeutics (Ropero, 2007). HDACs are inhibited by a range of compounds, encompassing four major groups: I. Short chain fatty acids (butyrate, valproic acid or valproate), II. Benzamides (MS-275, CI-994), III. Cyclic tetrapeptides with or without Zn2+ binding L-2-amino-8-oxo-9,10-epoxydecanoic acid (AOE). They inhibit class I and II HDACs by attacking Zn2+. Class III inhibitors affect NADH, sirtuin activity. IV. Category of HDACi is hydroxamates. They are known to buildup large quantity of acetylated histone proteins in normal cells as well as in tumorous tissues, in cutaneous T cell lymphoma (CTCL). HDACi cause cell differentiation, setting cell cycle in growth phase either in G1 and/ or G2 phase, or encourage cell death i.e. apoptosis in tumor/ altered cells. Molecular mechanism displays p21 (WAF/CIP1) initiation on exposure of tumor cells to HDACi. Xenografts establish the fact, HDACi portray antitumor actions with minimal side effects. When clinical trials of HDACi were given in bearable concentrations, a noteworthy antitumor behavior was observed. This remarkable and promising contribution has revolutionized cancer treatment, the fact emerged as an amalgamation of HDACi, cytotoxic drugs and differentiation-inducing agents possess antitumor effect (Mei, 2004). HDACs control transcription factors like Ef2, Stat3, p53, NF-κB, TFIIE and retinoblastoma protein (Lin et al, 2006). HDACs bring deacetylation misregulating cellular homeostasis, cell-cycle, cell differentiation and cell death i.e. apoptosis (Minucci, 2006). The amount of chromatin compression (formation of heterochromatin) and folding is greatly supported by H4 acetylation at the lysine 16 position, responsible for gene transcription (Mona, 2007). Different classes of HDCAs depending on the progression characteristics and province association encompass (Dokmanovic, 2007): Class I: Homologous to Rpd3 in yeast encompass HDAC1, HDAC2, HDAC3, HDAC8 present in nucleus, universally expressed in human tissues. HDAC 1, 2, 3 are highly expressed in renal cancer cells (Fritasche, et al, 2008), in gastric cancer (Weichert, 2008) Class II: Homologous to yeast HDA1, IIa: HDAC4, HDAC7, HDAC9; localized to nucleus IIb encmpass HDAC6, HDAC10. They are specific to tissues in their expression and can oscillate between cytoplasm and nucleus, displaying some role in acetylation of non-histone proteins. This class of HDAC has specific role in human breast cancer cells (Vanessa et al). Its expression is constrained to smooth muscle, heart, pancreas and brain. Class III: Homologs of Sir2 in yeast S. cerevisiae Sirtuns in mammals (SIRT1-7) and require NAD+ cofactor and is found to be associated with aging, neurodegenerative disorders, HIV and cancer. Class IV: HDAC11. Structural homology with Rpd3 and HDA1 Class I and II are susceptible to trichostatin A (TSA) a HDAC inhibitor on the other hand Class III is insensitive to it (Ropero, 2007). Inability to deacetylate by Class I HDACs cause transcription suppression enhanced cellular growth propagation, thereby seizing G1 checkpoint to stop any kind of DNA repair, cellular senescence and contact inhibition. Also, improper deacetylation by Class II HDACs causes delocalization of HDACs resulting in the onset of cancer (Wade, 2001). HDAC family members have Zn2+ cofactor- dependent catalytic activities and therefore any chemical targeting Zn-ion pocket can inhibit their activity. References 1. Ahmad, K., Henikoff, S. 2002. Histone H3 variants specify modes of chromatin assembly. Proc. Natl. Acad. Sci. USA, 10.1073/pnas.172403699. 2. Alberts, B., et al. 2001. Molecular Biology of the Cell- 4th Ed., Garland Science Publication, New York. 3. Aviles, F. J., Chapman, G. E., et al. 1978. The conformation of histone H5. Isolation and characterisation of the globular segment, Europ. J. Biochem., 88, 363-371. 4. Chadwick, B. P., Willard, H. F., 2002. Cell cycle-dependant localization of macroH2A in chromatin of the inactive X Chromosome. J. Cell Biol., 157, 1113-1123. 5. Dame, R. T. 2005. The role of nucleoid-associated proteins in the organization and compaction of bacterial chromatin. Mol. Microbiol. 56 (4), 858–70. 6. Dokmanovic, M., Clarke, C., Marks, P. A. 2007. Histone deacetylase inhibitors: overview and perspectives. Mol. Cancer Res. 5 (10), 981–9. 7. Esteller, M. 2002. CpG island hypermethylation and tumor suppressor genes: a booming present, a brighter future. Oncogene, 21, 5427–5440. 8. Fritasche, et al, 2008. Class I histone deacetylases 1, 2 and 3 are highly expressed in renal cell cancer. BMC Cancer, 8, 381 9. Goll, M. G., Bestor, T. H. 2002. Histone modification and replacement in chromatin activation. Genes Dev., 16, 1739-1742. 10. Iizuka, M., Smith, M.M. 2003. Functional consequences of histone modifications. Curr. Opin. Genet. Dev. 13, 154–160. 11. Johnstone, R.W. 2002. Histone-deacetylase inhibitors: novel drugs for the treatment of cancer. Nat. Rev. Drug Discov. 1, 287–299. 12. Kastan, M. B., Bartek, J. 2004. Cell-cycle checkpoints and cancer. Nature, 432, 316-323. 13. Kornberg, R. D. 1974. Chromatin structure: A repeating unit of histones and DNA. Science, 184, 868-871. 14. Lin, H.Y., Chen, C. S., Lin, S.P., Weng, J. R., Chen, C.S. 2006. Targeting histone deacetylase in cancer therapy. Med. Res. Rev. 26, 397–413. 15. Lunger, K., Mander, A. W., et al. 1997. Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature, 389, 251-260. 16. Mei, S., Ho, A. D., Mahlknecht, U. 2004. Role of histone deacetylases inhibitors in the treatment of cancer (Review). Int J Oncol. 25(6), 1509-19. 17. Minucci, S., Pelicci, P.G., 2006. Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer. Nat. Rev. Cancer, 6, 38–51. 18. Molecular Biology Images http://www.molecularstation.com/molecular-biology-images/502-dna-pictures/60-nucleosome.html. 19. Shahbazian, M, D., Grunstein, M., 2007. Functions of Site-Specific Histone Acetylation and Deacetylation. Annual Review of Biochemistry, 76, 75-100. 20. Polo, S. E., Almouzni, G. 2005. Histone metabolic pathways and chromatin assembly factors as proliferation markers. Cancer Lett. 220, 1–9. 21. Redon, C., et al. 2002. Histone H2A variants H2AX and H2AZ. Curr. Opin. Genet. Dev., 12, 162-169. 22. Ropero, S., Esteller, M. 2007. The role of histone deacetylases (HDACs) in human cancer. Molecular Oncology. 1(1), 19-25. 23. Vidanes, G. M., Bonilla, C. Y., Toczyski, D. P. 2005. Complicated tails: histone modifications and the DNA damage response. Cell, 121, 973–976. 24. Vanessa, D., Bret, C., Altucci, L., et al Specific Activity of Class II Histone Deacetylases in Human Breast Cancer Cells. Molecular Cancer Research. Available at http://mcr.aacrjournals.org/content/6/12/1908.abstract. [Accessed on 29th October 2009]. 25. Weichert W, Röske A, Gekeler V, Beckers T, Ebert MP, Pross M, Dietel M, Denkert C, Röcken C. 2008. Association of patterns of class I histone deacetylase expression with patient prognosis in gastric cancer: a retrospective analysis. Lancet Oncol, 9(2), 91-3. 26. Wade, P. A. (2001). Transcriptional control at regulatory checkpoints by histone deacetylases: molecular connections between cancer and chromatin. Human Molecular Genetics. 10(7), 693-98. Part B Antitumor activity of suberoylanilide hydroxamic acid against human oral squamous cell carcinoma cell lines in vitro and in vivo. (Marks, P. A., Richon, V. M., Rafkind, R. A., 2000. Histone Deacetylase Inhibitors: Inducers of Differentiation or Apoptosis of Transformed Cells. JNCI. 92(15), 1210-1216) Histone deacetylases (HDACs) have established relation with pathogenesis of cancer and other diseases. HDACi induce differentiation, apoptosis, prevents cellular growth in transformed cells both in vitro and in vivo. HDACi, Suberoylanilide Hydroxamic acid (SAHA), hydroxamic acid-based hybrid polar compounds (HPCs), is capable of inducing cellular differentiation even at micromolar concentration and is expected to be the most promising antitumor agent displaying negligible toxicity to normal cells. X-ray crystallographic studies of HDACs display their catalytic site with tubular site incorporating zinc ion. Hydroxamic moiety of SAHA binds to zinc ion encouraging accumulation of acetylated histones in tumor and non-tumor cells, a marker for HDACi action. SAHA inhibits HDAC1, 3 and 4 suppress growth in erythroleukemia, prostrate, bladder, ovarian cancer, lymphoma and endometrium. SAHA induces growth arrest, differentiation and / or apoptotic cell death in cultured tumor cells by inhibiting their growth in various animal models in non-toxic doses. Reagents and Antibodies- SAHA was dissolved in DMSO and stored at -20°C. Anti-acetyl histone H3 Antibody, Anti-p53 and antibodies for p16, p21, p27, cyclin D1, cyclin D2, cyclin E and β-actin were all collected from different sources (Tatsuhito et al., 2009). To carry out the study, OSCC cell lines SAS, HSC-2, HSC_4 and CA9-22 were preserved in a single layer (Monolayer) culture using Dulbecco’s MEM incorporated with a protein source of 10% fetal bovine serum and antibiotics along with the incubation performed at 37° in 5% CO2. Methodology Crystal violet mitogenic assay was performed in a 96-well flat bottom plates with 2500 cells/well. SAHA treatment was given after 24 hrs of incubation at varied concentrations keeping the control treated with DMSO. Cells were treated with PBS for washing at frequent intervals and hen stained with 0.1% w/v crystal violet. Detaining of the cell surface was performed using water for 3hrs, followed by air drying. The CV dye was eluted with Sorenson’s solution and absorbance was read at 595nm. OSCC cell lines showed inhibition of growth in vitro culture due to SAHA treatment. The final concentration that was estimated to cause 50% inhibition was at 48hr, 1.3µM for SAS, 0.7 µM for CA9-22, 1.7 µM for HSC-2 and 0.8 µM for HSC-4. Cell-cycle analysis was done using trypsinization method, processing was done using Cycle TEST PLUS DNA Reagent Kit and then flow cytometry was performed. Cell cycle phase distribution was done with the help of software. In vitro OSCC cells, cell treatment was performed with SAHA and cell-cycle was analyzed for the evaluation of DNA content. SAHA arrest the cell-cycle at G1 phase, this shows a remarkable decline in the S-phase cells. Western blot analysis was done to measure the protein concentration using Hybond PVDF membranes, which were then blocked with bovine serum albumin/ PBS and this was reacted with primary antibody for 90 minutes at room temperature. The blots obtained were visualized using Western Lightening Plus. Western blot studies were used to examine the overall acetylation of the cells. When compared to control acetylation of H3 was very less than those of OSCC cell lines. H4 did not show any remarkable difference between the control and experimental cells. Moreover, a constitutive and stable expression of p53 was observed in all cells indicating that SAHA was capable of inducing hyperacetylation of p53. Animal treatment was performed with SAHA with subcutaneous injection with 2X106 SAS cell suspension. After 1 week the tumor grew and animals were then peritoneally injected with SAHA (50mg/kg body weight). Tumor volume was determined by direct measurements with calipers and calculated by the formula: π/6 × (large diameter) × (small diameter) Animal treatment with SAHA-It was also observed that when nude mice were treated with SAHA, growth of xenograft SAS tumors was blocked without any big side effects. Part C How would you study whether the induction of p21 protein was accompanied by elevated p21 mRNA levels? The figure displays that p21 is induced after DNA damage and is essential for the cell survival, DNA repair, cell-cycle arrest, suppression of apoptosis. Antiproliferative effect of SAHA is due to the suppression of HDAC, this caused hyper-acetylated nucleosomal accumulation and thereby activated the transcription of genes responsible for cell proliferation, cell cycle progression and for cell survival. SAHA stimulated the formation of p21 expression in almost all cell lines which is imperative for arresting growth at G1 and G2 phases; on the other hand cyclin D2 was down regulated which is essential in S-phase, up-regulation could be a counter-reaction to the p21 over expression which was out shown in SAS cells. Thus, association of enhanced expression of p21 and downregulation of cyclin D2 can be formulated. Also, expression of p21 is related to greater histone acetylation. HDACIs induce p21 expression mainly by activating the Sp1/Sp3 pathway independent of p53; a direct role for p53 in HDAC-associated p21 expression has also been reported. Acetylated p53 was considerably encouraged after SAHA treatment. The expression of p21 was much higher in SAS than the other two cell lines. Northern blot for mRNA estimation and Western Blot for Protein estimation can be used to establish a correlation between highly expressed p21 protein to the elevated level of p21 mRNA. Read More