Basal c-Jun NH
2
-terminal protein kinase activity is essential
for survival and proliferation of T-cell acute
lymphoblastic leukemia cells
Jian Cui, Qingyang Wang, Jing Wang, Ming Lv,
Ning Zhu, Yan Li, Jiannan Feng, Beifen Shen,
and Jiyan Zhang
Department of Molecular Immunology, Institute of Basic Medical Sciences, Beijing, People's Republic of China
Abstract
Hyperactivation of c-Jun NH2-terminal protein kinase (JNK) has been found in various malignant lymphocytes and inhibition of JNK activity leads to cell cycle arrest and apoptosis. However, the role of JNK activity in the oncogenic growth of T-cell acute lymphoblastic leukemia (T-ALL) cells remains largely unknown. Here, we report that treatment of T-ALL cells with JNK inhibitors led to cell cycle arrest and apoptosis and increased sensitivity to Fas-mediated apoptosis, whereas weak ectopic expression of MKK7-JNK1 fusion protein, which shows constitutive JNK activity, in T-ALL cells resulted in accelerated cell cycle progression and resistance to Fas-mediated apoptosis. The protein levels of c-Myc and Bcl-2 were reduced in the presence of JNK inhibitors but were enhanced with MKK7-JNK1. Small interfering RNA against JNK1, but not JNK2, exhibited similar effects to JNK inhibitors. These findings suggest that targeting JNK, especially JNK1 isoform, may have some important therapeutic im-plications in the treatment of T-ALL. Further exploration revealed that JNK protein and basal JNK activity in T-ALL cells showed aberrant subcellular localization, but no hyperactivation of JNK was observed. Thus, our work sug-gests that there might be novel mechanism(s) other than hyperactivation underlying the protumorigenic role of JNK activity. [Mol Cancer Ther 2009;8(12):3214–22]
Introduction
c-Jun NH2-terminal protein kinase (JNK) is a member of the mitogen-activated protein kinase (MAPK) superfamily, which also includes the extracellular signal-regulated kinase and p38 family of kinases (1). JNK has two ubiqui-tously expressed isoforms, JNK1 and JNK2, and a tissue-specific isoform, JNK3, with different splicing forms (p54 and p46; refs. 2–4). JNK is activated by sequential protein phosphorylation through a MAPK module [MAPK kinase kinase→ MAPK kinase (or MKK) → MAPK] in response to a variety of extracellular stimuli (4, 5). Two MAPK nases, MKK4 and MKK7, and several MAPK kinase ki-nases are involved in JNK activation (4). Once activated, JNK phosphorylates and regulates the activity of several transcription factors, such as c-Jun and c-Myc, as well as nontranscription factors, such as members of the Bcl-2 family proteins (4, 6). Overwhelming evidence shows that JNK activity has a central role in regulating many cellular activities from cell cycle progression to apoptosis (2–4).
JNK activity has been suggested to play a context-depen-dent role in tumorigenesis (3). Several studies have linked JNK activity to tumor suppression. JNK1-null mice, which are deficient in JNK1 activity, exhibit enhanced sensitivity to skin tumorigenesis (7). Loss-of-function mutations in the MKK4 gene are found in∼5% of human tumors from a variety of tissues (8). A recent study shows that MKK4 and MKK7 activity suppresses metastasis by inhibiting the ability of disseminated tumor cells to colonize the lung (9). In contrast to studies that linked JNK activity to tumor sup-pression, other studies show protumorigenic roles for JNK activity, especially in malignant lymphocytes. For example, JNK1 activity is required in Bcr/Abl-mediated transforma-tion of pre-B cellsin vitro and in vivo (10). B-lymphoma and multiple myeloma cells constitutively express high levels of the activated form of JNK and inhibition of JNK activity with an anthrapyrazolone inhibitor, SP600125, or JNK-specific small interfering RNA (siRNA), or JNK-JNK-specific antisense oligonucleotides leads to cell cycle arrest and apoptosis (11, 12). Furthermore, it has been shown that on-cogenic kinase, nucleophosmin-anaplastic lymphoma ki-nase, promotes cell cycle progression through activation of JNK/c-Jun signaling in anaplastic large-cell lymphoma (13). The human T-cell lymphotropic virus oncoprotein Tax represses transforming growth factor-β1 signaling in human T cells via JNK/c-Jun hyperactivation, which may play a critical role in adult T-cell leukemia leukemogenesis (14). JNK activity is also required for vascular endothelial growth factor expression in cutaneous T-cell lymphoma (15). T-cell acute lymphoblastic leukemia (T-ALL) is repre-sented in only a small portion of hematologic malignancies Received 5/15/09; revised 9/15/09; accepted 10/8/09; published
OnlineFirst 12/8/09.
Grant support: National Natural Science Foundation of China grant 30973547 and 973 project 2010CB911904.
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 18 U.S.C. Section 1734 solely to indicate this fact.
Note: Supplementary material for this article is available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).
J. Cui and Q. Wang contributed equally to this work.
Requests for reprints: Jiyan Zhang or Jiannan Feng, Department of Molecular Immunology, Institute of Basic Medical Sciences, 27 Taiping Road, Beijing 100850, People's Republic of China. Phone: 86-10-68159436; Fax: 86-10-68159436.
E-mail: zhangjy@nic.bmi.ac.cn or fengjn@nic.bmi.ac.cn Copyright © 2009 American Association for Cancer Research. doi:10.1158/1535-7163.MCT-09-0408
but with very poor prognosis. The role of JNK activity in the oncogenic growth of T-ALL cells remains largely un-known. Here, we report that basal JNK activity, especially JNK1 activity, plays a pivotal role in the proliferation and survival of T-ALL cells via, at least partially, maintaining the protein levels of c-Myc and Bcl-2. These findings sug-gest that targeting JNK, especially JNK1 isoform, may have some important therapeutic implications in the treatment of T-ALL. Further exploration revealed that JNK protein and basal JNK activity in T-ALL cells showed aberrant subcellular localization, but no hyperactivation of JNK was observed. Thus, our work suggests that there might be novel mechanism(s) other than hyperactivation under-lying the protumorigenic role of JNK activity.
Materials and Methods
Reagents
JNK inhibitors SP600125 and AS601245 were purchased from Sigma and dissolved in DMSO. Whenever these inhi-bitors were used, DMSO was supplemented to keep concen-trations of DMSO (<0.1%) equal in all the wells. Antibodies against JNK, total JNK, JNK2, MKK7, phospho-p38, and phospho–extracellular signal-regulated kinase were from Cell Signaling Technology. Annexin V kit and antibodies against actin, JNK1, p38, phospho–c-Jun, c-Jun, c-Myc, Bcl-2, histone H1, and IκBα were from Santa Cruz Biotechnology. Antibodies against Bax, Bcl-XL, and Bim were from eBioscience. Antibody against human Fas (CH11) was obtained from BD Bioscience. Lipofectamine 2000 and neomycin were from Invitrogen.
Cells and Mice
The human T-ALL cell lines, CEM, HPB-ALL, HSB2, Jur-kat, Molt3, and Molt4, as well as two human B-lymphoma cell lines, Daudi and Raji, were gifts from Dr. Ben Chen (Division of Hematology and Oncology, Department of Internal Medicine and Karmanos Cancer Institute, Wayne State University School of Medicine) and were grown in RPMI 1640 containing 10% fetal bovine serum, 2 mmol/L L-glutamine, 100 units/mL penicillin, and 100μg/mL strep-tomycin. These cells were obtained and characterized in 1995 according to antigen/receptors expression. After that, the cells were frozen with multiple stocks until use in this work. For each cell line used in this work, cell stock was made in 5 to 7 days after resuscitation. The continuous pas-sage time was always kept within 1 to 2 months. Human peripheral blood mononuclear cells (PBMC) were isolated from the heparinized peripheral blood of healthy volunteers by density gradient centrifugations. C57BL/6 mice ages 4 to 6 weeks were purchased from the Institute of Experimental Animals, Academy of Chinese Medical Sciences. Normal T lymphocytes were isolated from human PBMCs or splenic cells of C57BL/6 mice with Pan T-cell isolation kits (Milte-nyi Biotech) according to the manufacturer's protocols.
Growth Assay and Soft-Agar Assay
The growth of T-ALL cells was measured by the MTT in-corporation assay. After the cells were cultured in 96-well plates for 68 h, 10μL MTT (5 mg/mL) was added to each
well. After 4 h, 100 μL of 10% SDS-10 mmol/L HCl were added to each well to dissolve the blue crystals of formazan. The samples were then measured atA570 nmusing an ELISA reader (Dynatech Laboratories). Soft-agar cloning assay was done as described previously (16). The cells were seeded in-to 6-well plates with a density of 5 × 103per well. On day 10, MTT incorporation was included to make the colonies visible. Each assay was repeated for three times.
Immunoblotting Analysis and Flow Cytometry The cells were treated under various conditions as indi-cated in the figure legends and were subjected to immuno-blotting analysis or cell cycle assay as described previously (17). To determine the extent of apoptosis, the cells were stained with FITC-conjugated Annexin V and propidium io-dide using the Annexin V kit according to the manufac-turer's protocol. Flow cytometry was carried out on a Becton Dickinson FACSCalibur machine (BD Biosciences).
Transfection of Plasmid and siRNAs into T-ALL Cells pcDNA3.1 MKK7-JNK1 was constructed according to a previous report (18) and verified by DNA sequencing. MKK7-JNK1 stable transfectants and mock control were generated by Lipofectamine 2000–mediated transfection of Jurkat cells with pcDNA3.1 MKK7-JNK1 and pcDNA3.1, respectively, and selected against neomycin (800 μg/mL). siRNAs that target human JNK1 and JNK2 mRNAs were designed based on nucleotides 1,013 to 1,031 (JNK1) and 461 to 479 (JNK2) relative to the translation start sites, respectively, and purchased from Dharmacon. Cells were transfected with siRNAs using Amaxa nucleofection kits according to the manufacturer's protocols.
Nuclear Cytoplasmic Fractionation
Cells were collected in 50 mmol/L glycerophosphate (pH 7.3), 1 mmol/L EDTA, 1 mmol/L EGTA, and 1 mmol/L DTT, centrifuged, and lysed in 40 mmol/L HEPES (pH 7.5), 5 mmol/L EGTA, 0.1% NP-40, 5 mmol/L MgCl2, 1 mmol/L DTT, 1 mmol/L sodium orthovanadate, and 1 mmol/L benzamide. The lysates were vortexed vigorously and centrifuged to obtain the cytoplasmic fraction as super-natant. Nuclei were resuspended in 50 mmol/L glyceropho-sphate (pH 7.3), 0.2 mmol/L EDTA, 420 mmol/L NaCl, 1.5 mmol/L MgCl2, 1 mmol/L DTT, and 25% glycerol, son-icated briefly on ice, vortexed, and centrifuged. The superna-tant was harvested as the nuclear fraction.
Immunofluorescence
Cells were dispersed on slides by cytospin, washed twice in PBS, fixed with 4% (w/v) paraformaldehyde in PBS for 10 min at room temperature, permeabilized with 0.5% Tri-ton X-100 in PBS for 15 min, and then treated with 0.1 mol/L glycine in PBS for 15 min. The nonspecific sites were blocked by incubation with 1% bovine serum albumin in PBS for 30 min at room temperature. Cells were then rinsed in PBS containing 0.05% Tween 20 for 5 min and incubated with mouse monoclonal antibody against human JNK1 di-luted in blocking buffer for 1 h at room temperature or over-night at 4°C. After being washed for three times in PBS containing 0.05% Tween 20, the cells were incubated with FITC-conjugated goat anti-mouse IgG for 45 min at room temperature. The cells were washed again as stated above,
incubated with 1μg/mL 4′,6-diamidine-2-phenylindole, and then observed under a laser scanning confocal micros-copy (RADIANCE 2100; Bio-Rad).
Results
JNK Inhibitors SP600125 and AS601245 Induce a Dose-Dependent Reduction in the Growth of T-ALL Cells
It has been reported that JNK activity contributes to the oncogenic growth of various malignant lymphocytes such as B-lymphoma cells. To investigate whether JNK activity plays a similar role in T-ALL cells, two human T-ALL cell lines, HPALL and Jurkat, as well as two human B-lymphoma cell lines, Daudi and Raji, were treated with varying concentrations of JNK selective inhibitor SP600125 (19). A dose-dependent reduction in the growth of human B-lymphoma cells, Daudi and Raji, was observed (Fig. 1A) as reported earlier (11). Under the same conditions, SP600125 showed more significant inhibitory effect on the growth of HPB-ALL and Jurkat cells (Fig. 1A). Consistently, soft-agar assay revealed that SP600125 suppressed the colony-forming ability of HPB-ALL and Jurkat cells more
significantly than Daudi and Raji cells in a dose-dependent manner (Fig. 1B). Besides HPB-ALL and Jurkat cells, SP600125 also suppressed the growth of other T-ALL cells, CEM, HSB2, Molt3, and Molt4 (Fig. 1C). Furthermore, another JNK inhibitor, AS601245 (20, 21), significantly sup-pressed the growth of T-ALL cells at the concentration of 10μmol/L (Fig. 1D). Because SP600125 and AS601245 in-hibited the phosphorylation of c-Jun, but not extracellular signal-regulated kinase or p38 MAPK (Supplementary Fig. S1), at the concentration of 10μmol/L they significantly suppressed the growth, the JNK inhibitors were efficient to inhibit JNK activity and showed selectivity against closely related kinases in T-ALL cells.
JNK Inhibitors Induce Cell Cycle Arrest and Apoptosis and Increased Sensitivity to Fas-Mediated Apoptosis in T-ALL Cells
Previous studies have revealed that the decreased growth of various tumor cells induced by inhibition of JNK activity was due to cell cycle arrest and/or apoptosis. Hence, we determined whether JNK inhibitors suppressed the growth of T-ALL cells via the same mechanism. Cell cycle assay revealed that HPB-ALL and Jurkat cells underwent G2-M arrest in the presence of either SP600125 or AS601245 Figure 1. JNK inhibitors induce a dose-dependent reduction in the growth of T-ALL cells.A,Daudi, Raji, HPB-ALL, and Jurkat cells were cultured for 72 h with various concentrations of SP600125. The growth of the cells was measured by MTT incorporation assay. Results were expressed as percentage of basal growth (mean ± SD of triplicate cultures) when compared with cells that were not treated with SP600125.B,Daudi, Raji, HPB-ALL, and Jurkat cells were cultured for 10 d in soft agar with indicated concentrations of SP600125. The colony-forming ability is calculated as the number of colonies in the treated group divided by the number of colonies in the control group times 100. Right, mean ± SD (n = 3); left, representative of three independent experiments.C,effect of SP600125 on the growth of CEM, HSB2, Molt3, and Molt4 cells was measured as described inA.D,effect of 10μmol/L AS601245 on the growth of Jurkat, HPB-ALL, CEM, HSB2, Molt3, and Molt4 cells was measured as described inA.
(Fig. 2A). Furthermore, HPB-ALL and Jurkat cells showed a significant increase in sub-G1 population, an indicator of apoptotic cells, with the treatment of JNK inhibitors (Fig. 2A). These data suggest that JNK inhibitors suppress the growth of T-ALL cells via cell cycle arrest and apoptosis. Impairment of Fas-mediated apoptosis has been sug-gested to contribute to tumorigenesis (22). Despite that JNK plays a proapoptotic role in response to various death stimuli, JNK activation is not required for Fas-mediated ap-optosis (23, 24). By contrast, it is reported that inhibition of JNK activity sensitizes some tumor cells to Fas-induced ap-optosis (25). Therefore, it is of importance to investigate whether it is the case in T-ALL cells. Apoptosis assay re-vealed that treatment of Jurkat cells with 50 ng/mL anti-Fas antibody (CH11) alone for 2 h induced up to ∼25% apoptosis (Fig. 2B). Pretreatment of Jurkat cells with 10μmol/L SP600125 for 12 h resulted in ∼12% apoptosis, whereas the basal level of apoptosis under the same condi-tion was∼4% (Fig. 2B). These data confirm that inhibition of
JNK activity induces apoptosis in T-ALL cells. Combination of SP600125 pretreatment and CH11 incubation led to∼56% apoptosis, a percentage much higher than the sum of 25% and 12% (Fig. 2B). Similar effects were also seen in CEM and Molt4 cells (Fig. 2B), suggesting that JNK activity acts to antagonize Fas-mediated apoptosis in T-ALL cells.
Weak Ectopic Expression of MKK7-JNK1 Fusion Protein, Which Shows Constitutive JNK Activity, in T - A L L C e l l s R e s u l t s i n A c c e l e r a t e d C e l l C y c l e Progression and Resistance to Fas-Mediated Apoptosis
If JNK activity indeed contributes to the oncogenic growth of T-ALL cells, selective activation of JNK in T-ALL cells should lead to accelerated cell cycle progression and resis-tance to Fas-mediated apoptosis. To test this scenario, Jurkat cells were stably transfected with a mammalian expression vector encoding MKK7-JNK1, because it has been shown that MKK7-JNK1 fusion protein linked via a short peptide can ex-press profound JNK activity in the absence of any stimulus (18). Immunoblotting analysis revealed weak expression of
Figure 2. JNK inhibitors induce cell cycle arrest and apoptosis and increased sensitivity to Fas-mediated apoptosis in T-ALL cells.A,HPB-ALL and Jurkat cells were treated with 10μmol/L SP600125 (SP) or AS601245 (AS) for 24 or 72 h or left untreated. Cell cycle assay was done on these cultured cells by propidium iodide staining. One representative of two independent experiments with very similar results is shown.B,after pretreatment with 10μmol/L SP600125 (Jurkat) or AS601245 (CEM and Molt4) for 12 h, Jurkat cells were treated with 50 ng/mL anti-human Fas antibody (CH11) for 2 h, CEM cells were treated with 150 ng/mL CH11 for 2 and 6 h, and Molt4 cells were treated with 250 ng/mL CH11 for 2 and 6 h. The cells were subjected to apoptosis assay. Right, mean ± SD (n = 3); left, representative of three independent experiments.
MKK7-JNK1 fusion protein in the two individual stable transfectants tested, Jurkat-y1 and Jurkat-y2 (Fig. 3A). As pected, Jurkat-y1 and Jurkat-y2 underwent faster growth, ex-hibited less G0-G1cells, and were resistant to Fas-mediated apoptosis compared with mock control (Fig. 3B-D). These da-ta further suggest that JNK activity contributes to the onco-genic growth of T-ALL cells.
Protein Levels of c-Myc and Bcl-2 Are Regulated by JNK Activity in T-ALL Cells
Several studies have shown that dysregulated expression of the oncoprotein c-Myc is critical for the development of T-ALL (26, 27), whereas the protein level of Bcl-2 determines the sensitivity to Fas-mediated apoptosis in various types of cells (28, 29). JNK activity has been shown to regulate the protein levels of c-Myc and Bcl-2 family members in certain cell context (3, 10, 11, 30). Therefore, it is of great importance to investigate whether levels of c-Myc and Bcl-2 family members in T-ALL cells are affected by variation of JNK ac-tivity. Immunoblotting analysis revealed that treatment of HPB-ALL and Jurkat cells with 10 μmol/L SP600125 for 12 to 24 h led to a significant reduction in Bcl-2 and c-Myc protein levels (Fig. 4A). Under the same conditions, SP600125 showed much weaker effect on the protein levels of Bax, Bcl-XL, and Bim (Fig. 4A, note that Bim was unde-tectable in HPB-ALL cells). Reduction in the protein levels of Bcl-2 and c-Myc was also observed in the presence of 10μmol/L AS601245 (Fig. 4B). By contrast, Bcl-2 and
c-Myc levels were increased in Jurkat-y1 and Jurkat-y2 cells compared with mock control (Fig. 4C), which was reversed by 10μmol/L SP600125 and AS601245 (Fig. 4D). The con-centration (10μmol/L) at which JNK inhibitors led to reduced protein levels of Bcl-2 and c-Myc is associated with significant inhibition of growth (Fig. 1), significant inhibition of JNK activity, and selectivity against closely related kinases in T-ALL cells (Supplementary Fig. S1). Taken together, these data suggest that the protein levels of c-Myc and Bcl-2 are regulated by JNK activity in T-ALL cells. siRNA against JNK1, but not JNK2, Exhibits Similar Effects to JNK Inhibitors
In mammals, there are three JNK genes,Jnk1, Jnk2, and Jnk3, with different splicing forms (p54 and p46; refs. 2–4). JNK1 and JNK2 are ubiquitously expressed, whereas expression of JNK3 is restricted to brain, heart, and testis (2–4). To test which isoform plays a major role in the protu-morigenic effects of JNK activity in T-ALL cells, knockdown of individual kinases, JNK1 and JNK2, was pursued. By us-ing different antibodies that specifically recognize either JNK1 or JNK2, we were able to show successful silencing of JNK1 and JNK2 specifically and independently in CEM cells (Fig. 5A). Reduction in the protein levels of JNK1 or JNK2 resulted in significant less p46 JNK activity and p54 JNK activity, respectively (Fig. 5A). JNK1 siRNA, but not JNK2 siRNA, led to reduced protein levels of Bcl-2 and c-Myc (Fig. 5A). Consistently, there was significant growth
Figure 3. Weak ectopic expression of MKK7-JNK1 fusion protein in Jurkat cells results in accelerated cell cycle progression and resistance to Fas-mediated apoptosis.A,lysates prepared from two individual MKK7-JNK1 stable transfectants (Jurkat-y1 and Jurkat-y2) and mock control were subjected to immunoblotting (IB) to detect the expression of MKK7-JNK1 fusion protein with antibody against MKK7. The same cell lysates were used to detect the expression of actin.B,Jurkat-y1 and Jurkat-y2 cells and mock control were seeded into 6-well plates at a density of 1 × 105/mL. Cell proliferation was determined by counting viable cells every 24 h.C,cell cycle distribution of Jurkat-y1 and Jurkat-y2 cells and mock control was determined by propidium iodide staining. Right, mean ± SD percentages of G0-G1cells (n = 3); left, representative of three independent experiments. *, P < 0.05 (Student’s t test). D,Jurkat-y1 and Jurkat-y2 cells and mock control were treated with or without 50 ng/mL CH11 for 6 h. The cells were subjected to apoptosis assay. Bottom, mean ± SD (n = 3); top, representative of three independent experiments.
inhibition in CEM cells transfected with JNK1 siRNA but not JNK2 siRNA (Fig. 5B). The growth inhibition was accompanied by an increase in G0-G1 phase and slightly more basal apoptosis (Fig. 5C and D). Knockdown of JNK1, but not JNK2, also augmented Fas-mediated apopto-sis (Fig. 5D). Similar growth-inhibitory and proapoptotic effects of JNK1 siRNA, but not JNK2 siRNA, were also observed in Jurkat cells (Supplementary Fig. S2). Thus, our data suggest that JNK1, but not JNK2, is the major player in the protumorigenic role of JNK activity in T-ALL cells.
JNK Protein and Basal JNK Activity in T-ALL Cells Show Aberrant Subcellular Localization, but No Hyperactivation of JNK Is Observed
Hyperactivation of JNK has been found in various malig-nant lymphocytes and inhibition of JNK activity leads to cell cycle arrest and apoptosis. Now that JNK activity also con-tributes to the oncogenic growth of T-ALL cells, it is reason-able to suppose that T-ALL cells exhibit higher levels of JNK activity than primary T cells. To test this scenario, HPB-ALL and Jurkat cells were serum-starved for 24 h, and the cells were stimulated with 20% fetal bovine serum for 15 min or left untreated. The levels of JNK phosphorylation were com-pared with those in human PBMC T cells. Immunoblotting analysis revealed that both HPB-ALL and Jurkat cells exhib-ited detectable levels of basal JNK phosphorylation even af-ter serum starvation, and fetal bovine serum treatment for 15 min significantly activated JNK in these cells (Fig. 6A), consistent with the finding that JNK activity contributes to the oncogenic growth of T-ALL cells. However, the levels of
JNK phosphorylation in both serum-starved and fetal bo-vine serum–stimulated HPB-ALL and Jurkat cells were sur-prisingly lower than in human PBMC T cells (Fig. 6A). Furthermore, the basal JNK activity in all the six T-ALL cell lines used in this work was lower than in murine splenic T cells and murine thymocytes despite that all the T-ALL cells expressed detectable levels of the activated form of JNK (Fig. 6B; data not shown). These data are contradictory to our hypothesis and suggest that JNK activity contributes to the oncogenic growth of T-ALL cells independent of hyperactivation.
JNK phosphorylates a broad spectrum of substrates dis-tributed throughout different subcellular compartments (31). The balance among these site-specific components of JNK signaling determines the biological outcomes (31). Because JNK activity contributes to the oncogenic growth of T-ALL cells via maintaining the protein levels of c-Myc and Bcl-2, it most likely does so in the nucleus by regulating transcription. Therefore, the basal JNK activity in the nucle-us is a more important factor. To compare the subcellular distribution of basal JNK activity between primary T cells and T-ALL cells, nuclear cytoplasmic fractionation was ap-plied to human PBMC T cells, Jurkat cells, and Molt4 cells. It is found that the basal JNK activity in primary T cells exhib-ited exclusive cytoplasm localization, whereas T-ALL cells showed aberrant nuclear accumulation of basal JNK activity (Fig. 6C). The discrepancy might result from the different subcellular distribution of JNK protein in primary T cells and T-ALL cells because JNK protein in primary T cells Figure 4. Protein levels of c-Myc and Bcl-2 are regulated by JNK activity in T-ALL cells.AandB,HPB-ALL and Jurkat cells were treated with 10μmol/L SP600125 (A) or AS601245 (B) for various periods or left untreated. The expression of Bcl-2, Bax, Bcl-XL, Bim, c-Myc, and actin was determined by immunoblotting. REL, relative expression level.C,expression of Bcl-2, c-Myc, and actin in Jurkat-y1 and Jurkat-y2 cells and mock control was determined by immunoblotting.D,Jurkat-y1 and Jurkat-y2 cells were treated with 10μmol/L SP600125 or AS601245 or DMSO of equal volume for 24 h. The expression of Bcl-2, c-Myc, and actin was determined by immunoblotting.
was cytoplasmic, whereas JNK protein in T-ALL cells was both nuclear and cytoplasmic (Fig. 6C). Immunofluores-cences confirmed the aberrant subcellular localization of JNK protein in all the six T-ALL cell lines used in this work, in contrast to the exclusive cytoplasm localization of JNK protein in both murine splenic T cells and human PBMC T cells (Fig. 6D; Supplementary Fig. S3). Taken together, our data suggest that JNK activity contributes to the oncogenic growth of T-ALL cells via aberrant subcellular localization but not hyperactivation.
Discussion
Hyperactivation of JNK has been found in various malig-nant lymphocytes. As for T-ALL cells, however, it is appar-ently not the case. In this work, T-ALL cells were found to express detectable levels of basal JNK phosphorylation. However, the basal JNK activity in all the six T-ALL cell lines tested was surprisingly lower than in primary T cells. An intriguing fact is that the weak basal JNK activity was
essential for survival and proliferation of T-ALL cells. Thus, hyperactivation is not a prerequisite for JNK activity to ex-hibit a protumorigenic role. There might be novel mecha-nism(s) other than hyperactivation underlying the protumorigenic role of JNK activity.
Our data revealed that the basal JNK activity in primary T cells exhibited exclusive cytoplasm localization, whereas T-ALL cells showed aberrant nuclear accumulation of basal JNK activity. The discrepancy might result from the differ-ent subcellular distribution of JNK protein in primary T cells and T-ALL cells because JNK protein in primary T cells was cytoplasmic, whereas JNK protein in T-ALL cells was both cytoplasmic and nuclear. Although some published data re-vealed the importance of JNK nuclear localization for the induction of cell death in certain cell context (32, 33), accu-mulation of basal JNK activity in the nucleus was seen in tumor cells (34, 35). Our preliminary data (Supplementary Fig. S4) also showed more nuclear entry of JNK protein in malignant B cells compared with its normal counterparts. Meanwhile, it has been established that JNK plays a
protu-Figure 5. JNK1 siRNA, but not JNK2 siRNA, shows growth-inhibitory and proapoptotic effects in CEM cells. CEM cells were transfected with JNK1 siRNA or JNK2 siRNA or the nontargeting negative control siRNA and cultured for 48 h.A,cell lysates were prepared and phosphorylation of JNK and expression of JNK1, JNK2, actin, Bcl-2, and c-Myc were determined by immunoblotting. Ctrl, control; P-JNK, phospho-JNK.B,growth of these cells was measured as described in Fig. 1A.C,cell cycle assay was done by propidium iodide staining. The percentages of G0-G1cells are shown as mean ± SD (n = 3).D,cells were treated with 150 ng/mL CH11 for 2 h or left untreated. The cells were subjected to apoptosis assay. Right, mean ± SD (n = 3); left, representative of three independent experiments.
morigenic role in malignant B cells. These observations sug-gest a protumorigenic role for basal nuclear JNK activity in tumor cells. T-ALL cells might make use of JNK aberrant subcellular localization to maintain enough JNK activity in the nucleus for oncogenic growth under the condition that there is no hyperactivation of JNK. It is of importance to elu-cidate whether translocation of JNK to the nucleus plays any role in cell cycle progression and resistance to apoptosis. However, little is known about the mechanism of JNK local-ization (36). It is not possible yet to specifically block the translocation of JNK to the nucleus to show whether the ab-errant subcellular localization of JNK protein and basal JNK activity in T-ALL cells contribute to the protumorigenic role of JNK. Further studies are required to clarify these issues.
Nevertheless, T-ALL cells retained some JNK activity, and our data show that JNK activity in a low range contributed to the survival and proliferation of T-ALL cells in a dose-dependent manner. These findings suggest that targeting
JNK may have some important therapeutic implications in the treatment of T-ALL. For this purpose, an important point to consider is the role of JNK isoforms and/or their ratio. Different JNK isoforms possess structural and bio-chemical similarities. All of them contribute to JNK activity and have many overlapping functions (2–4). However, JNK1 and JNK2 have recently been shown to exhibit some differences in their substrate specificities and gene targets (3, 4, 37). Our data suggest that, in T-ALL cells, phosphor-ylation of p46 JNK mainly comes from JNK1, whereas phos-phorylation of p54 JNK mainly comes from JNK2. JNK1 siRNA, but not JNK2 siRNA, showed growth-inhibitory and proapoptotic effects in T-ALL cells, although the effects of JNK1 siRNA were weak compared with JNK inhibitors because of partial knockdown. These data further support a protumorigenic role for JNK activity in T-ALL cells and suggest that targeting JNK should focus on JNK1, but not JNK2, for the treatment of T-ALL.
Figure 6. JNK protein and basal JNK activity in T-ALL cells show aberrant subcellular localization, but no hyperactivation of JNK is observed.A,human PBMC T cells were purified. HPB-ALL and Jurkat cells were serum-starved for 24 h, and the cells were stimulated with 20% fetal bovine serum for 15 min or left untreated. Phosphorylation of JNK and expression of JNK and actin were determined by immunoblotting.B,cell lysates were prepared from growing T-ALL cell lines and purified murine splenic T cells. Phosphorylation of JNK and expression of JNK and actin were determined by immunoblotting. ns, nonspecific band.C,nuclear cytoplasmic fractionation was applied to purified human PBMC T cells, Jurkat cells, and Molt4 cells. The subcellular distri-bution of phospho-JNK and JNK was determined by immunoblotting with IκBα as cytoplasm (C) marker and histone H1 as nucleus (N) marker.D,purified human PBMC T cells, Jurkat cells, and Molt4 cells were stained with JNK1-specific antibody, which was then revealed by a FITC-conjugated secondary antibody (left). Nuclei were counterstained for DNA by 4′,6-diamidine-2-phenylindole (middle). Right, merged image.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.Acknowledgments
We thank Ming Yu and Dr. Zhiyi Zhang for technical assistance. References
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