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BTG2TIS21/PC3 Induces Neuronal Differentiation and Prevents

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BTG2TIS21/PC3 Induces Neuronal Differentiation and Prevents Oncogene (2002) 21, 6772 – 6778 ª 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 1De´partement d’Oncologie Fondamentale et Applique´e, INSERM Unite´ 453. Centre Le´on 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 Ge´ne´tique Mole´culaire et Biochimie Clinique, Faculte´ de Pharmacie, 69008, Lyon, France The p53-transcriptional target, BTG2TIS21/PC3, was enhanced in response to genotoxic stress through a p53- previously identified as an antiproliferative gene. However, dependent mechanism (Rouault et al., 1996; Cortes et al., the precise biological functions of the protein product 2000). BTG2TIS21/PC3 is also transiently expressed in remain to be elucidated. BTG2TIS21/PC3 expression is response to signals such as epidermal growth factor induced in vivo during neurogenesis, and the gene is (EGF), tumor promoter agent (TPA), or the addition of transiently expressed in vitro in rat pheochromocytoma serum to starved cells (Bradbury et al., 1991; Fletcher et PC12 cells after induction of neuronal differentiation by al., 1991). However, several in vivo and in vitro addition of nerve growth factor (NGF). These observations observations suggest that it may play a role in neuronal suggest that BTG2TIS21/PC3 is functionally significant differentiation. In vivo, BTG2TIS21/PC3 is considered as a during the neuronal differentiation process. To test this marker of neuronal birth (Iacopetti et al., 1994). Indeed, hypothesis, a vector that expressed BTG2TIS21/PC3 under the BTG2TIS21/PC3 gene is specifically expressed in the control of an inducible promoter was introduced into neuroepithelial cells that will generate postmitotic PC12 cells. Growth arrest and differentiation in response neurons (Iacopetti et al., 1999). Its overexpression affects to NGF were greatly enhanced by BTG2TIS21/PC3 over- the pattern of cell division of cortical precursors in rats expression. Furthermore, an antisense oligonucleotide (Malatesta et al., 2000). In vitro, BTG2TIS21/PC3 is complementary to BTG2TIS21/PC3 mRNA, which was able activated by NGF at the onset of neuronal differentiation to inhibit endogenous BTG2TIS21/PC3 expression, triggered in PC12, a cell line derived from a murine adrenal medulla programmed cell death in differentiated PC12 cells. These tumor (Bradbury et al., 1991). In the PC12nn25 mutant observations confirm that BTG2TIS21/PC3 expression cell line, characterized by a defect in NGF receptor- promotes neuronal differentiation and that it is required mediated endocytosis, BTG2TIS21/PC3 response to NGF is for survival of terminally differentiated cells. completely abolished and cells remain undifferentiated Oncogene (2002) 21, 6772 – 6778. doi:10.1038/sj.onc. (Altin et al., 1991). Interestingly, the BTG2TIS21/PC3 1205888 protein physically interacts with and activates the PRMT1 protein-arginine-N-methyltransferase (Lin et Keywords: BTG2TIS21/PC3; PC12 cells; cell differentia- al., 1996) whose function is required for PC12 differentia- tion; apoptosis tion 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 Introduction inducible BTG2TIS21/PC3 expression vector. We show that BTG2TIS21/PC3 overexpression enhances NGF-induced The human BTG2TIS21/PC3 gene is a member of a newly neuronal differentiation of PC12 cells. Inhibition of identified family of six independent antiproliferative BTG2TIS21/PC3 mRNA with specific antisense oligonu- genes, namely, BTG1, ANA/BTG3, PC3B, TOB, TOB2 cleotide promotes programmed cell death in NGF (Rouault et al., 1992; Matsuda et al., 1996; Guehenneux et differentiated cells and in cells overexpressing al., 1997; Yoshida et al., 1998; Ikematsu et al., 1999; BTG2TIS21/PC3. We therefore conclude that the over- Buanne et al., 2000; Tirone, 2001). Although the expression of BTG2TIS21/PC3 is required for the survival of biological functions of the BTG2TIS21/PC3 protein remain differentiated PC12. to be elucidated, several studies have attempted to identify the different cues capable of inducing its expression. We Results previously reported that BTG2TIS21/PC3 expression is Generating and characterizing stable PC12 lines containing an inducible BTG2TIS21/PC3 construct *Correspondence: A Puisieux, Centre Le´ on Be´ rard, 28 rue Lae¨ nnec, 69008, Lyon, France; E-mail: [email protected] Clonal PC12 cell lines were generated with TIS21/PC3 Received 29 April 2002; revised 4 July 2002; accepted 18 July 2002 BTG2 cDNA under the control of the Lac BTG2TIS21/PC3 triggers differentiation of PC12 cells F el-Ghissassi et al 6773 repressor protein. Expression was induced by adding addition, then lasted 6 days after IPTG was removed iso propyl-b-D-thiogalactoside (IPTG), a non toxic from the medium (results not shown). lactose analog, to the culture medium. Using the Lac switch system, it was possible to generate stable cell Growth of PC12 cells containing an inducible lines in which BTG2TIS21/PC3 expression was induced BTG2TIS21/PC3 construct following the addition of IPTG to the cell culture media. The 1100 bp fragment which was used The proliferative status of PC12-18 and PC12-27 cells contained the entire coding region but lacked the cultured in the absence and in the presence of IPTG 3’untranslated region, making it possible to discrimi- was investigated. Similar data were obtained for both nate it from the endogenous messenger. Forty-three clones. As shown on Figure 2c, IPTG-induced different clonal lines were screened. In all of them, BTG2TIS21/PC3 expression caused a significant decrease as expected, endogenous BTG2TIS21/PC3 mRNA in cell growth whereas neither cell viability nor cell was almost undetectable. Ten showed inducible morphogenesis were significantly affected (data not 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 2 Characterization of inducible BTG2TIS21/PC3 expression Figure 1 Characterization of stable PC12 sublines containing in- and effect on cell growth. (a) Northern blot analysis of IPTG-in- ducible BTG2TIS21/PC3 expression. Northern blot analysis of ecto- duced BTG2TIS21/PC3 mRNA expression in PC12 cells (clone pic BTG2TIS21/PC3 mRNA in PC12 cells (clone PC12-18 and clone PC12-27) stably transfected with an inducible BTG2TIS21/PC3 ex- PC12-27) stably transfected with an inducible BTG2TIS21/PC3 ex- pression vector. Ten mg of total cell RNA were analysed after pression vector. Ten mg of total RNA were analysed after 12-h in- 0.5, 1, 2 and 3 days incubation with 3 mM IPTG. (b) Western blot TIS21/PC3 cubation with 3 mM IPTG. Clone PC12-2 is a negative control analysis of ectopic BTG2 protein expression in PC12 cells clone exhibiting no ectopic BTG2TIS21/PC3 expression even after (clone PC12-27) under the same conditions as (a). One hundred IPTG addition. The band at approximately 1.1 kb represents mg of total cell proteins were separated by SDS – PAGE and transfected BTG2TIS21/PC3 (T-BTG2TIS21/PC3), while the band at probed with BTG2TIS21/PC3 antibody. (c) Proliferation of cells fol- approximately 2.5 kb represents endogenous BTG2TIS21/PC3 lowing BTG2TIS21/PC3 induction was studied in PC12-27 cell line, TIS21/PC3 TIS21/PC3 mRNA (E-BTG2 ). Transfected BTG2 lacks ap- either untreated (green box), or following exposure to 3 mM proximately 1400 base pairs of the 3’untranslated region con- IPTG (pink box). Quantification was performed by counting the tained in endogenous BTG2TIS21/PC3. 18S ribosomal RNA is cell number at a given time. Data are expressed as means (+stan- shown to demonstrate equal loading of total RNA dard deviation) Oncogene BTG2TIS21/PC3 triggers differentiation of PC12 cells F el-Ghissassi et al 6774 shown). Cell cycle progression was then analysed using Discussion flow cytometry to measure changes in the DNA content. BTG2TIS21/PC3 overexpression in PC12-27 cells To understand the different aspects of neuronal caused partial growth inhibition within 1 day of IPTG differentiation, the PC12 rat pheochromocytoma cell exposure, with a slight decrease in the number of cells line has frequently been used as an in vitro model. in S phase (8 versus 13% in control cells) and a slight Undifferentiated PC12 cells proliferate in the presence increase in the number of cells in G1 (63 and 58% in of serum; however, the addition of NGF results in a induced and control cells, respectively). sympathetic-like neuronal differentiation, including complete mitotic arrest (Rudkin
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