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1 November 2001, Volume 20, Number 50, Pages 7386-7397
Table of contents    Previous  Article  Next   [PDF]
Original Paper
Expression of the oncogenic NPM-ALK chimeric protein in human lymphoid T-cells inhibits drug-induced, but not Fas-induced apoptosis
Catherine Greenland1, Christian Touriol1, Grégory Chevillard1, Stephan W Morris2, Renyuan Bai3, Justus Duyster3, Georges Delsol1 and Michèle Allouche1

1CNRS-UPCM, UPR 2163, CHU Purpan, 31059 Toulouse Cedex 03, France

2Department of Pathology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA

3Laboratory of Leukemogenesis, Dept. of Internal Medicine III, Technical University of Munich, Munich, Germany

Correspondence to: M Allouche, CNRS-UPCM, UPR2163, CHU Purpan, avenue de Grande Bretagne, 31059 Toulouse Cedex 03, France; E-mail: allouche@cict.fr


Anaplastic large cell lymphomas (ALCLs) are frequently associated with the t(2;5)(p23;q35) translocation, leading to the expression of NPM-ALK, a fusion protein linking nucleophosmin and anaplastic lymphoma kinase, a receptor tyrosine kinase. In ALCLs, dimerization of NPM-ALK leads to constitutive autophosphorylation and activation of the kinase, necessary for NPM-ALK oncogenicity. To investigate whether NPM-ALK, like other oncogenic tyrosine kinases, can inhibit drug-induced apoptosis, we permanently transfected NPM-ALK into Jurkat T-cells. As in ALCLs, NPM-ALK was expressed as a constitutively kinase-active 80 kDa protein, and could be detected by immunocytochemistry in nucleoli, nuclei and cytoplasm. Doxorubicin-induced apoptosis (assessed by cell morphology and annexin V-FITC binding) was significantly inhibited in two independent NPM-ALK-expressing clones (5.2±1.8 and 7.5±0.8% apoptosis), compared to control vector-transduced cells (36±6.7%). Similar results were observed with etoposide. In contrast, Fas-induced apoptosis was not inhibited. Cytochrome c release into the cytosol was delayed in doxorubicin-, but not anti-Fas-treated transfectant cells, indicating that apoptosis inhibition occurred upstream of mitochondrial events. Using NPM-ALK mutants, we demonstrated that inhibition of drug-induced apoptosis: (1) requires functional kinase activity, (2) does not involve phospholipase C-bold gamma, essential for NPM-ALK-mediated mitogenicity and (3) appears to be phosphoinositide 3-kinase independent, despite a strong Akt/PKB activation observed in wild type NPM-ALK-expressing cells. These results suggest that the NPM-ALK antiapoptotic and mitogenic pathways are distinct. Oncogene (2001) 20, 7386-7397.


anaplastic large cell lymphoma; ALK; tyrosine kinase; chemotherapy; apoptosis


Anaplastic large cell lymphomas (ALCLs) are lymphoid tumors of T or null phenotype expressing the CD30 antigen, which affect mostly children (Stein et al., 1985; Delsol et al., 1988; Lamant et al., 1996). A characteristic feature is their frequent association with the (2;5)(p23;q35) chromosomal translocation, fusing the ALK (anaplastic lymphoma kinase) gene at 2p23 and the nucleophosmin (NPM) gene at 5q35 (Lamant et al., 1996). The t(2;5) results in the expression of NPM-ALK, a fusion protein formed by the N-terminal region of NPM, a constitutively expressed nucleolar phosphoprotein (Chan et al., 1989; Borer et al., 1989), and the C-terminal region of ALK, containing a kinase domain (Morris et al., 1994). Molecular cloning of the ALK cDNA revealed that it encodes a receptor of the insulin receptor family, composed of an extracellular putative ligand-binding domain, a transmembrane segment and an intracellular region with tyrosine kinase activity. Interestingly, ALK is specifically expressed in the nervous system, especially during development, but never in lymphoid cells (Iwahara et al., 1997; Morris et al., 1997). In ALCL cells, dimerization of NPM-ALK (through the oligomerization domain of NPM) leads to the constitutive autophosphorylation, and thus activation, of the kinase (Bischof et al., 1997).

The constitutive activation of tyrosine kinases often leads to oncogenicity due to deregulated cell signaling (Ullrich and Schlessinger, 1990; Korsmeyer, 1992; Porter and Vaillancourt, 1998). The most common mechanism for activation of tyrosine kinases is fusion of the catalytic domain of the tyrosine kinase to an N-terminal partner that contains a dimerization or oligomerization domain as a consequence of balanced chromosomal translocations. For example, the t(9;22)(q34;q11) translocation that gives rise to the BCR-ABL fusion protein associated with chronic myeloid and acute lymphoblastic leukemias, activates the intracellular kinase ABL due to BCR-mediated oligomerization (reviewed in Warmuth et al., 1999; Chopra et al., 1999). Similar mechanisms may involve receptor tyrosine kinases, such as the receptors for platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF), to generate the leukemogenic fusion proteins TEL-PDGFR (Golub et al., 1994), HIP1-PDGFR (Ross and Gilliland, 1999), ZNF198-FGFR (Reiter et al., 1998; Xiao et al., 1998), FIM-FGFR1 (Ollendorff et al., 1999), and FOP-FGFR1 (Popovici et al., 1999). Similarly, the (2;5) translocation of ALCL generates the chimeric protein NPM-ALK, activating the ALK receptor tyrosine kinase and leading to cell transformation, as shown both in vitro and in vivo (Fujimoto et al., 1996; Bischof et al., 1997; Wellmann et al., 1997; Kuefer et al., 1997).

Increased or uncontrolled cell proliferation is required for lymphomagenesis, but tumor growth can also result from a decrease in the rate of cell death. Illustrating this latter point, McGahon et al. (1994) and Amarante-Mendes et al. (1998) suggested that the BCR-ABL activated kinase mediates resistance to apoptosis induced by cytotoxic drugs in chronic myeloid leukemia cells. In the present study, we asked whether the expression of NPM-ALK in lymphoid cells could protect these cells against apoptosis. Thus, we chose the human lymphoblastic T-cell line Jurkat transfected with NPM-ALK cDNA as a model, because ALCL most often present a T-cell phenotype. Our results demonstrate that stable expression of NPM-ALK in Jurkat cells leads to a significant inhibition of apoptosis induced by two antineoplastic drugs, doxorubicin and etoposide, but not by an anti-Fas antibody. In experiments with NPM-ALK mutants, we show that an intact ATP-binding site, thus a functional ALK tyrosine kinase, is required for this inhibition. In contrast, mutating the Y664 binding site of phospholipase C-gamma (PLC-gamma), involved in NPM-ALK-mediated mitogenicity (Bai et al., 1998), does not impair this antiapoptotic effect.

Because ALK is a receptor tyrosine kinase with high homology to LTK (leukocyte tyrosine kinase), we hypothesized that NPM-ALK, like LTK, might activate phosphoinositide 3-kinase (PI3K) (Kozutsumi et al., 1994; Fujimoto et al., 1996). A widely distributed isoform of PI3K is composed of a catalytic (p110) subunit and a regulatory (p85) subunit. The latter binds, through its src homology 2 (SH2) domain, to the phosphorylated tyrosine residues of tyrosine kinase receptors (Leevers et al., 1999). As PI3K has been involved in cell signaling of both proliferation and survival by receptor tyrosine kinases (Roche et al., 1994; Yao and Cooper, 1995), we presently investigated the role of PI3K signaling in the antiapoptotic effect of NPM-ALK. Our results suggest that NPM-ALK activates PI3K, as indicated by the phosphorylation of Akt/PKB (protein kinase B), a downstream effector of PI3K that has been implicated in survival signaling (reviewed by Downward (1998)), but that PI3K activation is not necessary for the inhibition of doxorubicin-induced apoptosis.


Characterization of NPM-ALK expression in transfected Jurkat cells

Jurkat T-lymphoid cells, transfected with NPM-ALK (designated JNA cells) or with control vector (Jurkat/neo cells), were selected with geneticin for stable gene expression. Immunohistochemical analysis with the ALK-specific monoclonal antibody (mAb) ALK1 revealed a strong staining of nucleoli, and a weaker nuclear and cytoplasmic staining, in all JNA cells (Figure 1b). This cellular localization is also found in patients' ALCL cells (Pulford et al., 1997). We also noted a very strong labeling of all three cellular compartments in approximately 10% of the cells (Figure 1b). In contrast, no ALK positivity was observed in Jurkat/neo (Figure 1a), or parental Jurkat cells (not shown). To further characterize the protein expressed in JNA cells, a Western blot was performed using another anti-ALK mAb, ALKc. A protein band of approximately 80 kDa was detected in two JNA clones, F2.3 and F2.6, consistent with the size of NPM-ALK expressed in the t(2;5)-positive ALCL cell line, SU-DHL1 (Figure 1c). Because of the weak cytoplasmic staining, we thought important to estimate the relative amount of NPM-ALK protein in the various cellular compartments. Western blotting revealed equal amounts of ALK-reactive protein in the nuclear (including nucleoli) and cytoplasmic fractions of JNA cells (Figure 1d). Neither Jurkat nor Jurkat/neo cells expressed the protein. Thus, JNA cells stably expressed the NPM-ALK fusion protein, although at substantially lower levels than in SU-DHL1 cells (Figure 1c).

NPM-ALK expressed in Jurkat cells is phosphorylated on tyrosine residues

In ALK-positive ALCL tumors, NPM-ALK is constitutively activated through dimerization and autophosphorylation of the ALK tyrosine kinase domain (Bischof et al., 1997). We therefore investigated whether NPM-ALK expressed in Jurkat cells is likewise tyrosine phosphorylated. Immunoprecipitation of NPM-ALK from JNA F2.3 and F2.6 cells with ALK1 mAb, followed by Western blot analysis with antiphosphotyrosine antibody 4G10, revealed that NPM-ALK was phosphorylated on tyrosine residues in vivo, exactly as in the t(2;5)-positive ALCL cell line, SU-DHL1. Tyrosine phosphorylated NPM-ALK was also detected on a total lysate of JNA F2.3 cells blotted with 4G10 mAb. As expected, no ALK-reactive protein could be immunoprecipitated from Jurkat/neo cells (Figure 2a).

NPM-ALK expressed in Jurkat cells has a functional kinase activity

The finding that NPM-ALK is tyrosine phosphorylated in JNA cells suggests that this modification results, mostly, from autophosphorylation due to a functional ALK kinase domain. This was demonstrated by incubating anti-ALK immunoprecipitates from NPM-ALK-expressing clones with [gamma-32P]ATP. As shown in Figure 2b, the 80 kDa NPM-ALK protein immunoprecipitated from JNA F2.3 and F2.6 cells was phosphorylated, revealing in vitro autokinase activity. As expected, no ALK1-reactive protein could be immunoprecipitated from Jurkat/neo and parental cells, and no signal was detected in the kinase assay.

Drug-induced apoptosis is decreased in NPM-ALK expressing Jurkat cells

We next investigated the effect of NPM-ALK expression in Jurkat cells on apoptosis induced by two antineoplastic drugs currently used in the treatment of ALCL, doxorubicin and etoposide. Parental Jurkat cells, Jurkat/neo cells and NPM-ALK-expressing clones JNA F2.3 and F2.6 were treated with 1 muM doxorubicin, 10 muM etoposide or culture medium for 14 or 18 h. Apoptosis was evaluated by a flow cytometric assay in which annexin V-FITC-positive cells, that continued to exclude propidium iodide, were quantified. No spontaneous apoptosis was observed in any of the cell lines incubated with culture medium in the absence of drug (not shown). As shown in Figure 3a, doxorubicin treatment of JNA F2.3 and F2.6 clones for 14 h induced 7.5±0.8 and 5.2±1.8% annexin-positive cells, respectively, as opposed to 36±6.7% in Jurkat/neo cells. This decrease of apoptosis was highly statistically significant (P<0.001 for both clones). Similarly etoposide-induced apoptosis was significantly reduced in NPM-ALK-expressing cells (27.8±2.1% annexin-positive cells for clone F2.3, 17.6±0.7% for clone F2.6), compared to control Jurkat/neo cells (41.8±3.0%) (P<0.01 and P<0.001 for F2.3 and F2.6, respectively) (Figure 3c). After 18 h of treatment with either drug, a smaller, yet significant difference in the percentage of annexin-positive cells between NPM-ALK-expressing and Jurkat/neo cells was still observed (P<0.05-0.001, Figure 3a; P<0.01-0.001, Figure 3c). The significant inhibition of drug-induced apoptosis after 18 h of incubation was confirmed for both doxorubicin and etoposide by enumeration of apoptotic cells following May-Grünwald-Giemsa (Figure 3b,d, P<0.05-0.01) or Hoechst 33348 and propidium iodide (not shown) staining.

In order to more precisely define this decrease of drug-induced apoptosis, we performed time- and dose-dependent studies. Figure 3e shows that the inhibition of doxorubicin-induced apoptosis in NPM-ALK-expressing cells was clearly dose-dependent. In contrast, the kinetics of drug-induced apoptosis could not be precisely followed, as significant apoptosis induced by either drug was not detectable before 14 h of incubation, and after 24 h, most control (Jurkat/neo) cells had died from (post-apoptotic) necrosis. As shown in Figure 3a,c, the proportion of NPM-ALK-expressing apoptotic cells increased with time, and cells eventually died after 72 h of culture in the presence of drugs (not shown).

Fas-induced apoptosis is not modulated by NPM-ALK expression in Jurkat cells

Fas-induced apoptosis, initiated by interaction with its ligand, is a physiological process that downregulates immune responses and is involved in T-cell-mediated cytotoxicity (Nagata and Golstein, 1995). Recent controversial reports have suggested that apoptosis induced by antitumor drugs can be mediated through Fas-Fas ligand interactions, at least in some cell types (Friesen et al., 1996; Scaffidi et al., 1998). In order to determine whether NPM-ALK expression could also decrease Fas-induced apoptosis, we performed a time-dependent analysis, using both annexin V-FITC binding and cell morphology. As shown in Figure 3f,g, treatment with the anti-Fas mAb CH11 induced similar levels of apoptosis in NPM-ALK-transfected and control Jurkat/neo cells, at all times analysed (from 6 to 19 h of treatment). The differences observed between JNA clones and control Jurkat/neo or parental cells were not significant. Thus, NPM-ALK expression does not inhibit Fas-induced apoptosis in Jurkat cells.

NPM-ALK decreases cytochrome c release into the cytosol of doxorubicin-, but not Fas-treated Jurkat cells

A number of recent reports underline the central role of mitochondria in the cellular apoptotic machinery through release of cytochrome c and subsequent activation of caspases (reviewed by Green and Reed, 1998; Kroemer et al., 1998). In drug-induced apoptosis, accumulating evidence shows that cytochrome c release from mitochondria into the cytosol is an early event preceeding the activation of caspases (Sanchez-Alcazar et al., 2000). In contrast, in most cells, Fas and other death receptors directly activate caspases-8 and -3 via a mitochondria-independent pathway. In Jurkat cells however, Fas ligation can activate both the mitochondria-dependent and independent apoptotic pathways (Scaffidi et al., 1998, 1999).

To elucidate the stage of the apoptotic pathway inhibited by NPM-ALK, we analysed the presence of cytochrome c in mitochondrial and cytosolic fractions from control (Jurkat/neo) and NPM-ALK-expressing (JNA F2.3) cells after 3 and 6 h of incubation with either doxorubicin or anti-Fas mAb. Figure 3h shows that the amount of cytochrome c released into the cytosol was decreased in doxorubicin-, but not anti-Fas-treated NPM-ALK-expressing cells compared to Jurkat/neo cells after 6 h of treatment. Identical findings were observed as soon as 3 h post induction (not shown). This result suggests that NPM-ALK-mediated inhibition of drug-induced apoptosis occurs upstream of the mitochondrial apoptotic events.

An intact ATP-binding site is necessary for NPM-ALK-mediated inhibition of drug-induced apoptosis

As we have shown above, the NPM-ALK kinase is functional in transfected Jurkat cells. To determine whether NPM-ALK-associated inhibition of drug-induced apoptosis is dependent on its kinase activity, Jurkat cells were stably transfected with a cDNA encoding NPM-ALK(K210R), a kinase-defective mutant, that has a mutated ATP-binding site (Bischof et al., 1997) (Table 1). NPM-ALK(K210R)-expressing cells were treated with 1 muM doxorubicin or 10 muM etoposide for 18 h. In contrast to cells transfected with wild-type NPM-ALK, NPM-ALK(K210R)-expressing cells showed no statistically significant decrease in the percentage of annexin V-positive cells as compared to control Jurkat/neo cells (Figure 4a,b). This loss of antiapoptotic activity was not due to the relatively low level of expression of the (K210R) mutant (Figure 4c). Indeed an uncloned JNA cell population (JNA pool), displaying strong antiapoptotic activity (Figures 3e and 4a), had a similar level of NPM-ALK expression as (K210R)-expressing cells (Figure 4c). These results strongly suggest that a functional ALK kinase domain is necessary for NPM-ALK-mediated antiapoptotic effects.

The tyrosine 664 PLC-italic gamma-binding site is not necessary for NPM-ALK-mediated inhibition of drug-induced apoptosis

It has previously been shown that mutation of tyrosine 664 of NPM-ALK to phenylalanine abrogates: (1) the association of PLC-gamma with NPM-ALK, (2) the subsequent activation of PLC-gamma by tyrosine phosphorylation, and (3) the cellular transforming effects of NPM-ALK (Bai et al., 1998) (Table 1). To determine the potential involvement of PLC-gamma in the antiapoptotic effects of NPM-ALK, Jurkat cells were permanently transfected with the NPM-ALK(Y664F) mutant, and treated with doxorubicin or etoposide for 18 h. Apoptosis was significantly reduced in NPM-ALK(Y664F)-transfected cells as compared to Jurkat/neo control cells, as in wild type NPM-ALK-expressing cells (Figure 4a,b). The level of NPM-ALK expression in (Y664F)-transfected cells was similar to that in the JNA (wild type) clone F2.3 (Figure 4c). This result suggests that the tyrosine 664 PLC-gamma binding site is not necessary for transduction of antiapoptotic signals by NPM-ALK in Jurkat lymphoid cells.

PI3K is not involved in NPM-ALK-mediated inhibition of drug-induced apoptosis despite a strong Akt activation in transfected Jurkat cells

It is now well established that PI3K is involved in survival signaling cascades (reviewed by Downward (1998)), and is often found in association with oncogenic tyrosine kinases, such as BCR-ABL (Skorski et al., 1995). To investigate the potential role of PI3K in NPM-ALK-mediated apoptosis inhibition, we used two NPM-ALK mutants (Table 1). NPM-ALK tyrosine 418 is a potential autophosphorylation site comprised within a consensus binding motif for the PI3K p85 SH2-domain (pYXXM, here pY418RIM). Mutation of this tyrosine (Y418F mutant, Table 1) did not abolish the inhibitory activity of NPM-ALK on drug-induced apoptosis (Figure 4a for doxorubicin and 4b for etoposide). Insulin receptor substrate-1 (IRS-1) binds to tyrosine 156 of NPM-ALK (Fujimoto et al., 1996). Because IRS-1 has been shown to recruit the p85 subunit of PI3K (Backer et al., 1992), we speculated that NPM-ALK may activate PI3K by recruiting IRS-1. However, doxorubicin-induced apoptosis was significantly inhibited (P<0.05) in cells expressing an NPM-ALK double mutant of the IRS-1- and p85-binding sites (Y156F-Y418F), as efficiently as in wild type NPM-ALK-expressing (JNA pool or F2.3 clone) cells (Figure 4a). This first result suggested a non-involvement of PI3K in our system. However, it has recently been shown, in transfected murine BaF3 cells, that the mutation of tyrosines 418 and/or 156 does not abolish NPM-ALK association with p85 (Bai et al., 2000). Thus these two mutants could still be capable of recruiting and thereby activating PI3K.

To investigate this possibility, we examined Akt activation in Jurkat cells transfected with wild type and mutant forms of NPM-ALK. Indeed Akt has been described as a downstream effector of PI3K in a number of models that involve activation of PI3K in survival signaling (reviewed by Downward (1998)). As shown in Figure 5a, we found a strong Akt phosphorylation in wild type NPM-ALK-expressing cells, which was abrogated by the specific PI3K inhibitor LY294002, demonstrating that this activation was PI3K-dependent. In addition, Akt was phosphorylated in the (Y418F) and (Y156F-Y418F) mutants (Figure 5a), confirming the fact that these point mutations are not sufficient to abolish the association and consequent activation of PI3K by NPM-ALK.

In order to determine whether inhibition of PI3K could reverse the antiapoptotic phenotype of NPM-ALK-expressing cells, cells were incubated with 25 muM LY294002, prior to drug treatment. Unexpectedly, LY294002 significantly increased doxorubicin-induced apoptosis, to the same extent, in all cell lines tested, i.e., vector-, wild type and mutant NPM-ALK-expressing Jurkat cells (P<0.05 or less for all cell lines, Figure 5b), indicating that this effect was not specifically due to NPM-ALK. It should also be noted that the increasing effect of LY294002 on doxorubicin-induced apoptosis was roughly additive: in JNA pool cells for example, doxorubicin and LY294002 separately induced 16.9±0.3 and 4.1±0.3% annexin-positive cells, respectively, while the combination of both agents induced apoptosis in 25.9±1.0% of these cells (Figure 5b).

Taken together, our results suggest that NPM-ALK activates PI3K (as indicated by Akt phosphorylation), but that PI3K is not involved in the inhibition of doxorubicin-induced apoptosis.


In recent years, it has become clear that apoptosis is an important mechanism of cell death initiated by anticancer drugs in chemosensitive malignant cells (Hickman, 1992; Hannun, 1997). The purpose of this study was to determine whether the expression of NPM-ALK, the fusion protein associated with the t(2 ;5) in ALCL, could alter apoptotic responses in human lymphoid cells. In this report, we show for the first time that NPM-ALK, a lymphoma-associated oncogenic tyrosine kinase, can delay apoptosis induced by anticancer drugs, but not by anti-Fas, in human lymphoid cells.

The stable expression of NPM-ALK in transfected Jurkat cells was confirmed using specific anti-ALK antibodies through Western blotting and immunocytochemistry. We found that the localization of NPM-ALK in JNA cells was similar to that observed in ALCL (Pulford et al., 1997): all the transfected cells displayed strong nucleolar ALK positivity and a weaker nuclear and cytoplasmic staining. In ALCL cells, these subcellular localizations are explained by the fact that the N-terminal portion of NPM present in the fusion lacks a nuclear localization signal but has an intact oligomerization domain (Bischof et al., 1997). Thus, oligomerization of NPM-ALK with normal NPM, a nucleolar-cytoplasmic shuttle protein, drives the complexes to the nucleus and nucleolus (Bischof et al., 1997), whereas NPM-ALK homodimers remain in the cytoplasm. It has been shown that the nucleolar localization of NPM-ALK is not required for the oncogenic effects of NPM-ALK (Bischof et al., 1997; Mason et al., 1998). As ALK is a receptor tyrosine kinase, the localization of NPM-ALK in the cytoplasm is probably important to allow autophosphorylation and activation of the kinase, and thus intracellular signaling. However, we noted that the ALK cytoplasmic staining of NPM-ALK-transfected Jurkat cells was much weaker than that of ALCL cells. Thus we fractionated the cells, showing by Western blotting that the protein was equally expressed in the cytoplasms and nuclei of NPM-ALK-transfected cells. This result, together with the recent description of ALCL tumors showing cytoplasmic-restricted ALK staining, in which the ALK gene is fused to partners other than NPM, (Lamant et al., 1999; Hernandez et al., 1999; Ma et al., 2000; Trinei et al., 2000; Colleoni et al., 2000; Touriol et al., 2000), suggests that ALK activation through dimerization occurs in the cytoplasm, where it initiates proliferative and antiapoptotic signaling.

As the transforming ability of NPM-ALK is linked to its constitutive activation, it was important to demonstrate that the NPM-ALK protein expressed in transfected Jurkat cells, despite a lower expression level than in ALCL, was activated. Indeed, we show that NPM-ALK is tyrosine phosphorylated in JNA cells, as in ALCL cell lines (Bischof et al., 1997). Moreover, the chimeric protein exhibits functional autokinase activity in vitro, suggesting that the tyrosine phosphorylation detected in vivo is due to dimerization and activation of the kinase within the cell, allowing the transduction of signaling events.

An increasing number of reports have suggested that aberrations in transduction of both proliferation and apoptosis signals are involved in the development of hematopoietic malignancies, including lymphomas (Guo and Bruce, 1999). Studies showing the strong antiapoptotic potential of the BCR-ABL oncogenic tyrosine kinase (McGahon et al., 1994; Gambacorti-Passerini et al., 1997; Amarante-Mendes et al., 1998), and of receptor tyrosine kinases of the insulin receptor family as a consequence of their overexpression (O'Connor et al., 1997; Kulik et al., 1997), led us to hypothesize that NPM-ALK could also act as an antiapoptotic gene.

Our findings demonstrate that NPM-ALK expressed in Jurkat cells significantly decreases apoptosis induced by two drugs used in the treatment of ALCL, doxorubicin and etoposide. This antiapoptotic effect was specifically mediated by NPM-ALK, as it was observed in both cloned and bulk cultures of JNA cells, but not in control vector-transduced cells. In addition, we found that inactivation of the critical ATP-binding residue K210 abolished the antiapoptotic activity of NPM-ALK. According to the work of Bischof et al. (1997), this site is also indispensable for NPM-ALK mitogenic and transforming activity. The ATP-binding site is therefore required for NPM-ALK inhibition of drug-induced apoptosis, strongly suggesting that the tyrosine kinase activity of the chimeric protein is necessary for its antiapoptotic effect. This result is not unexpected, given that intact kinase activity has also been found to be required for the antiapoptotic effects of other receptor tyrosine kinases, such as the insulin growth factor-1 receptor (IGF-1R), which confers protection against UVB radiation-induced apoptosis in murine fibroblasts upon ligand stimulation (Kulik et al., 1997).

In the IGF-1R system, receptor mutants have been used in order to study the domains required for the transmission of antiapoptotic signals from the receptor, and to compare these domains with those required for mitogenesis or transformation (O'Connor et al., 1997; Romano et al., 1999). The 664 tyrosine residue of NPM-ALK binds PLC-gamma, which has been implicated in proliferative and transforming NPM-ALK signaling pathways (Bai et al., 1998). We found that mutation of this tyrosine (Y664) did not affect NPM-ALK-mediated inhibition of drug-induced apoptosis, suggesting that the proliferative, but not antiapoptotic activity of NPM-ALK depends on PLC-gamma signaling.

In an attempt to elucidate the mechanism of NPM-ALK-mediated inhibition of apoptosis, we investigated the role of PI3K in our system. PI3K has been implicated in ligand-dependent survival signaling by receptor tyrosine kinases after growth factor deprivation (Yao and Cooper, 1996) or DNA damage (Fan et al., 2000). It is also involved in transformation and proliferation induced by oncogenic tyrosine kinases such as BCR-ABL (Skorski et al., 1997; Jain et al., 1996). Here we show that PI3K is specifically activated in wild type NPM-ALK-expressing Jurkat cells, as indicated by the phosphorylation of Akt. In agreement with our findings, two recent studies also demonstrated a permanent activation of Akt in transfected BaF3 murine hematopoietic cells, as well as in t(2;5)-positive ALCL cell lines (Bai et al., 2000; Slupianek et al., 2001). In these studies, PI3K activation appeared essential for the mitogenic and transforming effect of NPM-ALK. It has previously been shown that, in the BaF3 model, NPM-ALK confers interleukin 3-independence (Bai et al., 1998). In the absence of this growth factor, treatment of transfected cells with the PI3K inhibitor LY294002 alone induced apoptosis (Slupianek et al., 2001). This antiapoptotic effect of PI3K is in apparent contradiction with our findings, which show that incubation of transfected Jurkat cells with LY294002 prior to doxorubicin treatment does not reverse the antiapoptotic effect of NPM-ALK, despite PI3K activation. These diverging results could be explained by the differences inherent to the choice of cell model and apoptosis induction systems.

In contrast to its strong inhibitory effect on drug-induced apoptosis, we found that NPM-ALK did not modify Fas-induced apoptosis in transfected Jurkat cells. Two main pathways of apoptosis signal transduction have been identified, one initiated by cell surface death receptors of the tumor necrosis factor receptor family, such as Fas, the other triggered by external stimuli such as cytotoxic drugs. Fas signaling is initiated at the DISC (death inducing signaling complex) by activation of caspase 8 and subsequently caspase 3, and may or may not involve the release of mitochondrial cytochrome c (Scaffidi et al., 1998, 1999). In contrast, drugs primarily engage the mitochondrial machinery, the release of cytochrome c being an early event preceeding activation of caspases (reviewed by Green and Reed, 1998; Kroemer et al., 1998). We found that in Jurkat cells, apoptosis induced by Fas ligation and doxorubicin both involve the mitochondrial pathway. There is indeed a controversy as to whether apoptosis induced by cytotoxic drugs is mediated by Fas/Fas ligand interaction (Friesen et al., 1996; Eischen et al., 1997; Villunger et al., 1997; McGahon et al., 1998; Wesselborg et al., 1999). Our results however show that, in NPM-ALK-transfected Jurkat cells, cytochrome c release is delayed only after doxorubicin, but not anti-Fas treatment, suggesting that the Fas/Fas ligand pathway is probably not involved in drug-induced apoptosis of Jurkat cells. Most interestingly, these results allowed us to determine that the inhibition of drug-induced apoptosis by NPM-ALK occurs upstream of mitochondrial events.

Inhibition of drug-induced apoptosis by NPM-ALK was potent and reproducible. However, NPM-ALK delayed rather than completely abrogated cell death. Interestingly, known antiapoptotic genes, such as Bcl-2 (Yin and Schimke, 1995; Allouche et al., 1997) and BCR-ABL (Dubrez et al., 1998) also induce a delay in drug-induced apoptosis. Therefore, our results indicate that NPM-ALK acts as an antiapoptotic gene, as suggested also by its ability to confer cytokine-independent growth to the murine interleukin 3-dependent BaF3 cell line (Bai et al., 1998). The overexpression of NPM-ALK seems to confer a survival advantage to cells rather than a definitive protection. It is likely that this advantage can result in the expansion of a tumor by modifying the balance between the pools of living/dividing and dying cells. Alternatively, NPM-ALK may prolong the survival of a subpopulation of tumor cells after treatment with cytotoxic drugs. In agreement with the latter hypothesis, recent clinical studies indicate that, although ALCLs are highly chemosensitive tumors, a third of ALCL patients experience multiple relapses (Brugieres et al., 1998, 2000), suggesting that a few resistant NPM-ALK-expressing cells may be selected during remission induction by cytotoxic drugs.

Materials and methods

Reagents and antibodies

Doxorubicin (doxorubicine DakotaR pharm) was obtained from Sanofi Winthrop (Gentilly, France), geneticin (G418 sulphate) from GIBCO BRL (Life Technologies, Grand Island, NY, USA), and [gamma-32P]ATP from Amersham (Les Ulis, France). All other chemicals were from Sigma Chemical Co. (St Louis, MO, USA).

The ALK1 (Pulford et al., 1997) and ALKc (Falini et al., 1998) mAbs, which recognize the intracellular, kinase-containing region of ALK, have been described previously. The anti-Fas (CD95, clone CH11) and anti-phosphotyrosine (clone 4G10) mAbs were purchased from Upstate Biotechnology (Lake Placid, NY, USA), anti-actin mAb (clone AC-10) from Sigma, anti-cytochrome c mAb from Pharmingen (San Diego, CA, USA), anti-cytochrome oxidase subunit II mAb from Molecular Probes (Eugene, OR, USA), polyclonal rabbit anti-human phosphorylated Akt (serine 473) antibody from New England Biolabs (Beverly, MA, USA), and peroxidase-conjugated rabbit anti-mouse and pig anti-rabbit Ig antisera from Dako (Glostrup, Denmark).

Cell lines and culture

Jurkat (clone E6.1, TIB 152) lymphoblastic leukemia cell line was purchased from the ATCC (Rockville, MD, USA). SU-DHL1 was established from a t(2;5)-positive ALCL (Morgan et al., 1989). Jurkat cells were maintained in RPMI1640 (GIBCO BRL) containing 10% fetal calf serum (Myoclone, GIBCO BRL), and SU-DHL1 in Iscove's-modified Dulbecco's medium (GIBCO BRL) supplemented with 20% fetal calf serum. All culture media contained 2 mM glutamine, 100 Units/ml penicillin, 100 mug/ml streptomycin and 1 mM sodium pyruvate (GIBCO BRL). Transfected Jurkat cells were cultured in the continuous presence of 2 mg/ml G418.

DNA transfection

pcDNA3 expression vector (In Vitrogen, Groningen, The Netherlands), empty or containing the cDNA for wild-type NPM-ALK (Morris et al., 1994), or mutants of that gene, namely NPM-ALK (K210R), (Y664F), (Y418F) and (Y156F-Y418F) (Bai et al., 1998) was transfected into Jurkat cells by electroporation with a Biorad (Ivry-sur-Seine, France) Gene Pulser apparatus at 270 V, 960 muF. After 48 h of culture, transfected cells were selected and further cultured with 2 mg/ml G418. Cells transfected with wild-type NPM-ALK ('JNA pool' cells) were further cloned by limiting dilution in 96-well plates, generating two clones, JNA F2.3 and F2.6. Cells transfected with control vector (Jurkat/neo) or with NPM-ALK mutant cDNAs were used as pools.

Immunohistochemical analysis

Cells were cytospun onto glass slides coated with silane, fixed in acetone for 10 min, and stained using ALK1 (Pulford et al., 1997) or ALKc (Falini et al., 1998) mAb in a three stage immunoperoxidase technique (Benharroch et al., 1998).

Western blot analysis

Total cellular proteins were extracted by sonication at 4°C in lysis buffer A containing 20 mM Tris HCl, pH 7.5, 0.5 mM EDTA, 0.5 mM EGTA, 1 mM DTT, 10% v/v glycerol, 10 mug/ml leupeptin, 2 mug/ml aprotinin, 2 mug/ml pepstatin A, 1 mM 4-2 aminoethyl-benzenesulfonyl fluoride (AEBSF), 1 mM sodium orthovanadate and 4 mM sodium fluoride. Subcellular fractionation was done by two methods. For isolation of nuclear and cytoplasmic fractions, cells were lysed by four passages through a 25 gauge needle in lysis buffer B containing 10 mM HEPES-KOH, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT and protease inhibitors at 4°C. Nuclear (pellet) and cytoplasmic (supernatant) fractions were separated by centrifugation for 30 s at 12000 g. The integrity of cell nuclei was checked by microscope examination. The nuclear pellet was resuspended in lysis buffer A and sonicated at 4°C. In separate experiments, mitochondrial and cytosolic fractions were extracted by 20 passages through a dounce homogenizer, using the ApoAlert cell fractionation Kit (Clontech, Palo Alto, CA, USA).

Total cellular proteins (5-50 mug), or subcellular fractions were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, and transferred onto nitrocellulose membranes. NPM-ALK and tyrosine phosphorylated proteins were detected using the ALKc (1/100) and 4G10 (1/1000) mAbs, respectively, followed by a horseradish peroxidase-coupled rabbit anti-mouse Ig antiserum (1/5000). Phosphorylated Akt was detected with a polyclonal anti-phosphorylated Akt (serine 473) rabbit antibody (1/1000), followed by a horseradish peroxidase-coupled pig anti-rabbit Ig antiserum (1/1000). Cytochrome c and cytochrome oxidase subunit II were detected using specific mAbs, both at 1 mug/ml, followed by a horseradish peroxidase-coupled rabbit anti-mouse Ig antiserum (1/3000). Signal detection was performed with an enhanced chemoluminescence kit (ECL, Amersham).


Cell pellets were suspended in lysis buffer (50 mM Tris HCl, 150 mM NaCl, 1% Triton X100) containing 1 mM sodium orthovanadate, 4 mM sodium fluoride, 10 mug/ml leupeptin, 2 mug/ml aprotinin, 2 mug/ml pepstatin A, and 1 mM AEBSF at 4°C for 30 min. Samples were precleared at 4°C for 1 h using prewashed protein G-Sepharose beads. The supernatant was then incubated with ALK1-precoated Sepharose beads at 4°C for 1 h. After five washes in lysis buffer, the beads were heated at 95°C for 4 min. Protein separation was performed by SDS-PAGE, followed by Western blot analysis with the anti-phosphotyrosine or ALKc mAb.

In vitro kinase assay

Immunoprecipitation with ALK1 mAb was carried out as described above. Sepharose-bound immune complexes in lysis buffer were washed and resuspended in kinase buffer (20 mM HEPES, pH 7.4, 10 mM MnCl2, 10 mM sodium fluoride, 1 mM sodium orthovanadate), before incubation with 5 muCi [gamma-32P]ATP (Redivue, Amersham) at 25°C for 15 min. Samples were boiled at 95°C for 4 min and separated on a 10% gel by SDS-PAGE prior to autoradiography.

Induction of apoptosis and analysis by cell morphology

Exponentially growing cells were seeded at 4´105 cells/ml, and incubated for various periods of time with different inducers of apoptosis or with culture medium for controls. Doxorubicin (1 muM) or etoposide (10 muM) was added for 14 or 18 h, and anti-Fas mAb (5 ng/ml) for 6-19 h. Cell viability was determined by Trypan blue dye exclusion. At the end of the incubations, cells were washed with PBS, cytocentrifuged onto microscope slides, stained with May-Grünwald-Giemsa dye (Merck, Darmstadt, Germany) and examined under a light microscope. Alternatively, cells were stained with Hoechst 33348 (5 mug/ml) and propidium iodide (10 mug/ml) prior to examination under a fluorescent microscope. The percentage of apoptotic cells was determined by enumerating cells undergoing a reduction of cell volume with chromatin condensation or micronuclear fragmentation in a total of at least 300 cells.

Quantification of apoptosis by annexin V-FITC labeling

Annexin V-FITC binding to phosphatidylserine, a membrane phospholipid exposed at the surface of apoptotic cells (Koopman et al., 1994), was measured at the end of apoptosis induction. Cells were washed twice in PBS and resuspended in 100 mul annexin buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 5 mM CaCl2) containing annexin V-FITC (1/50) (Roche Diagnostics, Meylan, France) and 1 mug/ml propidium iodide, a DNA-intercalating agent. Propidium iodide allows discrimination of apoptotic from necrotic cells, which bind annexin due to loss of membrane integrity. After incubation for 15 min at room temperature, annexin buffer (400 mul) was added to samples and cell fluorescence was immediately analysed on an XL4C Coulter (Beckman Coulter, Hialeah, FL, USA) flow cytometer (excitation wavelength at 488 nm, and emission at 525 and 640 nm for FITC and propidium iodide, respectively). Cells labeled with annexin V-FITC and negative for propidium iodide were scored as apoptotic cells.

Statistical analysis

A paired Student's t-test was used to compare apoptosis values in NPM-ALK-expressing cells and in control vector-transfected or parental Jurkat cells. A P-value <0.05 was considered significant.


We are grateful to Georges Cassar from the Service Commun de Cytométrie de l'IFR Purpan for his help with flow cytometry analysis, to Drs Karen Pulford and Bruno Falini for the gift of ALK1 and ALKc mAbs, respectively, and to Dr Michael Cleary for the SU-DHL1 cell line. We also thank Dr Bent Rubin for his advice in transfection, and Dr Olivier Cuvillier for his helpful suggestions in cytochrome c experiments. This work was supported by grant 9937 (M Allouche) from the Association pour la Recherche sur le Cancer, by the ARECA network (Pole protéomique et cancer), by the National Cancer Institute grant CA69129 (SW Morris) and CORE grant CA21765, the American-Lebanese Syrian Associated Charities (ALSAC: SW Morris), St Jude Children's Research Hospital (SW Morris), and by a grant from the Wilhelm-Sander Stiftung (J Duyster).


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Figure 1 Characterization of NPM-ALK expressed in transfected Jurkat cells. Control vector-transfected Jurkat/neo cells (a) or NPM-ALK-transfected Jurkat clone JNA F2.3 (b), were cytospun onto glass slides. Immunohistochemical analysis of the NPM-ALK protein was done using the ALK1 mAb in a three stage immunoperoxidase technique. All NPM-ALK-transfected cells exhibit a strong nucleolar staining, and a weaker nuclear and cytoplasmic staining. In some cells, the cytoplasm, nucleoplasm and nucleoli display an intense ALK-specific labeling. Magnification,´800. (c) 50 mug (Jurkat, Jurkat/neo, JNA F2.3 and F2.6) or 5 mug (SU-DHL1) of protein from total cell lysates were separated by SDS-PAGE and analysed by Western blot using the ALKc mAb; the blots were reprobed with an anti-actin mAb without prior stripping. Molecular weight markers are indicated. (d) Western blot analysis of nuclear and cytoplasmic extracts from Jurkat/neo and JNA F2.3 cells with the ALKc mAb

Figure 2 NPM-ALK expressed in transfected Jurkat cells is phosphorylated on tyrosine residues and has a functional kinase activity. (a) 107 cells from Jurkat/neo, NPM-ALK-expressing clones (JNA F2.3, F2.6) and from the t(2;5)-positive cell line SU-DHL1 were lysed; NPM-ALK was immunoprecipitated using the ALK1 mAb, then analysed through Western blotting for tyrosine phosphorylation using the antiphosphotyrosine mAb 4G10. The second lane represents a total lysate of JNA F2.3 cells. Molecular weight markers are indicated. Immunoglobulin heavy and light chains are apparent on the blot. (b) In vitro kinase assay of anti-ALK immunoprecipitates from total cell extracts of NPM-ALK-expressing clones JNA F2.3 and F2.6, parental Jurkat, and Jurkat/neo cells

Figure 3 NPM-ALK expression decreases drug-induced, but not Fas-induced apoptosis in transfected Jurkat cells. Jurkat parental cells, Jurkat/neo and NPM-ALK-expressing clones JNA F2.3 and F2.6 were treated with 1 muM doxorubicin (a,b), 10 muM etoposide (c,d), or 5 ng/ml anti-human Fas mAb CH11 (f,g). A dose range of 0.25-1 muM doxorubicin was used in (e). AnnexinV-FITC-positive cells were quantified after 14 (black bars) or 18 (grey bars) hours of treatment with drugs (a,c), and after 6 and 19 h of anti-Fas treatment as indicated (f). Apoptosis was also determined using morphological criteria after May-Grünwald-Giemsa staining following an 18 h treatment with each drug (b, d), or a time range of 6-19 h with anti-Fas mAb (g). The mean±s.e.m. of at least three independent experiments is represented, except in (e), displaying one representative experiment of two. Statistical comparison of the percentage of apoptotic cells in JNA clones and Jurkat/neo cells was done using a paired Student's t-test. *P<0.05; **P<0.01; ***P<0.001. No significant difference was observed between the percentage of apoptotic cells in anti-Fas-treated JNA clones and Jurkat/neo cells at each time point. (h) Analysis of cytochrome c release into the cytosol of doxorubicin- and anti-Fas-treated control (Jurkat/neo) and NPM-ALK-expressing (JNA F2.3) cells after 6 h. Mitochondrial and cytosolic cell extracts (25 mug) were subjected to SDS-PAGE and analysed by Western blot with an anti-cytochrome c mAb. Mito control: mitochondrial fraction from untreated control cells. Western blotting with an anti-cytochrome oxidase (subunit II) was also performed as a control to exclude any contamination of the cytosolic fraction by mitochondria

Figure 4 Study of drug-induced apoptosis in Jurkat cells transfected with NPM-ALK mutants. Jurkat/neo, wild type NPM-ALK-expressing cells (uncloned JNA pool or clone JNA F2.3), and cells expressing the (K210R), (Y664F), (Y418F) or (Y156F-Y418F) NPM-ALK mutants (see Table 1) were treated with 1 muM doxorubicin (a) or 10 muM etoposide (b) for 18 h. AnnexinV-FITC-positive apoptotic cells were quantified by flow cytometry. Data shown represent the mean±s.e.m. of three independent experiments. The percentage of annexin-positive NPM-ALK-expressing cells was significantly reduced (P<0.05) in both wild type and mutants compared to Jurkat/neo cells, except for the (K210R) mutant. (c) Comparison of NPM-ALK expression in Jurkat cells transfected with wild type (JNA pool, JNA F2.3, JNA F2.6) and mutant (K210R, Y664F, Y156F-Y418F, Y418F) forms of NPM-ALK. Total cell lysates (top panel) or ALK1 mAb-immunoprecipitated cell extracts (bottom panel) were subjected to SDS-PAGE, and analysed by Western blotting using the ALKc mAb. The top panel blot was reprobed with an anti-actin mAb without prior stripping

Figure 5 Akt is activated in NPM-ALK-expressing Jurkat cells but PI3K is not involved in NPM-ALK-mediated inhibition of drug-induced apoptosis. (a) Western blot analysis of total cell extracts from Jurkat/neo, wild type (JNA pool and JNA F2.6) and mutant (K210R, Y156-Y418F and Y418F) NPM-ALK-expressing cells with a rabbit polyclonal anti-phosphorylated human Akt (serine 473) antibody. In lane 4, JNA F2.6 cells were also preincubated for 3 h with LY294002 (25 muM), as indicated. (b) Jurkat cells transfected with vector (Jurkat/neo), wild type (JNA pool, JNA F2.3) or mutant (K210R, Y664F, Y418F, Y156F-Y418F, see Table 1) NPM-ALK were preincubated for 30 min with 25 muM LY294002. Doxorubicin was then added for 18 h. AnnexinV-FITC-positive apoptotic cells were quantified by flow cytometry. Data shown represent the mean±s.e.m. of three independent experiments


Table 1 Summary of NPM-ALK mutant constructs

Received 7 November 2000; revised 25 July 2001; accepted 1 August 2001
1 November 2001, Volume 20, Number 50, Pages 7386-7397
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