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Expression of HAT1 and HDAC1, 2, 3 in Diffuse Large B-Cell Lymphomas, Peripheral T-Cell Lymphomas, and NK/T-Cell Lymphomas

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Expression of HAT1 and HDAC1, 2, 3 in Diffuse Large B-Cell Lymphomas, Peripheral T-Cell Lymphomas, and NK/T-Cell Lymphomas The Korean Journal of Pathology 2012; 46: 142-150 ▒ ORIGINAL ARTICLE ▒ http://dx.doi.org/10.4132/KoreanJPathol.2012.46.2.142 Expression of HAT1 and HDAC1, 2, 3 in Diffuse Large B-Cell Lymphomas, Peripheral T-Cell Lymphomas, and NK/T-Cell Lymphomas Soo Kee Min · Young Ho Koh1 Background: It has generally been proven that histone acetylation and deacetylation are involved Yunwoong Park1 · Hyo Jung Kim2 in the malignant transformation. To date, however, this has rarely been studied in cases of malig- Jinwon Seo · Hye-Rim Park nant lymphoma. Methods: We studied nine cases of reactive lymphoid hyperplasia, 78 cases of diffuse large B-cell lymphoma (DLBCL), 13 cases of peripheral T-cell lymphoma, not otherwise 3 4 Seong Jin Cho · In Sun Kim specified (PTCL-NOS), and 13 cases of extranodal NK/T-cell lymphoma, nasal type (NKTCL). Thus, we attempted to elucidate the associations of the degree of the expression of histone acetyltrans- Department of Pathology, Hallym University ferase 1 (HAT1), histone deacetylase (HDAC) 1, HDAC2, and HDAC3 with the clinical behaviors of Sacred Heart Hospital, Hallym University College above malignant lymphomas using the immunohistochemistry and a western blot analysis. of Medicine; 1Ilsong Institute of Life Science, Re- Hallym University; 2Department of Hemato- sults: The degree of the expression of HAT1 was higher in cases of DLBCL, PTCL-NOS or NKT- Oncology, Hallym University Sacred Heart CL as compared with reactive lymphoid hyperplasia (p< 0.05). The degree of the expression of Hospital, Hallym University College of Medicine, HAT1 was correlated with that of HDAC1 in cases of DLBCL or NKTCL (p< 0.05). The degree of Anyang; 3Department of Pathology, Hallym the expression of HAT1 and HDAC1 was correlated with a poor survival in cases of DLBCL or University Kangdong Sacred Heart Hospital, PTCL-NOS (p> 0.05). Conclusions: HAT1, HDAC1, and HDAC2 play a critical role in the develop- Hallym University College of Medicine, Seoul; ment of malignant lymphomas. Both HAT1 and HDAC1 might be indicators for a poor prognosis 4Department of Pathology, Korea University College of Medicine, Seoul, Korea in cases of DLBCL as cooperating factors. Received: December 27, 2011 Revised: March 10, 2012 Accepted: March 14, 2012 Corresponding Author Seong Jin Cho, M.D. Department of Pathology, Hallym University Kangdong Sacred Heart Hospital, 150 Seongan-ro, Gangdong-gu, Seoul 134-701, Korea Tel: +82-2-2224-2557 Fax: +82-2-2224-2214 E-mail: [email protected] Key Words: Acetylation; Deacetylation; Histone deacetylase inhibitors; Lymphoma Histone proteins bind to the DNA backbone to package the To date, it has been known that mutations, overexpression DNA into chromatin. Normal histone tails are positively charged and improper recruitment of HATs and HDACs develop ma- because of amine groups that are present on their lysine and ar- lignant tumors. Mutations in HATs could lead to increase of ginine residues, and bind to the DNA backbone with phos- histone acetylation.1 Histone acetylation may play an important phate groups that were negatively charged. Histone-related role in the pathogenesis of lymphoma with the up-regulation proteins (histone acetyltransferases [HATs] and histone deacety- of the recombination of the T-cell receptor gene segments.2 But lases [HDACs]) can influence the DNA transcription through histone hypoacetylation is also involved in the development of the balance between the histone acetylation and deacetylation. tumors through mutations, chromosomal translocations, or the Histone acetylation induces loose chromatin by HATs that cause increased activity of HDACs.3 Furthermore, the decrease in his- the lysine residue to lose the positive charge. This process is re- tone acetylation is also involved in tumor invasion and metasta- lated to the promotion of the DNA transcription. By contrast, sis.4 histone deacetylation induces condensed chromatin by HDACs HDACs normally function together with cofactors that re- that play a role in recovering the positive charge, which is asso- cruit HDACs to target genes.5 Their activity is associated with ciated with the gene repression. the development of several cancers in human, where more than pISSN 1738-1843 © 2012 The Korean Society of Pathologists/The Korean Society for Cytopathology 142 eISSN 2092-8920 HAT1 and HDAC1, 2, 3 in Malignant Lymphomas • 143 one mechanism is involved. The transcriptional repression of Briefly, IHC staining was performed as follows: 4-µm-tissue tumor suppressor-genes by the overexpression and improper re- sections were deparaffinized using EZ Prep solution. CC1 stan- cruitment of HDACs to their promoter region could be a com- dard (pH 8.4 buffer containing Tris/Borate/ethylenediaminetet- mon phenomenon in the development and progression of tu- raacetic acid) was used for antigen retrieval at 99˚C for 60 min- 3 mors. For example, chromosomal translocation is associated utes. i VIEW inhibitor (3% H2O2, endogenous peroxidase) was with the production of fusion proteins that recruit the HDAC blocked at 37˚C for four minutes. Slides were incubated with repressor complex with a high affinity to a specific promoter. primary antibodies (Table 1) at 42˚C for 32 minutes and sec- After that, these multi-protein complexes are involved in the ondary antibody to i VIEW biotinylated Ig at 37˚C for 8 min- development of the hematological malignancy by the repression utes. Slides were incubated in i VIEW streptavidin HRP at of genes that regulate normal differentiation and proliferation 37˚C for 8 minutes and then DAB+H2O2 substrate for 8 min- of hematopoietic cells.6-8 The aberrant recruitment of HDACs utes, which was followed by counterstaining with hematoxylin to the E-cadherin promoter may also have an important role in and bluing reagent at 37˚C. Reaction buffer (pH 7.6 Tris buf- the invasion and metastasis of tumor.9,10 HDAC inhibitors are fer) was used as a washing solution. currently intriguing many researchers who are trying to discov- er better anticancer agents. Suberoylanilide hydroxamic acid Interpretation of IHCs (vorinostat) has been approved by the US Food and Drug Ad- Antibodies to HAT1, HDAC1, HDAC2, HDAC3, and Ki- ministration (US FDA) for its indication in treating cutaneous 67 stained the nuclei. The IHCs associated with histone-related T-cell lymphoma (CTCL). proteins (HAT1, HDAC1, HDAC2, and HDAC3) were inter- To date, however, few studies have examined the expressions preted based on the intensity: 0, 1+, 2+, and 3+, where 0 is of HATs and HDACs in association with malignant lymphoma. negative, but Ki-67 based on the proportion (< 26%, 26-50%, Given the above background, we studied the expression of HAT1 51-75%, and>75%). In HAT1 and HDAC1 with an intensity and class 1 HDACs including HDAC1, HDAC2, and HDAC3 of 1+, the intensity of nearby endothelial cells was served as the in reactive lymphoid hyperplasia (RLH), diffuse large B-cell control one. In HAT1 and HDAC1 with an intensity of 3+, lymphomas (DLBCL), peripheral T-cell lymphomas, not other- however, the intensity of germinal center cells was served as the wise specified (PTCL-NOS) and extranodal NK/T-cell lympho- control one. Besides, in HDAC2 and HDAC3 with an intensity mas, nasal type (NKTCL) to identify the correlation between of 1+, the intensity of endothelial cells was served as the control the histone acetylation/deacetylation and clinical behavior of one. In HDAC2 and HDAC3 with an intensity of 3+, however, the tumor. the intensity of nearby histiocytes was served as the control one. Furthermore, 2+ was considered the moderate intensity between MATERIALS AND METHODS 1+ and 3+ (Figs. 1, 2). Finally, the intensity and proportion of histone-related proteins were compared using a Kaplan-Meier Materials survival and a Spearman correlation coefficient. This study was approved by the Institutional Ethics Commit- tee of our medical institution. We selected nine cases of RLH, 78 cases of DLBCL, 13 cases of PTCL-NOS, and 13 cases of Table 1. Antibodies used for this study NKTCL based on the criteria that the paraffin blocks were well Antibodies Dilution ratio Companies City, State, Nation preserved with a sufficient amount of tissue for evaluation. The IHCs clinical data, pathology reports and pathology slides were re- HAT1 polyclonal 1 : 200 Proteintech Chicago, IL, USA viewed. In addition, 3-mm tissue microarrays were made. HDAC1 monoclonal 1 : 4,000 Abnova Taipei, Taiwan HDAC2 polyclonal 1 : 2,000 Abnova Taipei, Taiwan HDAC3 polyclonal 1 : 200 Abnova Taipei, Taiwan Immunohistochemical (IHC) stains Ki-67 monoclonal 1 : 500 Lab vision Fremont, CA, USA IHC reactions were performed on paraffin tissue sections us- Western blot ing an automated IHC stainer (Ventana BenchMark XT, Ven- HAT1 polyclonal 1 : 5,000 Proteintech Chicago, IL, USA HDAC1 monoclonal 1 : 5,000 Abnova Taipei, Taiwan tana Medical Systems Inc., Tucson, AZ, USA) according to the β-actin monoclonal 1 : 10,000 Sigma-Aldrich St. Louis, MO, USA manufacturer’s protocol. Detection was done using the Ventana IHCs, immunohistochemical staining; HAT1, histone acetyltransferase 1; i VEIW DAB detection kit (Ventana Medical Systems Inc.). HDAC, histone deacetylase. http://dx.doi.org/10.4132/KoreanJPathol.2012.46.2.142 http://www.koreanjpathol.org 144 • Min SK, et al. A B C D Fig. 1. Immunohistochemical (IHC) stains for histone acetyltransferase 1 (A) and histone deacetylase (HDAC) 1 (B) show strong positive cells in the reactive germinal centers and are weak positive on the endothelial cells. IHC stains for HDAC2 (C) and HDAC3 (D) show more strong positive reaction for the histiocytes than for the endothelial cells. Western blot 24˚C for 30 seconds in Brasonic 8510R-DTH sonicator (Brason, Western blot analysis was available only for three cases of Danbury, CT, USA). This was followed by a 10-minute boiling RLH, four cases of DLBCL and three cases of PTCL-NOS. Pro- at 100˚C and a centrifugation at 15,000 ×g at 4˚C for 10 min- tein extraction was performed with the modified methods of utes.
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