CC Chemokine Ligand 25 Enhances Resistance to Apoptosis in CD4 T

[CANCER RESEARCH 64, 7579–7587, October 15, 2004] CC Chemokine Ligand 25 Enhances Resistance to Apoptosis in CD4؉ T Cells from Patients with T-Cell Lineage Acute and Chronic Lymphocytic Leukemia by Means of Livin Activation

Zhang Qiuping,1 Xiong Jei,1,2 Jin Youxin,2 Ju Wei,1 Liu Chun,1 Wang Jin,1 Wu Qun,1 Liu Yan,1 Hu Chunsong,3 Yang Mingzhen,4 Gao Qingping,5 Zhang Kejian,5 Sun Zhimin,6 Li Qun,3 Liu Junyan,1 and Tan Jinquan1,3 1Department of Immunology, and Laboratory of Allergy and Clinical Immunology, Institute of Allergy and Immune-related Diseases and Center for Medical Research, Wuhan University School of Medicine, Wuhan; 2The State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Science, Shanghai; 3Department of Immunology, College of Basic Medical Sciences, Anhui Medical University, Hefei; 4Department of Hematology, The Affiliated University Hospital, Anhui Medical University, Hefei; 5Department of Hematology, The First and Second Affiliated University Hospital, Wuhan University, Wuhan; and 6Department of Hematology, The Provincial Hospital of Anhui, Hefei, Peoples Republic of China

ABSTRACT intestine (8), providing the evidence for distinctive mechanisms of

-؉ lymphocyte recruitment. The importance of CCL25/TECK is to li We investigated CD4 and CD8 double-positive thymocytes, CD4 T cense effector/memory cells to access anatomic sites (9, 10). Thus, cells from typical patients with T-cell lineage acute lymphocytic leukemia CCL25/TECK is important for the homing, development, and home- (T-ALL) and T cell lineage chronic lymphocytic leukemia (T-CLL), and MOLT4 T cells in terms of CC chemokine ligand 25 (CCL25) functions of ostasis of T cells, particularly, mucosal T cells. The functional im- induction of resistance to tumor necrosis factor ␣ (TNF-␣)–mediated portance of CCR9/CCL25 in the physiologic and pathophysiologic apoptosis. We found that CCL25 selectively enhanced resistance to TNF- events of T-cell trafficking and selection is not fully understood .mediated apoptosis in T-ALL and T-CLL CD4؉ T cells as well as in despite a considerable body of investigations–␣ MOLT4 T cells, but CD4 and CD8 double-positive thymocytes did not. The inhibitor of apoptosis protein (IAP) family plays a key role in One member protein of the inhibitor of apoptosis protein (IAP) family, apoptosis regulation and has become increasingly prominent in the Livin, was selectively expressed in the malignant cells at higher levels, field of cancer (11). Thus far, eight human IAPs have been identified: ؉ particularly in T-ALL CD4 T cells, in comparison with the expression in c-IAP1, c-IAP2, NAIP, survivin, XIAP, Bruce, ILP-2, and Livin (11). CD4 and CD8 double-positive thymocytes. After stimulation with CCL25 IAPs can block apoptosis mainly through their ability to bind and and apoptotic induction with TNF-␣, the expression levels of Livin in these inhibit specific caspases such as caspase-3 and -7 (12, 13). A novel malignant cells were significantly increased. CCL25/thymus-expressed chemokine (TECK), by means of CC chemokine receptor 9 (CCR9) IAP member, designated Livin/ML-IAP/KIAP (14, 15), is suggested ligation, selectively activated Livin to enhance resistance to TNF-␣– to mediate suppression of cell death (14–16). A key function for Livin in the transformed phenotype suggests that this protein may be an mediated apoptosis in c-jun-NH2-kinase 1 (JNK1) kinase-dependent manner. These findings suggested differential functions of CCR9/CCL25 in important target for immune-mediated tumor destruction (17). distinct types of cells. CD4 and CD8 double-positive thymocytes used A common manifestation of T-cell lineage acute lymphocytic leu- CCR9/CCL25 for migration, homing, development, maturation, selection, kemia (T-ALL) and T-cell lineage chronic lymphocytic leukemia ؉ cell homeostasis, whereas malignant cells, particularly T-ALL CD4 T (T-CLL) is infiltration of various organs by leukemic cells (18). We cells, used CCR9/CCL25 for infiltration, resistance to apoptosis, and have reported that CCR9 is selectively and functionally overexpressed inappropriate proliferation. on T-ALL and T-CLL CD4ϩ T cells (19). Despite these findings, little is known about the exact mechanisms and molecule regulation in the homing, migration, inappropriate proliferation, and resistance to apo- INTRODUCTION ptosis of T-ALL and T-CLL T cells. CC chemokine ligand 25 (CCL25), also known as thymus-expressed chemokine (TECK), together with CC chemokine receptor 9 (CCR9), efficaciously induces chemotaxis of immature CD4ϩCD8ϩ MATERIALS AND METHODS double-positive, and mature CD4ϩ and CD8ϩ single-positive thymo- Patients and Cell Purification. All of the patients with T-ALL and T-CLL cytes, suggesting that CCL25/CCR9 interaction plays a pivotal role in were diagnosed according to The French-American-British (FAB) Cooperative T-cell migration in the thymus (1–4). CCL25/TECK delivers signals Group criteria (20) and to the guidelines of the National Cancer Institute through CCR9 for developing of thymocytes (5), and developing Working Group on B-CLL (21, 22), and informed consent according to Ϫ Ϫ and/or migrating of both ␣␤ and ␥␦ T cells (6). CCR9 activation institutional guidelines. CD4ϩ T cells were purified by a positive selection leads to phosphorylation of glycogen synthase kinase-3␤ (GSK-3␤) procedure of Dynabeads (Dynal A/S, Dynal, Norway). The malignancy of and forkhead transcriptional factor (FKHR) and provides a cell- purified T-ALL or T-CLL CD4ϩ T cells was checked by expression of CD25, survival signal (7). In the normal human, CCR9 is restrictedly ex- CD45RO, and HLA-DR (19). Human thymus tissue was aseptically obtained pressed at high levels on CD4ϩ and CD8ϩ T cells in the small after consent during surgical operative procedures involving the mediastinal region. Stromal cells were prepared by enzymatic digestion of thymus tissue, as described previously (23). CD4 and CD8 double-positive thymocytes were Received 2/21/04; revised 7/19/04; accepted 8/19/04. sorted with a FACSstarPlus. The cell lines were obtained from the American Grant support: This work was supported by the National Science Foundation of China (no. 39870674), Science Foundation of Anhui Province, China (no. 98436630), and Type Culture Collection, Manassas, VA (MOLT4 and human embryonic Education and Research Foundation of Anhui Province, China (no. 98JL063) and Re- kidney cells 293T). The anti-CCR9 monoclonal antibody and chemokines search Foundation from Health Department of Hubei Provincial Government, China (no. (CCL25/TECK, CXC (CC) chemokine ligand 12 (CXCL12)/SDF-1) were 301140344). purchased from R&D Systems, Abingdon, United Kingdom. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with Flow Cytometry. For detection of apoptosis, cells were stained in staining 18 U.S.C. Section 1734 solely to indicate this fact. buffer with 1 ␮g/mL propidium iodide for 30 minutes at 4°C, then stained with Note: Z. Qiuping and X. Jei contributed equally to this work. FITC-conjugated annexin V with binding buffer (BD PharMingen, San Diego, Requests for reprints: Tan Jinquan, Department of Immunology, Wuhan University CA) as described previously (7, 24). COULTER XL (Coulter, Miami, FL) was School of Medicine, Dong Hu Road 115, Wuchang 430071, Wuhan, Peoples Republic of China; Phone: 86-27-87331681; Fax: 45-35365326; E-mail: [email protected]. used for analyses. For detection of intracellular active caspases, cytofix/ ©2004 American Association for Cancer Research. cytoperm buffer (BD PharMingen) was used to permeabilize cells, and cells 7579

were subsequently stained with anti-active-capsase-3 or anti-active-caspase-8 under constant agitation. Immune complexes were allowed to bind to 20 ␮Lof monoclonal antibody (BD PharMingen). Data were analyzed by means of the protein A-Sepharose beads overnight, beads were washed three times with WinList program (The Scripps Research Institute, La Jolla, CA). lysis buffer, then suspended in 20 ␮L of kinase buffer containing 10 ␮Ci of Real-time Quantitative Reverse Transcription-PCR Assay. All real- [␥-32P]ATP and were incubated at 30°C for 30 minutes. The reaction was time quantitative reverse transcription-PCR (RT-PCR) reactions were done as stopped by the addition of 15 ␮Lof3ϫ sample buffer. Immunoprecipitates described elsewhere (24–26). Briefly, total RNA from purified cells (1 ϫ 105, were separated on SDS-12% polyacrylamide gels and were transferred to purity Ͼ99%) was prepared by using Quick Prep total RNA extraction kit nitrocellulose membranes. Filters were blocked with 5% nonfat milk in block- (Pharmacia Biotech, Hillerød, Denmark). RNA was reverse transcribed by ing buffer and were incubated with specified antibody for 2 hours, followed by

using oligo(dT)12–18 and Superscript II reverse transcriptase (Life Technolo- autoradiography. gies, Inc., Grand Island, NY). The real-time quantitative PCR was performed Chemotaxis Assay. The chemotaxis assay was performed in a 48-transwell with an ABI PRISM 7700 Sequence Detector Systems (Applied Biosystems, microchamber (Neuro Probe, Bethesda, MD) technique (7, 19). Briefly, che- Foster City, CA). By using SYBR Green PCR Core Reagents kit, fluorescence mokine (CCL25/TECK) in RPMI 1640 with 0.5% bovine serum albumin was signals were generated during each PCR cycle to provide real-time quantitative placed in the lower wells (25 ␮L). Cell suspension (25 ␮L, 2 ϫ 106 cells/mL) PCR information. The sequences of the specific primers were as follows: Livin was added to the upper well of the chamber, which was separated from the sense, 5Ј-GTCCCTGCCTCTGGGTAC-3Ј, and Livin antisense, 5Ј-CAGG- lower well by a 5 ␮m pore-size, polycarbonate, polyvinylpyrolidone-free GAGCCCACTCTGCA-3Ј. membrane (Nucleopore, Pleasanton, CA). The cells were CD4ϩ T cells that All unknown cDNAs were diluted to contain equal amounts of ␤-actin were positively selected by a procedure with anti-CD4 monoclonal antibody- cDNA. PCR retain conditions were 2 minutes at 50°C, 10 minutes at 95°C, 40 coated Dynabeads. The chamber was incubated for 60 minutes at 37°C and 5%

cycles with 15 seconds at 95°C, 60 seconds at 60°C for each amplification. CO2. The membrane was then carefully removed and was stained for 5 minutes Northern Blot Assays. For mRNA detection, 2 ␮g of total RNA obtained in 1% Coomassie Brilliant Blue. from each sample were electrophoresed under denaturing conditions, followed Gene Silencing Assay. Short hairpin RNAs (shRNAs) were produced in by blotting onto Nytran membranes, and were cross-linked by UV irradiation vitro as described previously (31) with chemically synthesized DNA oligonu- as described previously (27). Livin cDNA probe, labeled by [␣-32P]dCTP, was cleotide templates (Sigma). Transcription templates were designed such that obtained by PCR amplification of total RNA from human melanoma cell lines they contained U6 promoter sequences at the 5Ј end. shRNA transcripts (SK-Mel29). The membranes were hybridized overnight with 1 ϫ 106 cpm/mL subjected to in vitro dicer processing were synthesized with a Riboprobe kit of 32P-labeled probe, followed by intensive washing before being autoradio- (Promega, Madison, WI). Double-stranded DNA oligonucleotides encoding graphed. shRNAs with 21 bases of homology to the targeted Livin gene were ligated Plasmids and Cell Transfection. Plasmids encoding CCR9, c-IAP1, c- into the EcoRV site to produce expression constructs. Sequences inserted

IAP2, XIAP, survivin, Livin, and c-jun-NH2-kinase 1 (JNK1) had been de- immediately downstream of the U6 promoters were as follows: Livin sense, scribed previously (2, 16, 28, and 29). The cells were transiently transfected pU6hairpin21 GCGTCTGGCCTCCTTCTATGAtt. Cells were transfected with vectors encoding target genes as described elsewhere (2, 7, 16, 28, and with indicated amounts of shRNA and plasmid DNA with standard calcium 29). Briefly, the cells were cultured with DMEM containing 10% fetal calf phosphate procedures at 50 to 70% confluence in six-well plates. Cells were serum, penicillin, and streptomycin. Cells were grown to ϳ70% confluence in cultured in DMEM containing 10% of heat-inactivated fetal bovine serum, 60-mm dishes for 24 hours before transfection. The DNA construct of expres- penicillin, and streptomycin. Cells were harvested 2 days after the transfection. sion vectors (0.4 ␮g unless indicated) or vector only, was mixed with 12 ␮L of LipofectAMINE (Life Technologies, Inc.) in 2 mL of opti-DMEM serum- RESULTS free medium, was added to cells, and was incubated for 6 hours. The cells were further cultured in 2.5 mL of DMEM containing 10% fetal calf serum. CCL25/TECK Selectively Rescues T-ALL and T-CLL CD4؉ T Immune Complex Kinase Assay and Immunoblotting. Cell lysis was Cells from TNF-␣–Mediated Apoptosis. The data in Table 1 performed for 30 minutes at 4°C with lysis buffer (30). Expression of kinase showed that double-positive thymocytes, T-ALL and T-CLL CD4ϩ T proteins was semi-quantified after Western blot analysis (30). Lysates were cells and MOLT4 T cells expressed high levels of CCR9, which were centrifuged at 10,000 rpm for 5 minutes at 4°C. Protein concentration was ϳ ␮ in agreement with previous reports (1, 2, 7, 18, and 32). By knowing measured by Bio-Rad (Hercules, CA) protein assay. Protein ( 40 g) was ␤ loaded onto 16% SDS-PAGE, transferred onto polyvinylidene difluoride mem- that activation of CCR9 led to phosphorylation of GSK-3 and FKHR branes after electrophoresis, and incubated with the appropriate antibodies at and provided a cell survival signal (7), we examined the protective ␮ effects of CCL25/TECK and CXCL12/SDF-1 (7) on different types of 0.5 g/mL. All antibodies (Bcl-2, Bcl-X, c-FLIPL, c-IAP1, c-IAP2, XIAP, and survivin) were from Santa Cruz Biotechnology Inc., Santa Cruz, CA, except cells from tumor necrosis factor ␣ (TNF-␣)–mediated apoptosis. The anti-Livin was from Imgenex Corp. Sorrento Valley, San Diego, CA. The number of apoptotic and necrotic cells were significantly increased on membrane was blocked in 5% bovine serum albumin-Tris-buffered saline, culture of double-positive thymocytes in the presence of CCL25/ Myc-tagged proteins were immunoprecipitated with 20 ␮L of agarose-protein TECK (Fig. 1A-e), in comparison with that in malignant cells. The A (Pierce, Rockford, IL) preincubated with anti-Myc antibody (5 ␮g), and apoptotic responses in T-ALL and T-CLL CD4ϩ T cells as well as in ␮ FLAG-tagged proteins with 20 L of agarose conjugated with the M2 anti- MOLT4 T cells were significantly inhibited in the presence of FLAG monoclonal antibody (Sigma, St. Louis, MO). In vitro kinase assays CCL25/TECK (Fig. 1A-f, -g, and -h). CXCL12/SDF-1, to our sur- were performed as described elsewhere (29). For coimmunoprecipitations, cells were washed extensively and lysed in 200 ␮L of lysis buffer (29). After prise, could rescue malignant cells (Fig. 1A-j, -k, and -l), as well as incubation for 30 minutes on ice, cell lysates were centrifuged (10,000 rpm for double-positive thymocytes (Fig. 1A-i). Antibody against CCR9 could 10 minutes, 4°C) and the supernatants were recovered. Cell lysates were completely block the protective effect of CCL25/TECK (Fig. 1A-n, -o, precleared three times for 20 minutes at 4°C with 20 ␮L of protein A- and -p). As shown in Fig. 1B, the total dead cells (including apoptotic Sepharose beads and were mixed with specified antibodies for 3 hours at 4°C and necrotic) in different types of the cells tested were the same

Table 1 CCR9 expression in different T cells Thymocytes T-ALL T-CLL Peripheral Assay* (DP) CD4ϩ CD4ϩ MOLT4 CD4ϩ FL (%) 88 Ϯ 681Ϯ 545Ϯ 772Ϯ 46Ϯ 3 Real-time RT-PCR (ϫ 103) 7.3 Ϯ 0.8 7.1 Ϯ 0.4 4.1 Ϯ 0.4 6.8 Ϯ 0.7 0.8 Ϯ 0.2 NOTE. The cells were purified double-positive (DP) thymocytes, T-ALL CD4ϩ T cells, T-CLL CD4ϩ T cells, MOLT4 T-cell line, and purified peripheral CD4ϩ T cells. Data are mean values Ϯ SD of six experiments in each group. * Applied assays were flow cytometry (FL) and real-time quantitative RT-PCR (ϫ 103 mRNA copies/25 ng cDNA; ref. 19). 7580

motherapeutic drugs induced significant apoptosis when the cells were pretreated without or with low concentration of CCL25, whereas high concentration of CCL25 significantly prevented the malignant T cells from chemotherapy-induced apoptosis (Table 2). -Livin Expression in T-ALL CD4؉ T Cells Is Selectively In creased. To examine the mechanisms of cell-type selectivity of CCL25/TECK to rescue cells from TNF-␣–mediated apoptosis, we first examined the expression levels of some important substrates of apoptosis pathways in the distinct types of cells. The protein levels of

one antiapoptotic member Bcl-2 and c-FLIPL in freshly isolated different types of T cells were identical (Fig. 2A). Interestingly, Bcl-2 expression level was significantly elevated in double-positive thymocytes after stimulation with CCL25/TECK and apoptotic induction with TNF-␣, but not in the malignant cells (Fig. 2A). There were no

significant differences of c-FLIPL expression in various cell types before and after stimulation (Fig. 2A). We also observed a similar expression pattern of another antiapoptotic member, Bcl-X (data not shown). One antiapoptotic member protein in the IAP family, Livin, was selectively expressed at higher levels in the malignant cells, particularly in T-ALL CD4ϩ T cells, in comparison with the level in double-positive thymocytes (Fig. 2B). For a better comparison, samples from different un-stimulated cells were loaded in the same gel that was used for examining Livin expression (Fig. 2C). The expression levels of Livin in these malignant cells were significantly increased after stimulation with CCL25/TECK and TNF-␣ (Fig. 2B). Stimulation with TNF-␣ alone slightly changed (decreased) the expression of Livin in different types of cells. The antibody against CCR9 could significantly inhibit the elevation of expression level of Livin in these cells (data not shown). Another antiapoptotic member in the IAP family, survivin, expressed identical levels in the different conditions in various types of cells (Fig. 2B). The expression levels of other members in the IAP protein family (c-IAP1, c-IAP2, and XIAP) were not significantly changed in various cell types before and after stimulation with CCL25/TECK and TNF-␣ (data not shown). To further confirm elevated expression of Livin in T-ALL CD4ϩ T cells, we examined the expression of Livin mRNA in the distinct types of cells. The Livin mRNA was expressed at a very low level in freshly isolated double-positive thymocytes (1.1 ϫ 103 copies) and was not substantially altered after stimulation with CCL25/TECK and TNF-␣ (Fig. 3A). In contrast, Livin mRNA expression was significantly higher in T-ALL CD4ϩ T cells (3.2 ϫ 103 copies), whereas there were significant increases in T-ALL CD4ϩ T cells (6.2 ϫ 103 and 5.8 ϫ 103 copies, respectively) after stimulation with CCL25/TECK along or together with TNF-␣ (Fig. 3B). TNF-␣ stimulation alone slightly decreased the expression of Livin (2.5 ϫ 103 copies). The antibody against CCR9 could marginally inhibit the elevation in Livin mRNA expression (data not shown). The same patterns of Livin Fig. 1. Analysis of apoptotic and total dead cells. Flow cytometric analysis of apoptotic (A) and total dead cells (necrotic and apoptotic; B), the cells were purified CD4 and CD8 mRNA expression were seen in double-positive thymocytes (Fig. 3C) double-positive thymocytes, T-ALL or T-CLL CD4ϩ T cells, or MOLT4 cells, which and T-ALL CD4ϩ T cells (Fig. 3D) by Northern blot. For a better were pretreated at absence or presence of chemokine as indicated (100 ng/mL), after ␣ comparison, samples from unstimulated thymocytes and T-ALL stimulation with TNF- (100 ng/mL) for 24 hours at 37°C. The cells were analyzed by ϩ flow cytometry for propidium iodide (PI) and FITC-conjugated annexin V as described in CD4 T cells were loaded in the same gel that was used to examine Ϫ ϩ ϩ ϩ Materials and Methods. The percentages of PI annexin V cells and PI annexin V Livin mRNA (Fig. 3E). Elevated Livin mRNA expressions were also cells are indicated in the figure. The data (A) are from a single experiment that was ϩ representative of six experiments. The data for total dead cells (PIϪ annexin Vϩ ϩ pro- observed in other malignant cells (T-CLL CD4 T cells and MOLT4 pidium iodideϩ annexin Vϩ; B) are mean values Ϯ SD of six experiments. Statistically T cells) by real-time quantitative RT-PCR assay and Northern blot Ͻ significant differences as compared with controls (*, P 0.001). (data not shown). The pattern of Livin mRNA expression in normal peripheral CD4ϩCCR9ϩ T cells was similar to that in double-positive thymocytes (Fig. 3F). patterns as the results in Fig. 1A. These results were suggesting that The caspase-3 and caspase-8 expression is essential for this type of differential effects of CCR9 and TNF-␣ signaling existed between cell death (33). Expression levels of activated caspase-3 and caspase-8 double-positive thymocytes and malignant T cells. were measured by intracellular staining of double-positive thymocytes We examined the protective effects of CCL25/TECK on different (Fig. 4A) and T-ALL CD4ϩ T cells (Fig. 4B) with the use of cleavage- types of malignant T cells from chemotherapy-induced apoptosis. specific caspase-3 and caspase-8 antibody. Double-positive thymo- CCL25/TECK itself induced no apoptosis in malignant T cells. Che- cytes, normal peripheral CD4ϩCCR9ϩ T cells, and T-ALL CD4ϩ T 7581

Table 2 Apoptotic analysis in malignant T cells under different chemotherapeutic drug treatments in vitro T-ALL CD4ϩ T-CLL CD4ϩ MOLT4

Treatment 0 0.1 100 0 0.1 100 0 0.1 100 Nontreated 9 Ϯ 28Ϯ 19Ϯ 66Ϯ 310Ϯ 27Ϯ 28Ϯ 39Ϯ 311Ϯ 3 Comp. 62 Ϯ 12 55 Ϯ 17 13 Ϯ 657Ϯ 952Ϯ 12 9 Ϯ 567Ϯ 359Ϯ 610Ϯ 4 Cyclop. 65 Ϯ 862Ϯ 912Ϯ 265Ϯ 761Ϯ 11 10 Ϯ 559Ϯ 860Ϯ 10 14 Ϯ 6 NOTE. Cells were pretreated for 2 hours in the presence (at concentration of 0.1 or 100 ng/mL) or absence (0) of CCL25, then were treated in the presence of camptothecin (Comp., 5 ␮mol/L) or cyclophosphamide (Cyclop., 7.5 mmol/L; ref. 48) for 12 hours, followed by apoptotic analysis by flow cytometry. The data for apoptotic cells are mean values Ϯ SD of six experiments performed in each group. cells expressed substantially up-regulated cleaved caspase-3 and tions of pertussis toxin, an inhibitor of PKC; wortmannin, a potent caspase-8 in TNF-␣ stimulation, but CCL25/TECK could only stabi- PI3K inhibitor; PD98059, a MAPK inhibitor; or vehicle, DMSO. As lize caspase-3 and caspase-8 in T-ALL CD4ϩ T cells, but not in seen in Fig. 5A, pertussis toxin and wortmannin significantly inhibited double-positive thymocytes nor in normal peripheral CD4ϩCCR9ϩ T cell migration toward CCL25/TECK; PD98059 did not inhibit T-ALL cells (Fig. 4). We observed similar results in other malignant cell CD4ϩ T cell migration toward CCL25/TECK, which was in agree- types (T-CLL CD4ϩ T cells and MOLT4 T cells; data not shown), ment with those who observed similar results in MOLT4 T cells (7). which suggested that CCL25/TECK protection from apoptosis was We observed similar results in other malignant cell types (T-CLL limited in malignant cells. CD4ϩ T cells and MOLT4 T cells; data not shown). To determine Many signaling events of binding of certain chemokines to chemo- whether these kinases were responsible for CCL25/TECK-mediated kine receptors were included, such as phosphoinositide-3 kinase protection from apoptotic response, we pretreated the cells with the (PI3K), mitogen-activated protein kinase (MAPK), or protein kinase inhibitors mentioned above, As seen in Fig. 5B, only pertussis toxin C (PKC), which appeared to be involved in chemokine-mediated significantly inhibited the apoptotic protection effect in T-ALL CD4ϩ chemotaxis in certain cell types (7, 34–36). To determine whether T cells. Neither PD98059 nor wortmannin inhibited apoptotic protec- these kinases were responsible for CCL25/TECK-mediated chemo- tion effect in T-ALL CD4ϩ T cells, suggesting that CCL25/TECK taxis, T-ALL CD4ϩ T cells were pretreated with various concentra- went through different signaling pathway to carry out chemotaxis and

Fig. 2. Occurrence of Bcl-2, c-FLIPL, survivin, and Livin expression in distinct cells. The Bcl-2 and c-FLIPL (A), and survivin and Livin (B) were examined with Western blot analyses. The cells were purified CD4 and CD8 double-positive thymocytes, T-ALL or T-CLL CD4ϩ T cells, or MOLT4 cells that were pretreated as described in legend for Fig. 1. Different unstimulated cells were also loaded in the same gel for Livin test (C). Actins indicate the quantity of total cellular protein from the tested samples loaded in each lane. Ar- rows, markers used to verify equivalent molecular weights of appropriate proteins in each lane. The data were from a single experiment that was representative of six experiments.

7582

Fig. 3. Livin mRNA expression. The real-time quantitative detection of RT-PCR and Northern blot for mRNA of Livin mRNA in double-positive thymocytes (A, C), T-ALL CD4ϩ T cells (B, D), purified normal peripheral CD4ϩCCR9ϩ T cells (NOL, F). Differ- ent unstimulated cells were loaded in the same gel for Livin mRNA test (E). Cells were pretreated as described in legend for Fig. 1. The correlation coefficients were ϳ0.97 to 0.99. The showing bars (A, B, F) are representatives of eight similar experiments conducted. Total RNA from different cells as indicated were isolated, electrophoresed, and blotted. Top panels, the hybridization signals for Livin mRNA in different cells. Bottom panels, the 28S rRNAs (28S) confirm the comparable amounts of loaded total RNA. The data are from a single experiment that is representative of six experiments.

apoptotic protection in T-ALL CD4ϩ T cells. The inhibitors them- could not interfere in the interaction of CCR9 and JNK1, which might selves had no apoptotic effects on the T-ALL CD4ϩ T cells (Fig. be important for induction of protection of cells from TNF-␣–medi- 5B-g, -h, -i, and -j). Z-VAD-FMK rescued T-ALL CD4ϩ T cells from ated apoptosis. TNF-␣–mediated apoptosis. We observed similar results in other To be sure that the results of correlation between Livin activation malignant cell types (T-CLL CD4ϩ T cells and MOLT4 T cells; data and CCL25/TECK-induced resistance to TNF-␣–mediated apoptosis, not shown). we applied short hairpin RNA of Livin (shRNAlivin) to knockdown CCL25/TECK via CCR9 Activates Livin in JNK1 Kinase- Livin expression and activation in MOLT4 T cells before assay of Dependent Manner. The inhibitor of apoptosis family of proteins CCL25/TECK-induced resistance to TNF-␣–mediated apoptosis. The was found to protect against the broadest spectrum of apoptotic results (Fig. 8A) showed that culture with shRNAlivin at high con- signals (37, 38). We further investigated the ability of the IAP family centration completely abolished expression of Livin in MOLT4 T members to induce down-stream kinase activation for protection from cells at both mRNA and protein levels, whereas low-concentration apoptosis by CCL25/TECK in the cells. CCL25/TECK selectively shRNAlivin or DNAlivin or vector had no such effect. Only high- activated Livin (Fig. 6A). The phosphorylation of ATF-2 substrate concentration shRNAlivin significantly blocked the effects of CCL25/ was dose dependent on expression level of Livin in the system. In TECK on induction of resistance to TNF-␣–mediated apoptosis (Fig. contrast, none of c-IAP1, c-IAP2, XIAP, or survivin showed activa- 8B and C) and on stabilization of caspase-3 and caspase-8 in MOLT4 tion. In the system with JNK1 transfection lacking, Livin could not be T cells (Fig. 8D and E). We observed similar results for shRNAlivin activated, phosphorylation of ATF-2 substrate was also dose-depen- in T-ALL and T-CLL CD4ϩ T cells (data not shown). These results dent on expression level of JNK1 (Fig. 6B), suggesting CCL25/TECK in MOLT4 and in T-ALL and T-CLL CD4ϩ T cells confirmed activating Livin was JNK1 kinase-dependent. To further elucidate experimentally the observations that CCL25/TECK, via CCR9, acti- whether the activation of JNK1 was also important for the protective vated Livin to induce resistance to TNF-␣–mediated apoptosis. activity of the IAPs against TNF-␣-induced apoptosis,. All examined Thus, CCL25/TECK rescued T-ALL and T-CLL CD4ϩ T cells as kinases (p38, extracellular signal-regulated kinase (ERK2), JNK1, or well as MOLT4 T cells from TNF-␣–mediated apoptosis, but double- PI3K) were activated in MOLT4 T cells by CCL25/TECK, but they positive thymocytes did not. CCL25/TECK selectively activated were inhibited by stimulation with TNF-␣ (Fig. 7), except for JNK1, Livin to induce resistance to TNF-␣–mediated apoptosis in JNK1 which was constantly activated (Fig. 7C), suggesting that TNF-␣ kinase-dependent manner. 7583

Fig. 5. Inhibition of chemotaxis and TNF-␣—mediated apoptosis of cells. A, the migration of T-ALL CD4ϩ T cells toward CCL25/TECK (100 ng/mL). The cells were pretreated with various concentrations of pertussis toxin (PT), wortmannin (WT), PD98059, or vehicle DMSO for 1 hour. All of the results are expressed as chemotactic index (ϮSD). The illustrated data are mean values of six experiments. Statistically significant differences as compared with untreated cells are indicated (*, P Ͻ 0.001). B, T-ALL CD4ϩ T cells were serum starved overnight and treated with pertussis toxin (PT, 1 ␮g/mL), wortmannin (WT, 50 nmol/L), PD98059 (50 ␮mol/L), or vehicle DMSO for 1 hour, after culture in the presence of CCL25/TECK (ࡗ, 100 ng/mL) for 6 hours. As a control, the apoptotic effects of inhibitors were measured in the T-ALL CD4ϩ T cells treated with these inhibitors [DMSO (g), PT (h), PD98059 (i), and WT (j)]. Z-VAD-FMK (20 ␮mol/L) was used for blocking apoptosis. Numbers in boxes a-k, the percentages of propidium iodide (PI)Ϫ annexin Vϩ cells and PIϩ annexin Vϩ cells. The data are from a single experiment that is representative of five experiments.

DISCUSSION A majority of the G protein-coupled receptor supergene family has been shown to be capable of activating MAPK, which is an indication of providing proliferative or antiapoptotic signaling. Several chemo- Fig. 4. Activation of caspases. Flow cytometric analysis of active caspase-3 (A) and kines are able to activate MAPK to function as proliferative or caspase-8 (B) in double-positive thymocytes, normal peripheral CD4ϩ T cells (gated CCR9ϩ; antiapoptotic signals (39). For example, CXCL12/SDF-1 is an impor- NOL CD4ϩ), and T-ALL CD4ϩ T cells. Cells were pretreated as described in legend for Fig. 1. CCR9 monoclocal antibody blocking assay (CCR9B) was applied to some of the cells. tant cytokine that, acting together with thrombopoietin, enhances the Activated caspase-3–specific or caspase-8–specific fluorescence intensity was measured by development of megakaryocytic progenitor cells and activates circu- flow cytometry. Percentages of cells (n%) with activated caspase-3 or caspase-8 were quan- ϩ titated from relative-frequency histograms. Dished curves, isotype antibody controls. The data lating CD34 cells and platelets (40, 41). CXCL1 and CXCL4 are were from a single experiment which was representative of six experiments. able to support the survival of endothelial cells and monocytes, 7584

respectively (42, 43). Chemokine receptor signaling may be able to provide antiapoptotic activity to hematopoietic cells in a natural context (44). A solid report shows that CCR9/CCL25 interaction provides a cell survival signal to the receptor-expressing cells (7). However, there are some controversial, even contradictory, reports. For example, CXCR4 has induced programmed cell death of human peripheral CD4ϩ T cells, malignant T cells, and CD4/CXCR4 trans- fectants (45). The interaction between HIV R5 Env and CCR5 activates the Fas pathway and caspase-8, as well as triggering FasL production, ultimately causing CD4ϩ T cell death (46). CCR3 expression, induced by interleukin (IL)-2 and IL-4, functions as a death receptor for B cells (24). We have investigated four different types of cells: double-positive thymocytes, T-ALL and T-CLL CD4ϩ T cells, and MOLT4 T cells. We have found that, even through all types of cells are CCR9 rich-expressing cells, the responsiveness to CCL25/ TECK-mediated protection from apoptosis is quite differential; double-positive thymocytes use CCR9/CCL25 for migration, homing, development, maturation, selection, and cell homeostasis. Meanwhile, malignant cells, particularly T-ALL CD4ϩ T cells, take advantages of CCR9/CCL25 for infiltration, resistance to apoptosis, and inappropriate proliferation. To our knowledge, this study is the first report on differential functions of CCR9/CCL25 in distinct types of cells and is the direct evidence of the pathophysiologic activity of T-ALL and T-CLL CD4ϩ T cells induced by CCL25/TECK. A genetic change that leads to blocking apoptosis may allow a cell to acquire additional mutations, survive inappropriately and eventu- ally become malignant. Additionally, a defect in the inherent ability of a cell to undergo apoptosis may account for much of the resistance to different therapies observed in leukemic cells. Therefore, much effort has gone into understanding how apoptosis is altered in leukemia cells and how it can be modulated to overcome resistance and improve clinical outcomes. Recent reports that CCR9 is selectively and functionally overexpressed on T-ALL and T-CLL CD4ϩ T cells (19) prompted us to investigate more closely the additional functions of Fig. 6. CCL25/TECK selectively activates Livin protein. 293T cells were cotransfected CCR9 with regard to these malignant cells, which often manifest with vectors encoding CCR9 and JNK1 (400 ng each, A; or 200 ng, 400 ng, and 600 ng, inappropriate survival and resistance to various clinical therapies. We respectively, B) in the absence or presence of increasing concentrations of c-IAP1, c-IAP2, XIAP, survivin, or Livin (200 or 600 ng). The amount of transfected cDNA was have found that one antiapoptotic member protein in IAP family, kept constant in each sample by adding control pcDNA3 vector. Cells were pretreated at Livin, is selectively expressed at higher levels in the malignant cells, absence or presence of CCL25/TECK (100 ng/mL) before cell lysis. An in vitro kinase ϩ assay was performed with ATF-2 as substrate. Kinase activity was semiquantitated and is particularly in T-ALL CD4 T cells, in comparison with double- expressed to the basal level of phosphorylation of each MAPK. (Ultra Violet stimulation) positive thymocytes. After stimulation with CCL25/TECK and apo- was used as positive controls. Western blottings (WB) show equal expression levels of ptotic induction with TNF-␣, the expression levels of Livin in these JNK1 and CCR9 (A) or various expression levels of JNK1 and equal expression levels of Livin (B).

Fig. 7. TNF-␣ interferes with the activation of p38, ERK2, and PI3K, but not JNK1, by CCL25/TECK. MOLT4 T cells were used to examine activated signal transducers by CCL25/ TECK as follows: p38 (A), or ERK2 (B), or JNK1 (C), or PI3K (PI-3, D). were pretreated in absence or presence of CCL25/TECK (100 ng/mL) described in Materials and Meth- ods; some of cells were stimulated with TNF-␣ (100 ng/mL) for 2 hours at 37°C before cell lysis as indicated. Activated signal transducers were detected by respective phospho-specific antibodies (pho), and total proteins were examined by respective pan-antibodies (pan) in Western blottings. PMA, phorbol myristate acetate, used as positive control.

7585

Fig. 8. Blocking effects of shRNAlivin. MOLT4 T cells were cultured for 2 days in the presence or absence of shRNA as indicated. Vector (2 ␮g); DNAlivin, DNA Livin sequence (2 ␮g); shRNAlivin (l), low concentration (0.02 ␮g); shRNAlivin (h), high concentration (2 ␮g). Cells were then treated in presence of CCL25/ TECK (100 ng/mL), after stimulation with TNF-␣ (100 ng/mL) for 24 hours at 37°C. The Livin (A) were examined with Northern (top panel) and Western blot (bottom panel) analyses as described in legend for Fig. 3. Flow cytometric analyses of apoptotic (B) and total dead cells (C) were done as described in the legend for Fig. 1. The data (B) were from a single representative experiment of five performed. The data for total dead cells (C) were mean values Ϯ SD of five experiments performed. Statistically significant differences as compared with controls are indicated (*, P Ͻ 0.001). Flow cytometric analysis of active caspase-3 (D) and caspase-8 (E)in cells were done as described in the legend for Fig. 4. The data are from a single representative experiment of five performed.

cells are significantly increased (Fig. 2B). With sufficient and direct identifying Livin as antigen linked with immune-mediated tumor evidence, we have shown that Livin, but not other IAP members destruction (immunotherapy; ref. 17). (c-IAP1, c-IAP2, XIAP, and survivin), has been directly activated by PI3K, but not MAPK, is required for CCR9-mediated chemotaxis CCL25/TECK in JNK1-dependent manner and that, subsequently, (7). Costimulation of MOLT4 T cells with CCL25/TECK and T-ALL and T-CLL CD4ϩ T cells, as well as MOLT4 T cells, CXCL12/SDF-1 significantly blocks CHX-mediated apoptosis, inappropriately survive. Originally characterized in a number of mel- whereas stimulation with only CCL25/TECK only partially blocks anoma cell lines (14, 15), Livin is a potent antiapoptotic protein and Fas-mediated apoptosis. Akt, GSK-3␤, FKHR, and MAPK have been is not detectable in most normal adult tissues. Elevated expression of involved in cell survival signals in response to an array of death Livin renders melanoma cells resistant to apoptotic stimuli and po- stimuli. Our results showing that CCL25/TECK rescues T-ALL and tentially contributes to the pathogenesis of this malignancy (47). Our T-CLL CD4ϩ T cells, as well as MOLT4 T cells, from TNF-␣– findings, together with those of others, indicate that Livin may be one mediated apoptosis are largely in agreement with these observations. of the critical cellular factors the increased expression of which Of note, we have not been able to show that stimulation of CCR9 with confers resistance to apoptotic stimuli, thereby contributing to the CCL25/TECK can directly activate p38, ERK2, or PI3K in the pres- pathogenesis and progression of malignant cells, such as melanoma ence of stimulation with TNF-␣ (Fig. 7) in our applied experimental cells and T-ALL and T-CLL CD4ϩ T cells. The observation interest- system, except JNK1 is constantly activated (Fig. 7C). Taking into ingly indicates that Livin may be a suitable therapeutic target for gene account that CCL25/TECK can directly activate JNK1, and subse- therapy. Moreover, the results may strengthen the strategic ideal of quently Livin, to induce resistance to TNF-␣–mediated apoptosis, we 7586

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2004 American Association for Cancer Research. APOPTOTIC RESISTANCE VIA CCR9/LIVIN/JNK1 PATHWAY suggest that TNF-␣ can interfere with the activation of p38, ERK2, 21. Cheson BD, Bennett JM, Grever M, et al. National Cancer Institute-sponsored and PI3K, which is a necessary signal for cell survival. However, Working Group guidelines for chronic lymphocytic leukemia: revised guidelines for diagnosis and treatment. Blood 1996;87:4990–7. CCL25/TECK can bypass the obstacle, and directly activate JNK1. 22. Bennett JM, Catovsky D, Daniel MT, et al. Proposals for the classification of chronic With facilitation of the environment of activated JNK1, Livin can, (mature) B and T lymphoid leukemias. J Clin Pathol 1989;42:567–84. 23. Wu L, Li CL, Shortman K. Thymic dendritic cell precursors: relationship to the T therefore, be activated to initiate a rescuing mechanism in the cells. lymphocyte lineage and phenotype of the dendritic cell progeny. J Exp Med 1996; On the basis of our findings, we suggest that there are differential 184:903–11. functions of CCR9/CCL25 in distinct types of cells: Double-positive 24. Jinquan T, Jacobi HH, Jing C, et al. CCR3 expression induced by IL-2 and IL-4 functioning as a death receptor for B cells. J Immunol 2003;171:1722–31. thymocytes use CCR9/CCL25 for migration, homing, development, 25. Heid CA, Stevens J, Livak KJ, William PM. Real time quantitative PCR. Genome Res maturation, selection, and cell homeostasis. Meanwhile, the malignant 1996;6:986–94. cells use CCR9/CCL25 for infiltration, resistance to apoptosis, and 26. Kruse N, Pette M, Toyka K, Rieckmann P. Quantification of cytokine mRNA expression by RT PCR in samples of previously frozen blood. J Immunol Methods inappropriate proliferation. The observation indicates that Livin may 1997;210:195–203. be a suitable therapeutic target for gene therapy. 27. Sica A, Saccani A, Borsatti A, et al. Bacterial lipopolysaccharide rapidly inhibits expression of C-C chemokine receptors in human monocytes. J Exp Med 1997;185: 969–74. 28. Vucic D, Deshayes K, Ackerly H, et al. SMAC negatively regulates the anti-apoptotic REFERENCES activity of melanoma inhibitor of apoptosis (ML-IAP). J Biol Chem 2002;277: 12275–9. 1. Johansson-Lindbom B, Svensson M, Wurbel MA, Malissen B, Marquez G, Agace W. 29. Sanna MG, Duckett CS, Richter BW, Thompson CB, Ulevitch RJ. Selective activa- Selective generation of gut tropic T cells in gut-associated lymphoid tissue (GALT): tion of JNK1 is necessary for the anti-apoptotic activity of hILP. Proc Natl Acad Sci requirement for GALT dendritic cells and adjuvant. J Exp Med 2003;198:963–9. USA 1998;95:6015–20. 2. Youn BS, Kim CH, Smith FO, Broxmeyer HE. TECK, an efficacious chemoattractant 30. Massari P, Ho Y, Wetzler LM. Neisseria meningitidis porin PorB interacts with for human thymocytes, uses GPR-9–6/CCR9 as a specific receptor. Blood 1999;94: mitochondria and protects cells from apoptosis. Proc Natl Acad Sci USA 2000;97: 2533–6. 9070–5. 3. Zaballos A, Gutierrez J, Varona R, Ardavin C, Marquez G. Cutting edge: identifi- 31. Kawasaki H, Taira K. Short hairpin type of dsRNAs that are controlled by tRNAVal cation of the orphan chemokine receptor GPR-9–6 as CCR9, the receptor for the promoter significantly induce RNAi-mediated gene silencing in the cytoplasm of chemokine TECK. J Immunol 1999;162:5671–5. human cells. Nucleic Acids Res 2003;31:700–7. 4. Yu CR, Peden KW, Zaitseva MB, Golding H, Farber JM. CCR9A and CCR9B: two 32. Zabel BA, Agace WW, Campbell JJ. Human G protein-coupled receptor GPR-9– receptors for the chemokine CCL25/TECK/Ck beta-15 that differ in their sensitivities 6/CC chemokine receptor 9 is selectively expressed on intestinal homing T lympho- to ligand. J Immunol 2000;164:1293–305. cytes, mucosal lymphocytes, and thymocytes and is required for thymus-expressed 5. Norment AM, Bogatzki LY, Gantner BN, Bevan MJ. Murine CCR9, a chemokine chemokine-mediated chemotaxis. J Exp Med 1999;190:1241–56. receptor for thymus-expressed chemokine that is up-regulated following pre-TCR 33. Thornberry NA, Lazebnik Y. Caspases: enemies within. Science 1998;281:1312–6. signaling. J Immunol 2000;164:639–48. 34. Kampen GT, Stafford S, Adachi T, et al. Eotaxin induces degranulation and chemo- 6. Uehara S, Grinberg A, Farber JM, Love PE. A role for CCR9 in T lymphocyte taxis of eosinophils through the activation of ERK2 and p38 mitogen-activated development and migration. J Immunol 2002;168:2811–9. protein kinases. Blood 2000;15:1911–7. 7. Youn BS, Kim YJ, Mantel C, Yu KY, Broxmeyer HE. Blocking of c-FLIP(L)– 35. Boehme SA, Sullivan SK, Crowe PD, et al. Activation of mitogen-activated protein independent cycloheximide-induced apoptosis or Fas-mediated apoptosis by the CC kinase regulates eotaxin-induced eosinophil migration. J Immunol 1999;163:1611–8. chemokine receptor 9/TECK interaction. Blood 2001;98:925–33. 36. Wang JF, Park IW, Groopman JE. Stromal cell-derived factor-1alpha stimulates 8. Kunkel EJ, Campbell JJ, Haraldsen G, Pan J, Boisvert J, Roberts AI. Lymphocyte CC tyrosine phosphorylation of multiple focal adhesion proteins and induces migration of chemokine receptor 9 and epithelial thymus-expressed chemokine (TECK) expression hematopoietic progenitor cells: roles of phosphoinositide-3 kinase and protein kinase distinguish the small intestinal immune compartment: Epithelial expression of tissue- C. Blood 2000;95:2505–13. specific chemokines as an organizing principle in regional immunity. J Exp Med 37. Duckett CS, Nava VE, Gedrich RW, et al. A conserved family of cellular genes 2000;192:761–8. related to the baculovirus iap gene and encoding apoptosis inhibitors. EMBO J 9. Mora JR, Bono MR, Manjunath N, Weninger W, Cavanagh LL, Rosemblatt M. 1996;15:2685–94. Selective imprinting of gut-homing T cells by Peyer’s patch dendritic cells. Nature 38. Miller LK. An exegesis of IAPs: salvation and surprises from BIR motifs. Trends Cell 2003;424:88–93. Biol 1999;9:323–8. 10. Papadakis KA, Landers C, Prehn J, et al. CC chemokine receptor 9 expression defines 39. Ganju RK, Brubaker SA, Meyer J, et al. The alpha-chemokine, stromal cell-derived a subset of peripheral blood lymphocytes with mucosal T cell phenotype and Th1 or factor-1alpha, binds to the transmembrane G-protein-coupled CXCR-4 receptor and T-regulatory 1 cytokine profile. J Immunol 2003;171:159–65. activates multiple signal transduction pathways. J Biol Chem 1998;273:23169–75. 11. Salvesen GS, Duckett CS. IAP proteins: blocking the road to death’s door. Nat Rev 40. Hodohara K, Fujii N, Yamamoto N, Kaushansky K. Stromal cell-derived factor-1 Mol. Cell Biol 2002;3:401–10. (SDF-1) acts together with thrombopoitin to enhance the development of megakaryo- 12. Chai J, Shiozaki E, Srinivasula SM, et al. Structural basis of caspase-7 inhibition by cytic progenitor cells (CFU-MK). Blood 2000;95:769–75. XIAP. Cell 2001;104:769–80. 41. Lataillade JJ, Clay D, Dupuy C, et al. Chemokine SDF-1 enhances circulat-ing 13. Riedl SJ, Renatus M, Schwarzenbacher R, et al. Structural basis for the inhibition of CD34ϩ cell proliferation in synergy with cytokines: possible role in progenitor caspase-3 by XIAP. Cell 2001;104:791–800. survival. Blood 2000;95:756–68. 14. Kasof GM, Gomes BC. Livin, a novel inhibitor of apoptosis protein family member. 42. Han ZC, Lu M, Li J, et al. Platelet factor 4 and other CXC chemokines support the J Biol Chem 2001;276:3238–46. survival of normal hematopoietic cells and reduce the chemosensitivity of cells to 15. Lin JH, Deng G, Huang Q, Morser J. KIAP, a novel member of the inhibitor of cytotoxic agents. Blood 1997;89:2328–35. apoptosis protein family. Biochem Biophys Res Commun 2000;279:820–31. 43. Scheuerer B, Ernst M, Durrbaum-Landmann I, et al. The CXC chemokine platelet 16. Sanna MG, da Silva Correia J, et al. IAP suppression of apoptosis involves distinct factor 4 promotes survival and induce monocyte differentiation into macrophages. mechanisms: the TAK1/JNK1 signaling cascade and caspase inhibition. Mol Cell Blood 2000;95:1158–66. Biol 2002;22:1754–66. 44. Youn BS, Yu KY, Oh J, Lee J, Lee TH, Broxmeyer HE. Role of the CC chemokine 17. Schmollinger JC, Vonderheide RH, Hoar KM, et al. Melanoma inhibitor of apoptosis receptor 9/TECK interaction in apoptosis. Apoptosis 2002;7:271–6. protein (ML-IAP) is a target for immune-mediated tumor destruction. Proc Natl Acad 45. Berndt C, Mopps B, Angermuller S, Gierschik P, Krammer PH. CXCR4 and CD4 Sci USA 2003;100:3398–403. mediate a rapid CD95-independent cell death in CD4ϩ T cells. Proc Natl Acad Sci 18. Till KJ, Lin K, Zuzel M, Cawley JC. The chemokine receptor CCR7 and alpha4 USA 1998;95:12556–61. integrin are important for migration of chronic lymphocytic leukemia cells into lymph 46. Algeciras-Schimnich A, Vlahakis SR, Villasis-Keever A, et al. CCR5 mediates Fas- nodes. Blood 2002;99:2977–84. and caspase-8 dependent apoptosis of both uninfected and HIV infected primary 19. Qiuping Z, Qun L, Chunsong H, et al. Selectively increased expression and functions human CD4 T cells. AIDS 2002;16:1467–78. of chemokine receptor CCR9 on CD4ϩ T cells from patients with T-cell lineage acute 47. Vucic D, Stennicke HR, Pisabarro MT, Salvesen GS, Dixit VM. ML-IAP, a novel lymphocytic leukemia. Cancer Res 2003;63:6469–77. inhibitor of apoptosis that is preferentially expressed in human melanomas. Curr Biol 20. Bennett JM, Catowsky D, Daniel MT, et al. The French-American-British (FAB) 2000;10:1359–66. Cooperative Group. The morphological classification of acute lymphoblastic leukae- 48. Hamano Y, Sugimoto H, Soubasakos MA, et al. Thrombospondin-1 associated with mia: concordance among observers and clinical correlation. Br J Haematol 1981;47: tumor microenvironment contributes to low-dose cyclophosphamide-mediated endo- 553–8. thelial cell apoptosis and tumor growth suppression. Cancer Res 2004;64:1570–4.

7587

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2004 American Association for Cancer Research. CC Chemokine Ligand 25 Enhances Resistance to Apoptosis in CD4 + T Cells from Patients with T-Cell Lineage Acute and Chronic Lymphocytic Leukemia by Means of Livin Activation

Zhang Qiuping, Xiong Jei, Jin Youxin, et al.

Cancer Res 2004;64:7579-7587.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/64/20/7579

Cited articles This article cites 48 articles, 34 of which you can access for free at: http://cancerres.aacrjournals.org/content/64/20/7579.full#ref-list-1

Citing articles This article has been cited by 4 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.org/content/64/20/7579.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/64/20/7579. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.