BTG2TIS21/PC3 induces neuronal differentiation and ... - Nature

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Oncogene (2002) 21, 6772 – 6778 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc

BTG2TIS21/PC3 induces neuronal differentiation and prevents apoptosis of terminally differentiated PC12 cells Fatiha el-Ghissassi1, Sandrine Valsesia-Wittmann1, Nicole Falette1, Cyril Duriez1, Paul D Walden2,3 and Alain Puisieux*,1,4 1

De´partement d’Oncologie Fondamentale et Appliqueé, INSERM Unite´ 453. Centre León Be´rard, 28 rue Lae¨nnec, 69008, Lyon, France; 2Department of Biochemistry, NYU School of Medicine, 550 First Avenue, New York, NY 10016, USA; 3Department of Urology, NYU School of Medicine, 550 First Avenue, New York, NY 10016, USA; 4De´partement de Geńe´tique Molećulaire et Biochimie Clinique, Faculte´ de Pharmacie, 69008, Lyon, France

The p53-transcriptional target, BTG2TIS21/PC3, was previously identified as an antiproliferative gene. However, the precise biological functions of the protein product remain to be elucidated. BTG2TIS21/PC3 expression is induced in vivo during neurogenesis, and the gene is transiently expressed in vitro in rat pheochromocytoma PC12 cells after induction of neuronal differentiation by addition of nerve growth factor (NGF). These observations suggest that BTG2TIS21/PC3 is functionally significant during the neuronal differentiation process. To test this hypothesis, a vector that expressed BTG2TIS21/PC3 under the control of an inducible promoter was introduced into PC12 cells. Growth arrest and differentiation in response to NGF were greatly enhanced by BTG2TIS21/PC3 overexpression. Furthermore, an antisense oligonucleotide complementary to BTG2TIS21/PC3 mRNA, which was able to inhibit endogenous BTG2TIS21/PC3 expression, triggered programmed cell death in differentiated PC12 cells. These observations confirm that BTG2TIS21/PC3 expression promotes neuronal differentiation and that it is required for survival of terminally differentiated cells. Oncogene (2002) 21, 6772 – 6778. doi:10.1038/sj.onc. 1205888 Keywords: BTG2TIS21/PC3; PC12 cells; cell differentiation; apoptosis Introduction The human BTG2TIS21/PC3 gene is a member of a newly identified family of six independent antiproliferative genes, namely, BTG1, ANA/BTG3, PC3B, TOB, TOB2 (Rouault et al., 1992; Matsuda et al., 1996; Guehenneux et al., 1997; Yoshida et al., 1998; Ikematsu et al., 1999; Buanne et al., 2000; Tirone, 2001). Although the biological functions of the BTG2TIS21/PC3 protein remain to be elucidated, several studies have attempted to identify the different cues capable of inducing its expression. We previously reported that BTG2TIS21/PC3 expression is

enhanced in response to genotoxic stress through a p53dependent mechanism (Rouault et al., 1996; Cortes et al., 2000). BTG2TIS21/PC3 is also transiently expressed in response to signals such as epidermal growth factor (EGF), tumor promoter agent (TPA), or the addition of serum to starved cells (Bradbury et al., 1991; Fletcher et al., 1991). However, several in vivo and in vitro observations suggest that it may play a role in neuronal differentiation. In vivo, BTG2TIS21/PC3 is considered as a marker of neuronal birth (Iacopetti et al., 1994). Indeed, the BTG2TIS21/PC3 gene is specifically expressed in neuroepithelial cells that will generate postmitotic neurons (Iacopetti et al., 1999). Its overexpression affects the pattern of cell division of cortical precursors in rats (Malatesta et al., 2000). In vitro, BTG2TIS21/PC3 is activated by NGF at the onset of neuronal differentiation in PC12, a cell line derived from a murine adrenal medulla tumor (Bradbury et al., 1991). In the PC12nn25 mutant cell line, characterized by a defect in NGF receptormediated endocytosis, BTG2TIS21/PC3 response to NGF is completely abolished and cells remain undifferentiated (Altin et al., 1991). Interestingly, the BTG2TIS21/PC3 protein physically interacts with and activates the PRMT1 protein-arginine-N-methyltransferase (Lin et al., 1996) whose function is required for PC12 differentiation by NGF (Cimato et al., 1997). To directly investigate the role of BTG2TIS21/PC3 in neuronal differentiation, rat pheochromocytoma PC12 cells were transfected with an inducible BTG2TIS21/PC3 expression vector. We show that BTG2TIS21/PC3 overexpression enhances NGF-induced neuronal differentiation of PC12 cells. Inhibition of BTG2TIS21/PC3 mRNA with specific antisense oligonucleotide promotes programmed cell death in NGF differentiated cells and in cells overexpressing BTG2TIS21/PC3. We therefore conclude that the overexpression of BTG2TIS21/PC3 is required for the survival of differentiated PC12. Results Generating and characterizing stable PC12 lines containing an inducible BTG2TIS21/PC3 construct

*Correspondence: A Puisieux, Centre León Be´rard, 28 rue Lae¨nnec, 69008, Lyon, France; E-mail: [email protected] Received 29 April 2002; revised 4 July 2002; accepted 18 July 2002

Clonal PC12 cell lines were generated with BTG2TIS21/PC3 cDNA under the control of the Lac

BTG2TIS21/PC3 triggers differentiation of PC12 cells F el-Ghissassi et al

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repressor protein. Expression was induced by adding iso propyl-b-D-thiogalactoside (IPTG), a non toxic lactose analog, to the culture medium. Using the Lac switch system, it was possible to generate stable cell lines in which BTG2TIS21/PC3 expression was induced following the addition of IPTG to the cell culture media. The 1100 bp fragment which was used contained the entire coding region but lacked the 3’untranslated region, making it possible to discriminate it from the endogenous messenger. Forty-three different clonal lines were screened. In all of them, as expected, endogenous BTG2TIS21/PC3 mRNA was almost undetectable. Ten showed inducible BTG2TIS21/PC3 expression after IPTG addition. Three sublines were selected for further analysis (Figure 1): PC12718 and PC12-27 exhibiting high inducible BTG2TIS21/PC3 expression and as a control PC12-2 clone, which displayed no expression of BTG2TIS21/PC3, even after IPTG addition. As shown on Figure 2a, the expression of exogenous BTG2TIS21/PC3 mRNA was increased in PC12-18 and PC12-27 cells following exposure to IPTG for 12 h. BTG2TIS21/PC3 protein levels also increased after IPTG addition, with a kinetic profile similar to that of exogenous BTG2TIS21/PC3 mRNA (Figure 2b). Maximum expression levels were reached within 3 days of IPTG exposure. In PC12 sublines, the ectopic expression persisted up to 8 days following IPTG

Figure 1 Characterization of stable PC12 sublines containing inducible BTG2TIS21/PC3 expression. Northern blot analysis of ectopic BTG2TIS21/PC3 mRNA in PC12 cells (clone PC12-18 and clone PC12-27) stably transfected with an inducible BTG2TIS21/PC3 expression vector. Ten mg of total RNA were analysed after 12-h incubation with 3 mM IPTG. Clone PC12-2 is a negative control clone exhibiting no ectopic BTG2TIS21/PC3 expression even after IPTG addition. The band at approximately 1.1 kb represents transfected BTG2TIS21/PC3 (T-BTG2TIS21/PC3), while the band at approximately 2.5 kb represents endogenous BTG2TIS21/PC3 mRNA (E-BTG2TIS21/PC3). Transfected BTG2TIS21/PC3 lacks approximately 1400 base pairs of the 3’untranslated region contained in endogenous BTG2TIS21/PC3. 18S ribosomal RNA is shown to demonstrate equal loading of total RNA

addition, then lasted 6 days after IPTG was removed from the medium (results not shown). Growth of PC12 cells containing an inducible BTG2TIS21/PC3 construct The proliferative status of PC12-18 and PC12-27 cells cultured in the absence and in the presence of IPTG was investigated. Similar data were obtained for both clones. As shown on Figure 2c, IPTG-induced BTG2TIS21/PC3 expression caused a significant decrease in cell growth whereas neither cell viability nor cell morphogenesis were significantly affected (data not

Figure 2 Characterization of inducible BTG2TIS21/PC3 expression and effect on cell growth. (a) Northern blot analysis of IPTG-induced BTG2TIS21/PC3 mRNA expression in PC12 cells (clone PC12-27) stably transfected with an inducible BTG2TIS21/PC3 expression vector. Ten mg of total cell RNA were analysed after 0.5, 1, 2 and 3 days incubation with 3 mM IPTG. (b) Western blot analysis of ectopic BTG2TIS21/PC3 protein expression in PC12 cells (clone PC12-27) under the same conditions as (a). One hundred mg of total cell proteins were separated by SDS – PAGE and probed with BTG2TIS21/PC3 antibody. (c) Proliferation of cells following BTG2TIS21/PC3 induction was studied in PC12-27 cell line, either untreated (green box), or following exposure to 3 mM IPTG (pink box). Quantification was performed by counting the cell number at a given time. Data are expressed as means (+standard deviation) Oncogene


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shown). Cell cycle progression was then analysed using flow cytometry to measure changes in the DNA content. BTG2TIS21/PC3 overexpression in PC12-27 cells caused partial growth inhibition within 1 day of IPTG exposure, with a slight decrease in the number of cells in S phase (8 versus 13% in control cells) and a slight increase in the number of cells in G1 (63 and 58% in induced and control cells, respectively). Effects of BTG2TIS21/PC3 expression on PC12 differentiation It is known that BTG2TIS21/PC3 is expressed in PC12 cells after induction of neuronal differentiation by NGF. In order to demonstrate the role of BTG2TIS21/PC3 in NGF-induced differentiation, PC1218 and PC12-27 lines were treated, either with NGF alone or with a combination of IPTG and NGF (100 ng/ml). Neuronal differentiation was estimated by neurite outgrowth which was correlated with enhanced levels of the neuron-specific marker of differentiation aenolase (data not shown) (Vinores et al., 1981; Marz et al., 1997). After NGF treatment 20% of PC12-18 and PC12-27 lines developed neurites, whereas after IPTG/NGF treatment, up to 80% of cells differentiated (Figure 3a,b). These data suggested that induction of BTG2TIS21/PC3 expression, substantially enhanced responsiveness to NGF-induced neurite outgrowth suggesting that these cells differentiated more rapidly in the presence of BTG2TIS21/PC3. BTG2TIS21/PC3 is involved in the survival of NGF treated PC12 To confirm the role of BTG2TIS21/PC3 in the differentiation process, differentiation of NGF-treated PC12 cells was triggered in the presence of an antisense phosphorothioate oligodeoxynucleotide designed to interfere with BTG2TIS21/PC3 expression. After treatment with antisense oligonucleotide AS-BTG2, the expression of the BTG2TIS21/PC3 protein was reduced to levels lower than the basal level seen in untreated cells, as judged by Western blot analysis (Figure 4a). Conversely, BTG2TIS21/PC3 protein levels were unaffected by treatment with a non specific oligonucleotide (Figure 4a). In the absence of NGF treatment, ASBTG2 had no effect on PC12 cells (Figure 4c). However, the addition of NGF to PC12 cells treated by AS-BTG2 triggered a massive cell death, as evidenced by an increased number of floating cells. The percentage of apoptotic cells was then evaluated by the TUNEL method: 20% of apoptotic cells were observed 6 days after treatment with AS-BTG2, compared to 4% after treatment with a control non specific oligonucleotide (Figure 4b,c). This increase in apoptotic cell death was confirmed by cell-cycle analysis evidencing the emergence of a significant sub-G1 cell population (data not shown). Taken together these data strongly suggest that BTG2TIS21/PC3 is required for the survival of NGF treated PC12 cells. Oncogene

Discussion To understand the different aspects of neuronal differentiation, the PC12 rat pheochromocytoma cell line has frequently been used as an in vitro model. Undifferentiated PC12 cells proliferate in the presence of serum; however, the addition of NGF results in a sympathetic-like neuronal differentiation, including complete mitotic arrest (Rudkin et al., 1989; van Grunsven et al., 1996). The present study demonstrates that the forced expression of the antiproliferative BTG2TIS21/PC3 gene enhances NGF-induced neuronal differentiation and favors the survival of differentiated PC12 cells. Cell-cycle arrest is known to be an essential preliminary requirement for terminal differentiation. Other studies have shown that proliferation inhibitors combined with NGF treatment, enhance the differentiation rate of PC12 cells (Gupta et al., 1987). Similar findings resulted from culturing PC12 cells in serum-free medium prior to NGF addition (Rudkin et al., 1989). However, the in vivo mechanisms governing this process are not completely understood. Much attention has focused on the regulation of components known to control progression through cell cycle, including cyclins, cyclin-dependent kinases and cyclindependent kinase inhibitors (Erhardt and Pittman, 1998). In addition to previous reports, our data show that BTG2TIS21/PC3 expression may play a significant role on the control of cell growth. In vitro, BTG2TIS21/PC3 was known to trigger a G1 arrest in pheochromocytoma PC12 cells and in non-neuronal cells (Montagnoli et al., 1996; Guardavaccaro et al., 2000). However, the inducible overexpression of BTG2TIS21/PC3 led only to a partial G1-arrest in the absence of NGF (Figure 2), suggesting that its induction was not sufficient to trigger the G1 block that characterizes neuronal differentiation by NGF. In vivo, BTG2TIS21/PC3 expression was observed during neurogenesis, in the subset of neuroblasts differentiating into post-mitotic neurons (Iacopetti et al., 1999). The expression of the protein was sustained during the mitosis of neuron-generating neuroepithelial cells, and during a few days in post-mitotic neuronal daughter cells. This observation is consistent with the hypothesis that BTG2TIS21/PC3 induction does not trigger a complete cell-cycle arrest, but more probably reduces the cycling rate of neuroepithelial cells to below a certain threshold, thereby forcing the cells to switch from a proliferative symmetric mode (that generates two proliferating daughter neuroepithelial cells) to asymmetric division (that gives one post-mitotic neuron and one neuroepithelial cell) (Tirone, 2001; Malatesta et al., 2000). This function may be common to other members of the BTG/TOB family. Like BTG2TIS21/PC3, BTG3/ANA is expressed in the neuroepithelial cells of the ventricular zone of the developing central nervous system (Yoshida et al., 1998). BTG2TIS21/PC3 and BTG3/ANA may thus have overlapping roles in the growth arrest of neural precursor cells at the time when the commitment of the precursor cells to the neural or glial lineage occurs.


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Figure 3 Enhanced differentiation of PC12 cells after induction of BTG2TIS21/PC3 expression and NGF treatment. (a) Photomicrographs of undifferentiated and NGF-differentiated cells (clone 12-27) following 12 h pre-treatment with 3 mM IPTG (+ IPTG) or no treatment (control). (b) Measurement of neurite development of differentiating cells (clone PC12-27 + NGF) at 2, 3, 6 and 8 days, as well as in control and IPTG-treated cultures. The IPTG group was initially incubated with IPTG for 12 h prior to NGF treatment. In order to quantify differentiation discrepancies observed, cells with one or more neurite extensions of at least twice the cell body length were regarded as neurite positive. The number of cells with such outgrowth was counted from five randomly selected fields. Results are expressed as a percentage of the total number of cells. All cells on any individual dish were counted up to a total of 250 cells. Data represent means (+standard deviation) of three individual experiments. Representative photographs of cells were taken using an Axiophot2 camera attachment

Apart from the role of BTG2TIS21/PC3 as a signal transducer at the onset of neurogenesis, data of experiments using antisense phosphorothioate oligodeoxynucleotide suggest that BTG2TIS21/PC3 expression is required for the survival of post-mitotic neuronal daughter cells. This observation is highly reminiscent of the apoptotic cell death of differentiated PC12 cells after NGF withdrawal (Greene, 1978). Similarly, the inhibition of BTG2TIS21/PC3 expression may trigger programmed cell death as a consequence of terminally differentiated cells attempting to re-enter the cycle. Consistent with this role of BTG2TIS21/PC3 as a survival

factor in NGF treated cells, our team previously showed that the differentiated embryonic stem cells from which BTG2TIS21/PC3 had been ablated underwent apoptosis following DNA damage because of a failure in growth arrest (Rouault et al., 1996). Another key event in the growth arrest associated with the differentiation process is the induction of the p21Waf1 cyclin-dependent kinase inhibitor. p21Waf1 is upregulated during the NGF-induced cell cycle block in PC12 cells. The experimental overexpression of p21Waf1 is sufficient to induce differentiation-specific cell cycle events (Erhardt and Pittman, 1998). InterestOncogene


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2000). p21Waf1 and BTG2TIS21/PC3 are transcriptionally induced by p73 (Zhu et al., 1998), their expression increases during the differentiation of neuroblastoma cells (unpublished data). Studies are ongoing to assess whether p21Waf1 and BTG2TIS21/PC3 act as p73 downstream targets in this process. While this manuscript was under review, we learned that another, independent study reported that transient transfection of BTG2TIS21/PC3 was able to potentiate the effect of NGF on differentiation (Corrente et al., 2002). Combined data clearly demonstrate the role of BTG2TIS21/PC3 in neuronal differentiation. Materials and methods Cell culture PC12 cells were grown at 378C (5% CO2) in DMEM (4.5 g/l glucose) with 6% donor heat-inactivated horse serum, 6% heat-inactivated fetal bovine serum, 10 mM HEPES, 10 mM glutamine, 100 units/ml penicillin and 100 mg/ml streptomycin. Cells were cultured in plastic tissue culture dishes pre-coated with rat-tail collagen (0.25 mg/ml) (Sigma) and poly-L-lysine (10 mg/ml) (Sigma). Growth medium was supplemented with 200 mg/ml hygromycin B (Gibco-BRL) and 300 mg/ml G418 (Gibco-BRL) in cell lines transfected with Lac repressor and BTG2TIS21/PC3 expression plasmids. For induction of BTG2TIS21/PC3 in transfected lines, 3 mM IPTG (Boehringer Mannheim Biochemicals) were added to the medium. To induce differentiation, 100 ng/ml of nerve growth factor (NGF) (Sigma) were added to the same medium as above. Plasmids

Figure 4 Treatment with antisense oligonucleotide AS-BTG2 inhibits BTG2TIS21/PC3 protein expression and enhances cell death in NGF treated PC12 cells. PC12 cells were treated during 6 days with NGF in the absence of antisense oligonucleotide (control), in the presence of control non specific oligonucleotide (NSO) or with the anti-BTG2TIS21/PC3 antisense oligonucleotide (AS-BTG2). Then, proteins were extracted and samples were analysed by Western blotting (a). Detection and quantification of apoptotic cell were performed using the TUNEL staining method (b). Examples of apoptotic bodies (arrows) in cells treated with BTG2TIS21/PC3 antisense oligonucleotides. (c) Quantification of apoptotic bodies (values are means with standard deviation)

ingly, both BTG2TIS21/PC3 and p21Waf1 are known to be transcriptional targets of the tumor suppressor p53, in response to DNA damage (Rouault et al., 1996; elDeiry et al., 1993). In contrast to p53, the p53-homolog p73 plays a critical role in neuronal differentiation. P73-deficient mice show major neurological defects (Yang et al., 2000). In vitro, endogenous p73 levels increase in neuroblastoma cells induced to differentiate by retinoic acid. The experimental overexpression of p73 (but not p53) is sufficient to induce both morphological (neurite outgrowth) and biochemical markers of neuronal differentiation (de Laurenzi et al., Oncogene

For induction of BTG2TIS21/PC3 expression, plasmids from the Lac expression kit (Stratagene) were employed. Briefly, this system involves the constitutive expression of the Lac repressor protein and the introduction of a gene of interest (BTG2TIS21/PC3) under the control of a RSV promoter containing Lac repressor protein binding sites. Under normal conditions, the Lac repressor protein binds to Lac operator sites in the RSV promoter, thus inhibiting transcription. For induction of BTG2TIS21/PC3, isopropyl-b-D-thiogalactoside (IPTG) was added to the cells. IPTG binds to the Lac repressor protein, thus inhibiting interactions with DNA. The RSV promoter then initiates transcription, leading to ectopic BTG2TIS21/PC3 expression. The first plasmid, pCMV, contains the Lac repressor protein coding sequence and the hygromicin B resistance gene. The second plasmid, pOPRSV1, is an inducible expression plasmid bearing geneticin G418 resistance, in which a 1.1 kilobase human BTG2TIS21/PC3 cDNA fragment has been subcloned into the KpnI site. Both plasmids were cotransfected with lipofectin (Gibco-BRL) into PC12 cells according to the manufacturer’s instructions. The selection for the clonal expression of both the Lac repressor protein and the BTG2TIS21/PC3 construct was obtained by addition of hygromicin (200 mg/ml) (Gibco-BRL) and geneticin G418 (300 mg/ml) (Gibco-BRL). Lac repressor protein expression was tested by Western blot analysis and the expression inducible in clones was confirmed with Northern blot and Western blot analyses. Cell proliferation assay Cell growth was determined by the measurement of cell numbers after incubation with Trypan blue (0.06% in PBS).


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Unstained cells (living cells) were counted in three separate wells per experiment; five random fields were counted in each well. The relative cell number is the ratio of the cell count at a given time to the number of cells at time 0. RNA isolation and Northern blot analysis Total RNA from PC12 cells was isolated using the tri-reagent method (Sigma). RNA samples (10 mg) were electrophoresed on denaturating formaldehyde agarose gel, then transferred to nylon membranes (Hybond-N+; Amersham). Membranes were hybridized with a a-32P-dCTP-labeled BTG2TIS21/PC3 cDNA probe. Bands were visualized by autoradiography. Protein isolation and Western blot analysis Cells were lysed on ice in RIPA buffer (50 mM Tris-HCl, 0.1% SDS, 2 mM DTT, 0.5% NP40) containing 1 mM PMSF, 40 mg/ml leupeptin, 40 mg/ml aprotinin, 20 mg/ml pepstatin A. Total protein concentrations were determined by the Bio-Rad protein assay. Aliquots of each sample containing equal amounts of proteins were subjected to Western immunoblot analysis. Briefly, proteins were separated by electrophoresis on a 10% polyacrylamide gel, then transferred to Immobilon polyvinylidene difluoride transfer membrane (Millipore). Immunological studies For detection by Western blot, equal amounts of proteins were mixed with Laemmli sample buffer, resolved on 12% SDS-polyacrylamide gel, then transferred onto PVDF membranes (Millipore). Western blot analysis was performed using enhanced chemiluminescence in accordance with the manufacturer’s instructions. Specific antibodies to the BTG2TIS21/PC3 protein were generated by immunizing rabbits with six times His-tagged BTG2TIS21/PC3 protein, followed by affinity purification involving adsorption to, and elution from a column of BTG2 protein and by negative adsorption to a column of BTG1 protein (Walden et al., 1998). For the detection of Neuron Specific aEnolase, the primary antibody was Enolase C-19 (Santa Cruz). Peroxidase-conjugated antirabbit or anti-goat IgG (Dako) were used as secondary antibodies. Cell cycle analysis using flow cytometry (FACS) Cells were harvested at various times after the different treatments, then fixed with 70% ethanol for at least 20 min.

After centrifugation, the cell pellets were washed twice with PBS, then treated with 100 mg/ml DNase-free RNase A in PBS for 30 min. The nuclei were then stained with propidium iodide used at the concentration of 50 mg/ml. The DNA content of the stained nuclei was measured on a FACScalibur flow cytometer. The Cell Quest software (Becton Dickinson, USA) was used to determine cell cycle phase distribution. Antisense oligonucleotides Phosphorothioate oligonucleotides (100 mM) and lipofectin (1 mg/ml) (Gibco) were incubated for 15 min at 378C. The oligonucleotide-lipofectin mixture was diluted with serumcontaining medium, then added to the cells. In most cases, the dilution was 1 : 100, yielding a final oligonucleotideconcentration of 1 mM. Fresh oligonucleotide-containing medium was added to the cells each day. We used 22-mer antisense oligonucleotides complementary to the region of the BTG2TIS21/PC3 mRNA that encompasses the initiation codon. The oligonucleotides were purchased from GENSET (France). A non specific phosphorothioate 22-mer oligonucleotide (NSO) was used as control. Detection of apoptotic cell death The detection of apoptotic cell death was performed using the fluorescein-based in situ cell detection Kit (Enzo Roche). The assay was based upon the detection of DNA degradation using TUNEL (terminal dUTP-fluorescein nick end labeling) staining. The TUNEL assay was conducted on cytospin preparations of cells removed from dishes by trituration in the culture medium (to avoid loss of detached cells) as per manufacturer’s instructions. Apoptotic cells were visualized under a Zeiss axioplot 2 fluorescence microscope. The extent of apoptosis was expressed as the ratio of cells that showed positive TUNEL staining on the total number of cells (the total number of cells was calculated from cells stained with Hoechst 33342 (10 mg/ml)). Typically, at least 300 cells per treatment were counted in random fields. Three independent experiments were performed in each study.

Acknowledgements We thank MD Reynaud and N Borel for their help in preparing the manuscript. This work was supported by grants from INSERM and Association pour la Recherche sur le Cancer.

References Altin JG, Kujubu DA, Raffioni S, Eveleth DD, Herschman HR and Bradshaw RA. (1991). J. Biol. Chem., 266, 5401 – 5406. Bradbury A, Possenti R, Shooter EM and Tirone F. (1991). Proc. Natl. Acad. Sci. USA, 88, 3353 – 3357. Buanne P, Corrente G, Micheli L, Palena A, Lavia P, Spadafora C, Lakshmana MK, Rinaldi A, Banfi S, Quarto M, Bulfone A and Tirone F. (2000). Genomics, 68, 253 – 263. Cimato TR, Ettinger MJ, Zhou X and Aletta JM. (1997). J. Cell Biol., 138, 1089 – 1103. Corrente G, Guardavaccaro D and Tirone F. (2002). Neuroreport, 13, 417 – 422.

Cortes U, Moyret-Lalle C, Falette N, Duriez C, Ghissassi F, Barnas C, Morel AP, Hainaut P, Magaud JP and Puisieux A. (2000). Mol. Carcinog., 27, 57 – 64. De Laurenzi V, Raschella G, Barcaroli D, AnnicchiaricoPetruzzelli M, Ranalli M, Catani MV, Tanno B, Costanzo A, Levrero M and Melino G. (2000). J. Biol. Chem., 275, 15226 – 15231. el-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, Lin D, Mercer WE, Kinzler KW and Vogelstein B. (1993). Cell, 75, 817 – 825. Erhardt JA and Pittman RN. (1998). J. Biol. Chem., 273, 23517 – 23523.

Oncogene


6778

Fletcher BS, Lim RW, Varnum BC, Kujubu DA, Koski RA and Herschman HR. (1991). J. Biol. Chem., 266, 14511 – 14518. Greene LA. (1978). J. Cell Biol., 78, 747 – 755. Guardavaccaro D, Corrente G, Covone F, Micheli L, d’Agnano I, Starace G, Caruso M and Tirone F. (2000). Mol. Cell. Biol., 20, 1797 – 1815. Guehenneux F, Duret L, Callanan MB, Bouhas R, Hayette S, Berthet C, Samarut C, Rimokh R, Birot AM, Wang Q, Magaud JP and Rouault JP. (1997). Leukemia, 11, 370 – 375. Gupta M, England MA and Notter MF. (1987). Brain Res. Bull., 18, 555 – 561. Iacopetti P, Barsacchi G, Tirone F, Maffei L and Cremisi F. (1994). Mech. Dev., 47, 127 – 137. Iacopetti P, Michelini M, Stuckmann I, Oback B, AakuSaraste E and Huttner WB. (1999). Proc. Natl. Acad. Sci. USA, 96, 4639 – 4644. Ikematsu N, Yoshida Y, Kawamura-Tsuzuku J, Ohsugi M, Onda M, Hirai M, Fujimoto J and Yamamoto T. (1999). Oncogene, 18, 7432 – 7441. Lin WJ, Gary JD, Yang MC, Clarke S and Herschman HR. (1996). J. Biol. Chem., 271, 15034 – 15044. Malatesta P, Gotz M, Barsacchi G, Price J, Zoncu R and Cremisi F. (2000). Mech. Dev., 90, 17 – 28. Marz P, Herget T, Lang E, Otten U and Rose-John S. (1997). Eur. J. Neurosci., 9, 2765 – 2773. Matsuda S, Kawamura-Tsuzuku J, Ohsugi M, Yoshida M, Emi M, Nakamura Y, Onda M, Yoshida Y, Nishiyama A and Yamamoto T. (1996). Oncogene, 12, 705 – 713.

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Montagnoli A, Guardavaccaro D, Starace G and Tirone F. (1996). Cell Growth Differ., 7, 1327 – 1336. Rouault JP, Rimokh R, Tessa C, Paranhos G, Ffrench M, Duret L, Garoccio M, Germain D, Samarut J and Magaud JP. (1992). EMBO J., 11, 1663 – 1670. Rouault JP, Falette N, Guehenneux F, Guillot C, Rimokh R, Wang Q, Berthet C, Moyret-Lalle C, Savatier P, Pain B, Shaw P, Berger R, Samarut J, Magaud JP, Ozturk M, Samarut C and Puisieux A. (1996). Nat. Genet., 14, 482 – 486. Rudkin BB, Lazarovici P, Levi BZ, Abe Y, Fujita K and Guroff G. (1989). EMBO J., 8, 3319 – 3325. Tirone F. (2001). J. Cell. Physiol., 187, 155 – 165. Van Grunsven LA, Thomas A, Urdiales JL, Machenaud S, Choler P, Durand I and Rudkin BB. (1996). Oncogene, 12, 855 – 862. Vinores SA, Marangos PJ, Parma AM and Guroff G. (1981). J. Neurochem., 37, 597 – 600. Walden PD, Lefkowitz GK, Ficazzola M, Gitlin J and Lepor H. (1998). Exp. Cell Res., 245, 19 – 26. Yang A, Walker N, Bronson R, Kaghad M, Oosterwegel M, Bonnin J, Vagner C, Bonnet H, Dikkes P, Sharpe A, McKeon F and Caput D. (2000). Nature, 404, 99 – 103. Yoshida Y, Matsuda S, Ikematsu N, Kawamura-Tsuzuku J, Inazawa J, Umemori H and Yamamoto T. (1998). Oncogene, 16, 2687 – 2693. Zhu J, Jiang J, Zhou W and Chen X. (1998). Cancer Res., 58, 5061 – 5065.