Title: Selective Induction of Apoptosis by Histone Deacetylase Inhibitor SAHA in Cutaneous T-Cell Lymphoma Cells: Relevance to Mechanism of Therapeutic Action
Abstract: Suberoylanilide hydroxamic acid (SAHA), an orally administered inhibitor of histone deacetylases, is currently in phase II clinical trials for cutaneous T cell lymphomas (CTCL), but the mechanism of SAHA action is unknown. In this study, we investigated the anti-tumor effects of SAHA in CTCL cell lines and freshly isolated peripheral blood lymphocytes (PBL) from CTCL patients with high percentage of circulating malignant T cells. Three cell lines (MJ, Hut78, and HH) and PBL from 11 patients and three healthy donors were treated with SAHA (1, 2.5, and 5 μM) for 24 and/or 48 h. Apoptosis was determined by flow cytometry analysis of sub-G1 hypodiploid nuclei and/or annexin V binding populations. Acetylated histones and apoptosis-associated proteins were detected by Western blotting. SAHA at 1–5 μM for 24 and 48 h induced apoptosis in a concentration- and time-dependent manner in three cell lines: MJ (0%–7% and 1%–32%), Hut78 (4%–36% and 5%–54%), and HH (4%–67% and 8%–81%). SAHA at 1–5 μM for 48 h also induced more apoptosis of patients' PBL than healthy donors' (15%–32%versus 6%–13%, p<0.05). SAHA treatment caused an accumulation of acetylated histones (H2B, H3, and H4), an increase of p21WAF1 and bax proteins, a decrease of Stat6 and phospho-Stat6 proteins, and activation of caspase-3 in CTCL cells. Our data suggest that selective induction of malignant T cell apoptosis and modulation of acetylated histones, p21WAF1, bax, Stat6, and caspase-3 may underlie the therapeutic action of SAHA in CTCL patients. Suberoylanilide hydroxamic acid (SAHA), an orally administered inhibitor of histone deacetylases, is currently in phase II clinical trials for cutaneous T cell lymphomas (CTCL), but the mechanism of SAHA action is unknown. In this study, we investigated the anti-tumor effects of SAHA in CTCL cell lines and freshly isolated peripheral blood lymphocytes (PBL) from CTCL patients with high percentage of circulating malignant T cells. Three cell lines (MJ, Hut78, and HH) and PBL from 11 patients and three healthy donors were treated with SAHA (1, 2.5, and 5 μM) for 24 and/or 48 h. Apoptosis was determined by flow cytometry analysis of sub-G1 hypodiploid nuclei and/or annexin V binding populations. Acetylated histones and apoptosis-associated proteins were detected by Western blotting. SAHA at 1–5 μM for 24 and 48 h induced apoptosis in a concentration- and time-dependent manner in three cell lines: MJ (0%–7% and 1%–32%), Hut78 (4%–36% and 5%–54%), and HH (4%–67% and 8%–81%). SAHA at 1–5 μM for 48 h also induced more apoptosis of patients' PBL than healthy donors' (15%–32%versus 6%–13%, p<0.05). SAHA treatment caused an accumulation of acetylated histones (H2B, H3, and H4), an increase of p21WAF1 and bax proteins, a decrease of Stat6 and phospho-Stat6 proteins, and activation of caspase-3 in CTCL cells. Our data suggest that selective induction of malignant T cell apoptosis and modulation of acetylated histones, p21WAF1, bax, Stat6, and caspase-3 may underlie the therapeutic action of SAHA in CTCL patients. cyclin-dependent kinase cutaneous T cell lymphomas histone acetyltransferase histone deacetylase mycosis fungoides poly (ADP-Ribose) polymerase peripheral blood lymphocytes propidium iodide suberoylanilide hydroxamic acid Sézary syndrome signal transducers and activators of transcription Cutaneous T cell lymphomas (CTCL) are extranodal non-Hodgkin's lymphomas with pleomorphic skin lesions and distinct T-cell markers. Mycosis fungoides (MF), the most common and indolent form of CTCL, is characterized by malignant CD4+CD45RO+CLA+CCR+ T cells that also lack CD7 and/or CD26. MF may evolve into a leukemic variant, Sézary syndrome (SS), or transform to large cell lymphoma. As MF/SS advances, the clonal dominance of the malignant cells results in the expression of predominantly Th2 cytokines, progressive immune dysregulation in patients, and further tumor cell growth (Kim et al., 2005Kim E.J. Hess S. Richardson S.K. et al.Immunopathogenesis and therapy of cutaneous T cell lymphoma.J Clin Invest. 2005; 115: 798-812Crossref PubMed Scopus (344) Google Scholar). Early MF can often be controlled with skin directed topical steroids, retinoids, or chemotherapy with phototherapy, systemic biological response modifiers, or total skin electron beam irradiation. Although partial and complete remissions can be achieved, subsequent relapses are common. Since patients with refractory or transformed MF and SS have a poor prognosis and therapy is palliative (Duvic and Cather, 2000Duvic M. Cather J.C. Emerging new therapies for cutaneous T-cell lymphoma.Dermatol Clin. 2000; 18: 147-156Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar; Kim et al., 2005Kim E.J. Hess S. Richardson S.K. et al.Immunopathogenesis and therapy of cutaneous T cell lymphoma.J Clin Invest. 2005; 115: 798-812Crossref PubMed Scopus (344) Google Scholar), new therapies are needed. CTCL cells have defective apoptosis; therefore, reversing resistance to apoptosis may provide a new therapeutic approach (Kacinski and Flick, 2001Kacinski B.M. Flick M. Apoptosis and cutaneous T cell lymphoma.Ann N Y Acad Sci. 2001; 941: 194-199Crossref PubMed Scopus (28) Google Scholar; Meech et al., 2001Meech S.J. Edelson R. Walsh P. Norris D.A. Duke R.C. Reversible resistance to apoptosis in cutaneous T cell lymphoma.Ann N Y Acad Sci. 2001; 941: 46-58Crossref PubMed Scopus (36) Google Scholar; Ni et al., 2005Ni X. Zhang C. Talpur R. Duvic M. Resistance to activation-induced cell death and bystander cytotoxicity via the Fas/Fas ligand pathway are implicated in the pathogenesis of cutaneous T cell lymphomas.J Invest Dermatol. 2005; 124: 741-750Crossref PubMed Scopus (43) Google Scholar). Histone deacetylase (HDAC) inhibitors are a novel class of antineoplastic agents. By blocking deacetylation of histones, they promote an open chromatin structure and thereby alter the expression of genes regulating cell survival, proliferation, differentiation, and apoptosis (Rosato and Grant, 2003Rosato R.R. Grant S. Histone deacetylase inhibitors in cancer therapy.Cancer Bio Ther. 2003; 2: 30-37Crossref PubMed Scopus (1) Google Scholar; Mei et al., 2004Mei S. Ho A.D. Mahlknecht U. Role of histone deacetylase inhibitors in the treatment of cancer (Review).Int J Oncol. 2004; 25: 1509-1519PubMed Google Scholar; Drummond et al., 2005Drummond D.C. Noble C.O. Kirpotin D.B. et al.Clinical development of histone deacetylase inhibitors as anticancer agents.Annu Rev Pharmacol Toxicol. 2005; 45: 495-528Crossref PubMed Scopus (518) Google Scholar). Suberoylanilide hydroxamic acid (SAHA), an inhibitor of class I and II HDAC, induces cell growth arrest and apoptosis of a broad spectrum of transformed cells in in vitro and in vivo studies (de Ruijter et al., 2003de Ruijter A.J. van Gennip A.H. Caron H.N. Kemp S. van Kuilenburg A.B. Histone deacetylases (HDACs): Characterization of the classical HDAC family.Biochem J. 2003; 370: 737-749Crossref PubMed Scopus (2263) Google Scholar; Marks et al., 2003Marks P.A. Miller T. Richon V.M. Histone deacetylases.Curr Opin Pharmacol. 2003; 3: 344-351Crossref PubMed Scopus (334) Google Scholar; Mei et al., 2004Mei S. Ho A.D. Mahlknecht U. Role of histone deacetylase inhibitors in the treatment of cancer (Review).Int J Oncol. 2004; 25: 1509-1519PubMed Google Scholar; Drummond et al., 2005Drummond D.C. Noble C.O. Kirpotin D.B. et al.Clinical development of histone deacetylase inhibitors as anticancer agents.Annu Rev Pharmacol Toxicol. 2005; 45: 495-528Crossref PubMed Scopus (518) Google Scholar). SAHA is currently in phase I and II clinical trials for the treatment of various cancers and has shown promising anticancer activity at doses that are well tolerated by patients (Kelly et al., 2003Kelly W.K. Richon V.M. O'Connor O. et al.Phase I clinical trial of histone deacetylase inhibitor: Suberoylanilide hydroxamic acid administered intravenously.Clin Cancer Res. 2003; 9: 3578-3588PubMed Google Scholar; Piekarz and Bates, 2004Piekarz R. Bates S. A review of depsipeptide and other histone deacetylase inhibitors in clinical trials.Curr Pharm Des. 2004; 10: 2289-2298Crossref PubMed Scopus (170) Google Scholar). Clinical evaluation of SAHA in patients with CTCL has revealed that oral SAHA produces clinically meaningful partial responses in ten of 37 advanced, heavily pretreated patients associated with decreased pruritus. 1Duvic M, Talpur R, Zhang CL, Goy A, Richon, V, Frankel SR: Phase II trial of oral suberoylanilide hydroxamic acid (SAHA) for cutaneous T-cell lymphoma (CTCL) unresponsive to conventional therapy. J Clin Oncol:23(16S) 6571, 2005 (abstr).1Duvic M, Talpur R, Zhang CL, Goy A, Richon, V, Frankel SR: Phase II trial of oral suberoylanilide hydroxamic acid (SAHA) for cutaneous T-cell lymphoma (CTCL) unresponsive to conventional therapy. J Clin Oncol:23(16S) 6571, 2005 (abstr). SAHA's mechanism of action in CTCL has not yet been demonstrated. In this study, we performed in vitro translational studies to further define the anti-tumor effects of SAHA in well-established CTCL cell lines and freshly isolated peripheral blood lymphocytes (PBL) from SS/MF patients with high percentage of circulating malignant T-cells (Bernengo et al., 2001Bernengo M.G. Novelli M. Quaglino P. et al.The relevance of the CD4+CD26- subset in the identification of circulating Sezary cells.Br J Dermatol. 2001; 144: 125-135Crossref PubMed Scopus (169) Google Scholar). To determine whether SAHA inhibits CTCL cell growth, MJ, Hut78, and HH cell lines were treated with or without SAHA (1, 2.5, and 5 μM) for 24 and 48 h, and their viability was evaluated by CellTiter 96 AQ One Solution Cell Proliferation Assay. As the dose of SAHA increased from 1 to 5 μM over 24 and 48 h, growth inhibition of HH cells increased from 2% to 34% and from 21% to 70% in a concentration- and a time-dependent manner compared to vehicle control (n=3, p<0.05). Similar results were also seen in the MJ and Hut78 cell lines; however, MJ cells were less sensitive to SAHA (Figure 1a). These results demonstrated that SAHA is an inhibitor of CTCL cell line growth in vitro.FigureEffects of suberoylanilide hydroxamic acid (SAHA) on cell growth, cell cycle, and apoptosis in cutaneous T cell lymphomas (CTCL) cell lines. MJ, Hut78, and HH cell lines were treated with or without SAHA (1, 2.5, and 5 μM) for 24 and 48 h. (1). Inhibition of cell growth. (A) Cells were aliquoted into a 96-well culture plate and cell viability was determined by comparing untreated control to treated groups with MTS assay performed in triplicate. Each point represented mean±SD of triplicate determinations. (2). Increase of sub-G1 arrest. Cell-cycle distributions were determined by DNA content analysis with propidium iodide (PI) staining and flow cytometry. (B) Induction of sub-G1 population of HH cell line in a concentration- and time-dependent manner. (C) Each point represented the percent of cell-cycle distributions with different DNA content. Results were representative of one of three independent experiments. (3). Induction of annexin V binding. Annexin V binding was carried out with the Annexin V-FITC Detection Kit. (D) Induction of annexin V binding of HH cell line in a concentration- and time-dependent manner. (E) Each point represented the percentage of both annexin V+PI− and annexin V+PI+ cells (mean±SD of triplicate determinations). *Significantly different from vehicle control values (n=3; p<0.05).View Large Image Figure ViewerDownload (PPT) To determine whether growth inhibition of SAHA is due to cell-cycle arrest and/or to apoptosis in CTCL cell lines, MJ, Hut78, and HH cells were treated with or without SAHA (1, 2.5, and 5 μM) for 24 and 48 h. As the dose of SAHA increased from 1 to 5 μM over 48 h, the percent of HH cells with sub-G1 population increased from 11% to 88% in a concentration-dependent manner compared to vehicle control. As the incubation time of SAHA at 2.5 μM increased from 24 to 48 h, the percent of HH cells with sub-G1 population increased from 37% to 80% in a time-dependent manner compared to vehicle control (Figure 1b). Similar results were also seen in the MJ and Hut78 cell lines (Figure 1c). An increased percentage of cells in the sub-G1 population was accompanied by a loss of cells in the G1, S, and G2/M phases, suggesting cells are undergoing apoptosis (Zhang et al., 2002Zhang C.L. Hazarika P. Ni X. Weidner D.A. Duvic M. Induction of apoptosis by bexarotene in cutaneous T-cell lymphoma cells: Relevance to mechanism of therapeutic action.Clin Cancer Res. 2002; 8: 1234-1240PubMed Google Scholar). In parallel to the cell cycle analysis, we also investigated the effect of SAHA on annexin V binding in three CTCL cell lines. As the SAHA concentration increased from 1 to 5 μM for 24 h, the number of HH cells stained for annexin V increased from 3% to 63% in a concentration-dependent manner compared to vehicle control. As the incubation time of SAHA at 2.5 μM increased from 24 to 48 h, the number of HH cells stained for annexin V increased from 43% to 63% in a time-dependent manner compared to vehicle control (Figure 1d). Similar results were also seen in the MJ and Hut78 cell lines but again MJ cells were less sensitive to SAHA (Figure 1e). These results were consistent with analysis of cell-cycle distributions from PI staining, further confirming that CTCL cell lines undergo apoptosis following SAHA treatment. To confirm these findings, we also tested the pro-apoptotic effect of SAHA on freshly isolated PBL from 11 SS/MF patients with high percentage of circulating malignant T-cells and three healthy donors. As the SAHA concentration increased from 1 to 5 μM for 48 h, the number of patients' PBL stained for annexin V increased from 15% to 32% compared to vehicle control. As the SAHA concentration increased from 1 to 5 μM for 48 h, the number of healthy donors' PBL stained for annexin V increased from 6% to 13% compared to vehicle control. Thus, patients' PBL were more sensitive to SAHA treatment than healthy donors' PBL (p<0.05) (Table I).Table ISAHA selectively induced apoptosis of SS/MF patients' PBLPatient no.Age/genderDiagnosisCD4+CD26- (%)Apoptosis (%) (48 h)aApoptosis was determined by flow cytometry analysis of annexin V/PI staining. The values represented the percentage of annexin V+PI- and annexin V+PI+ binding from PBL treated with different concentrations of SAHA over vehicle control. SAHA, suberoylanilide hydroxamic acid; SS, Sézary syndrome; MF, mycosis fungoides; PBL, peripheral blood lymphocytes; M, male; F, female. Concentrations (μM)12.55175/FSS95193445269/FSS82355859361/FSS8381818444/FMF40335576/FSS91112424675/MSS84244553758/MSS67355556881/FMF37122320967/MSS61421291059/FSS79919241154/MSS9761618Statistic analysis Total (mean±SD) Patients (n=11)15±1129±1732±18 Healthy donors (n=3)6±211±313±5 p valuePatients versus healthy donorsp=0.03p=0.006p=0.009a Apoptosis was determined by flow cytometry analysis of annexin V/PI staining. The values represented the percentage of annexin V+PI- and annexin V+PI+ binding from PBL treated with different concentrations of SAHA over vehicle control.SAHA, suberoylanilide hydroxamic acid; SS, Sézary syndrome; MF, mycosis fungoides; PBL, peripheral blood lymphocytes; M, male; F, female. Open table in a new tab To understand the mechanism of SAHA-induced apoptosis, western blotting was used to examine the effects of SAHA on the accumulation of acetylated histones and expression of apoptosis-associated proteins in three CTCL cell lines and patients' PBL undergoing apoptosis. The acetylated histones (H2B, H3, and H4) were absent or weakly expressed in three CTCL cell lines and patients' PBL, and increased following SAHA treatment (Figure 2a). The cyclin-dependent kinase (CDK) inhibitor p21WAF1 was absent in Hut78, HH, and two patients' PBL but expressed in MJ, and induced following SAHA treatment. The tumor suppressor p53 protein was expressed in MJ, HH, and patients' PBL but not in Hut78 cells, and did not change following SAHA treatment. Stat3 and Stat6 proteins were expressed and Stat6 but not Stat3 decreased following SAHA treatment. Stat3 and Stat6 proteins were phosphorylated and phospho-Stat6 but not Stat3 decreased following SAHA treatment. SAHA did not change the level of anti-apoptotic protein bcl-2 but increased pro-apoptotic protein bax. Caspase-3 activation and poly (ADP-Ribose) polymerase (PARP) cleavage following SAHA treatment were evident as shown by the appearance of cleaved 20- and 17-, and 85-kDa fragments, respectively (Figure 2b). In CTCL, most effective treatments including phototherapy (Baron and Stevens, 2003Baron E.D. Stevens S.R. Phototherapy for cutaneous T-cell lymphoma.Dermatol Ther. 2003; 16: 303-310Crossref PubMed Scopus (50) Google Scholar), photopheresis (Zic, 2003Zic J.A. The treatment of cutaneous T-cell lymphoma with photopheresis.Dermatol Ther. 2003; 16: 337-346Crossref PubMed Scopus (109) Google Scholar) and rexinoids (Zhang et al., 2002Zhang C.L. Hazarika P. Ni X. Weidner D.A. Duvic M. Induction of apoptosis by bexarotene in cutaneous T-cell lymphoma cells: Relevance to mechanism of therapeutic action.Clin Cancer Res. 2002; 8: 1234-1240PubMed Google Scholar) induce apoptosis of T cells. In this study, we show that SAHA at 1–5 μM selectively causes apoptosis of CTCL cell lines and SS/MF patients' PBL compared to healthy donors' PBL. SAHA also induced an accumulation of acetylated histones, an increase of p21WAF1and bax, a decrease of Stat6 and phospho-Stat6, and activation of caspase3. Our study agrees with the finding that depsipeptide (FK228), another structural class of HDAC inhibitors, also induces apoptosis of Hut78 CTCL cells in vitro (Piekarz et al., 2004Piekarz R.L. Robey R.W. Zhan Z.R. et al.T-cell lymphoma as a model for the use of histone deacetylase inhibitors in cancer therapy: Impact of depsipeptide on molecular markers, therapeutic targets, and mechanisms of resistance.Blood. 2004; 103: 4636-4643Crossref PubMed Scopus (167) Google Scholar). An important attribute of HDAC inhibitors including SAHA is that they induce tumor cell apoptosis at concentrations to which normal cells are relatively resistant, making them well suited for cancer therapy (Marks and Jiang, 2005Marks P.A. Jiang X. Histone deacetylase inhibitors in programmed cell death and cancer therapy.Cell Cycle. 2005; 4: 549-551Crossref PubMed Scopus (134) Google Scholar). This difference in sensitivity to SAHA-induced apoptosis appears not to be caused by a difference in the ability to inhibit HDAC activity because accumulation of acetylated histones occurs in both tumor and normal cells (Ungerstedt et al., 2005Ungerstedt J.S. Sowa Y. Xu W.S. et al.Role of thioredoxin in the response of normal and transformed cells to histone deacetylase inhibitors.Proc Natl Acad Sci USA. 2005; 102: 673-678Crossref PubMed Scopus (430) Google Scholar). In this study, we also showed that SAHA at 1–5 μM selectively causes apoptosis of CTCL cell lines and patients' PBL compared to healthy donors' PBL. More recent studies showed that thioredoxin, a hydrogen donor for many protein targets and a scavenger of ROS, is an important determinant of resistance of cells to SAHA-induced apoptosis (Ungerstedt et al., 2005Ungerstedt J.S. Sowa Y. Xu W.S. et al.Role of thioredoxin in the response of normal and transformed cells to histone deacetylase inhibitors.Proc Natl Acad Sci USA. 2005; 102: 673-678Crossref PubMed Scopus (430) Google Scholar). In our study, there is also different sensitivity to SAHA in three cell lines and patients' PBL. Further exploration may be needed to understand the mechanism of sensitivity difference to SAHA in inducing apoptosis of different CTCL cell lines and patients' PBL. Chromatin, the basic structural unit of DNA, is composed of nucleosomes with approximately 146 bp of DNA wrapped around an octamer of core histones (two molecules of H2A, H2B, H3, and H4) (Mei et al., 2004Mei S. Ho A.D. Mahlknecht U. Role of histone deacetylase inhibitors in the treatment of cancer (Review).Int J Oncol. 2004; 25: 1509-1519PubMed Google Scholar; Drummond et al., 2005Drummond D.C. Noble C.O. Kirpotin D.B. et al.Clinical development of histone deacetylase inhibitors as anticancer agents.Annu Rev Pharmacol Toxicol. 2005; 45: 495-528Crossref PubMed Scopus (518) Google Scholar). Histone acetylation plays an important role in transcriptional regulation. The status of histone acetylation is a dynamic process depending on a balance between the activities of HDAC and histone acetyltransferases (HAT) (Mei et al., 2004Mei S. Ho A.D. Mahlknecht U. Role of histone deacetylase inhibitors in the treatment of cancer (Review).Int J Oncol. 2004; 25: 1509-1519PubMed Google Scholar; Drummond et al., 2005Drummond D.C. Noble C.O. Kirpotin D.B. et al.Clinical development of histone deacetylase inhibitors as anticancer agents.Annu Rev Pharmacol Toxicol. 2005; 45: 495-528Crossref PubMed Scopus (518) Google Scholar). HAT acetylate histone lysine substrates, opening up compacted chromatin and promoting binding of transcription factors and gene transcription. In contrast, HDAC decrease acetylation of histone lysine tails, thereby condensing chromatin structure and repressing gene transcription (Mei et al., 2004Mei S. Ho A.D. Mahlknecht U. Role of histone deacetylase inhibitors in the treatment of cancer (Review).Int J Oncol. 2004; 25: 1509-1519PubMed Google Scholar; Drummond et al., 2005Drummond D.C. Noble C.O. Kirpotin D.B. et al.Clinical development of histone deacetylase inhibitors as anticancer agents.Annu Rev Pharmacol Toxicol. 2005; 45: 495-528Crossref PubMed Scopus (518) Google Scholar). Abnormal expression, function or recruitment of HDAC and/or HAT is associated with neoplasia, including lymphoid and myeloid leukemia (Vigushin and Coombes, 2004Vigushin D.M. Coombes R.C. Targeted histone deacetylase inhibition for cancer therapy.Curr Cancer Drug Targets. 2004; 4: 205-218Crossref PubMed Scopus (87) Google Scholar). In this study we show that SAHA treatment at concentrations causing apoptosis also induces an accumulation of acetylated histones, which may represent a therapeutic marker of drug activity in CTCL patients. Despite wide distribution of HDAC in chromatin, HDAC inhibitors including SAHA alter the expression of only a small number of genes (2%–17%) by differential display and DNA microarray analysis of gene profiling (Lindemann et al., 2004Lindemann R.K. Gabrielli B. Johnstone R.W. Histone-deacetylase inhibitors for the treatment of cancer.Cell Cycle. 2004; 3: 779-788Crossref PubMed Scopus (126) Google Scholar). Among these genes, the cdk inhibitor p21WAF1 is one of the most commonly induced in several types of transformed cells by SAHA treatment (Richon et al., 2000Richon V.M. Sandhoff T.W. Rifkind R.A. Marks P.A. Histone deacetylase inhibitor selectively induces p21WAF1 expression and gene-associated histone acetylation.Proc Natl Acad Sci USA. 2000; 97: 10014-10019Crossref PubMed Scopus (977) Google Scholar; Gui et al., 2004Gui C.Y. Ngo L. Xu W.S. Richon V.M. Marks P.A. Histone deacetylase (HDAC) inhibitor activation of p21WAF1 involves changes in promoter-associated proteins, including HDAC1.Proc Natl Acad Sci USA. 2004; 101: 1241-1246Crossref PubMed Scopus (498) Google Scholar). The upregulation of p21WAF1 is responsible for not only cell cycle arrest but also apoptosis induced by HDAC inhibitors (Mei et al., 2004Mei S. Ho A.D. Mahlknecht U. Role of histone deacetylase inhibitors in the treatment of cancer (Review).Int J Oncol. 2004; 25: 1509-1519PubMed Google Scholar; Somech et al., 2004Somech R. Izraeli S. Simon A.J. Histone deacetylase inhibitors—a new tool to treat cancer.Cancer Treat Rev. 2004; 30: 461-472Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). In this study, we show that SAHA treatment at concentrations causing apoptosis induced the expression of p21WAF1 protein without cell cycle arrest in all three CTCL cell lines, suggesting that p21WAF1 is involved in SAHA-induced apoptosis of CTCL cells. Furthermore, p21WAF1 is a downstream target of the tumor suppressor p53 and can be regulated in a p53-dependent or -independent manner (Gartel and Tyner, 1999Gartel A.L. Tyner A.L. Transcriptional regulation of the p21 (WAF1/CIP1) gene.Exp Cell Res. 1999; 246: 280-289Crossref PubMed Scopus (566) Google Scholar). To address whether up-regulation of p21WAF1 protein is related to p53, we analyzed CTCL cells for protein expression of p53 following SAHA treatment. Our immunoblot results showed that there was no change of p53. Thus, up-regulation of p21WAF1 by SAHA is likely to be independent of a wild-type p53 status (Vrana et al., 1999Vrana J.A. Decker R.H. Johnson C.R. et al.Induction of apoptosis in U937 human leukemia cells by suberoylanilide hydroxamic acid (SAHA) proceeds through pathways that are regulated by Bcl-2/Bcl-XL, c-Jun, and p21CIP1, but independent of p53.Oncogene. 1999; 18: 7016-7025Crossref PubMed Scopus (257) Google Scholar; Huang et al., 2000Huang L. Sowa Y. Sakai T. 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After activated by phosphorylation in response to a series of cytokines, growth factors, and hormones, Stat dimerize and translocate to the nucleus where Stat bind to specific DNA promoter sequences and thereby regulate gene expression (Calo et al., 2003Calo V. Migliavacca M. Bazan V. Macaluso M. Buscemi M. Gebbia N. Russo A. STAT proteins: From normal control of cellular events to tumorigenesis.J Cell Physiol. 2003; 197: 157-168Crossref PubMed Scopus (498) Google Scholar). Stat3 is activated by a wide variety of extracellular and intracellular stimuli such as IL-2, IL-6, IL-7, IL-9, IL-10, and IL-15, and by intracellular tyrosine kinases such as src and abl (Calo et al., 2003Calo V. Migliavacca M. Bazan V. Macaluso M. Buscemi M. Gebbia N. Russo A. STAT proteins: From normal control of cellular events to tumorigenesis.J Cell Physiol. 2003; 197: 157-168Crossref PubMed Scopus (498) Google Scholar). Constitutive activation of Stat3 has been demonstrated to contribute to oncogenesis by stimulating cell proliferation and preventing apoptosis in a large number of solid tumors and hematological malignancies including CTCL (Sommer et al., 2004Sommer V.H. Clemmensen O.J. Nielsen O. et al.In vivo activation of STAT3 in cutaneous T-cell lymphoma. Evidence for an antiapoptotic function of STAT3.Leukemia. 2004; 18: 1288-1295Crossref PubMed Scopus (137) Google Scholar; Turkson, 2004Turkson J. STAT proteins as novel targets for cancer drug discovery.Expert Opin Ther Targets. 2004; 8: 409-422Crossref PubMed Scopus (252) Google Scholar; Mitchell and John, 2005Mitchell T.J. John S. Signal transducer and activator of transcription (STAT) signalling and T-cell lymphomas.Immunology. 2005; 114: 301-312Crossref PubMed Scopus (106) Google Scholar). In contrast, Stat6 is only primarily activated by IL-4 and IL-13 for the development of Th2 cells (Calo et al., 2003Calo V. Migliavacca M. Bazan V. Macaluso M. Buscemi M. Gebbia N. Russo A. STAT proteins: From normal control of cellular events to tumorigenesis.J Cell Physiol. 2003; 197: 157-168Crossref PubMed Scopus (498) Google Scholar). The activation of Stat6 in malignancies is little known. Constitutive activation of Stat6 has been demonstrated in adult T-cell leukemia/lymphoma (Takemoto et al., 1997Takemoto S. Mulloy J.C. Cereseto A. et al.Proliferation of adult T cell leukemia/lymphoma cells is associated with the constitutive activation of JAK/STAT proteins.Proc Natl Acad Sci USA. 1997; 94: 13897-13902Crossref PubMed Scopus (225) Google Scholar), leukemia associated with the p190bcr-abl (Ilaria and van Etten, 1996Ilaria R.L. van Etten R.A. P210 and P190BCR/ABL induce the tyrosine phosphorylation and DNA binding activity of multiple specific STAT family members.J Biol Chem. 1996; 271: 31704-31710Crossref PubMed Scopus (415) Google Scholar), and Hodgkin's lymphoma (Skinnider et al., 2002Skinnider B.F. Elia A.J. Gascoyne R.D. Patterson B. Trumper L. Kapp U. Mak T.W. Signal transducer and activator of transcription 6 is frequently activated in Hodgkin and Reed-Sternberg cells of Hodgkin lymphoma.Blood. 2002; 99: 618-626Crossref PubMed Scopus (219) Google Scholar). In addition, human lymphocytes with Stat6 null phenotype exhibit increased apoptosis (Galka et al., 2004Galka E. Thompson J.L. Zhang W.J. Poritz L.S. Koltun W.A. Stat6 (null phenotype) human lymphocytes exhibit increased apoptosis.J Surg Res. 2004; 122: 14-20Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). In this study, we found for the first time that SAHA at concentrations causing apoptosis decreases the expression of Stat6 and phospho-Stat6 but not Stat3 and phospho-Stat3 in three cell lines and patients' PBL. Thus, down-regulation of Stat6 and phospho-Stat6 may be involved in SAHA-induced apoptosis of CTCL cells. The balance between expression of anti-apoptotic protein bcl-2 and pro-apoptotic protein bax is critical in controlling the activation of caspases by regulating release of cytochrome c from mitochondria (Manion and Hockenb