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First published online 2 December 2008
doi: 10.1242/jcs.033316

Journal of Cell Science 122, 36-43 (2009)
Published by The Company of Biologists 2009
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BDNF splice variants from the second promoter cluster support cell survival of differentiated neuroblastoma upon cytotoxic stress

Baj Gabriele and Tongiorgi Enrico*

University of Trieste, BRAIN Centre for Neuroscience, Department of Biology, Via Giorgieri 10, 34127 Trieste, Italy


Figure 1
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  		Fig. 1. Schematic representation of human BDNF gene structure and its splicing variants. Open boxes represent exons. Lines connect two exons. Arrows indicate alternative polyadenylation sites (PolyA) in the 3'UTR and internal alternative splice sites in exons 2, 6, 7 and 9a (letters a, b, c and d). Exons are drawn to scale.

 

Figure 2
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  		Fig. 2. Retinoic acid differentiation increases production of TrkB protein and BDNF mRNA in human neuroblastoma cell lines SK-N-BE and SH-SY-5Y. (A) Western-blot analysis of TrkB expression in SK-N-BE and SH-SY-5Y cell lines at different days (d) of treatment with 5 µM retinoic acid (left; und=undifferentiated). Specificity of the anti-TrkB antibody was tested on rat brain homogenates and on untreated HEK293T cells, which do not express TrkB (negative) or in HEK293T cells transfected with rat full-length TrkB (positive). Densitometric quantification of protein expression revealed a biphasic expression curve in SK-N-BE cells and a progressive increase in SH-SY-5Y cells from day 1 to day 9 (each column represents the mean fold increase with respect to undifferentiated cultures ± s.e.m. from three immunoblots from three different cultures). The Ponceau-S-stained protein band at 45 kDa was used for normalisation. (B) Biphasic expression of BDNF mRNA in SK-N-BE and SH-SY-5Y cell lines and densitometric quantification of PCR reactions normalised to expression of GAPDH (each column represents the mean fold increase with respect to undifferentiated cultures ± s.e.m. from n=3 cDNAs from three different cultures). **P<0.01; *P<0.05, one-way ANOVA.

 

Figure 3
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  		Fig. 3. Retinoic acid differentiation increases viability of neuroblastoma cell lines against cytotoxic drugs. (A) Neuroblastoma cell line SK-N-BE treated with cisplatin (+CIS) in undifferentiated (und) and differentiated (dif) conditions. (B) Neuroblastoma cell line SH-SY-5Y treated with cisplatin (+CIS) in undifferentiated and differentiated conditions. (C) Quantification of the viability assay for SK-N-BE cells in retinoic-acid-differentiated and undifferentiated conditions using the three cytotoxic drugs cisplatin (CIS), etoposide (ETO) and doxorubicin (DOXO). Cells were stained with the nuclear marker Hoechst 33258 and counted (each column represents the mean percentage of survival ± s.e.m.; undifferentiated or differentiated cultures with no drug added were taken as 100%; n=3 cultures). (D) Quantification of the viability assay (n=3) for SH-SY-5Y cells in retinoic-acid-differentiated and undifferentiated conditions using the three cytotoxic drugs as above, performed by counting cells labelled with Hoechst 33258 (mean ± s.e.m.). (E,F) Viability assay of neuroblastoma cell lines under different stress conditions. SH-SY-5Y and SK-N-BE cells were plated and counted at different time points in the presence of cisplatin, in serum-free medium or in serum-free medium (SFM) with cisplatin. Quantification of viability (n=3) for cells was performed with the MTT assay (each column represents mean ± s.e.m.). The survival curves shown in E and F were fitted with linear functions and the corresponding m values are indicated. Statistical analysis was performed with ANOVA (*P<0.05; **P<0.01).

 

Figure 4
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  		Fig. 4. Increased resistance to cytotoxic stress depends upon TrkB activation. (A) Quantification of the viability (MTT assay) of neuroblastoma cell lines SH-SY-5Y and SK-N-BE treated with BDNF siRNA or with the Trk inhibitor K252a. Each column represents mean ± s.e.m. of n=3 independent experiments. (B) BDNF quantification in cell lysates and supernatant of samples treated as in A was carried out by ELISA (Promega). (C) Expression of full-length TrkB, detected by immunofluorescence, was unchanged in SK-N-BE cells treated with BDNF siRNA or K252a, whereas activated TrkB, detected with an anti-TrkB-P antibody, was markedly reduced by BDNF siRNA and completely abolished by K252a. Scale bar: 50 µm. Statistical analysis was performed with ANOVA (**P<0.01).

 

Figure 5
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  		Fig. 5. Silencing of endogenous BDNF reduced resistance of differentiated neuroblastoma cell lines to chemotherapeutic drugs. (A) Cell survival of the neuroblastoma SK-N-BE cell line treated for 24 hours with different drugs, either alone or in the presence of BDNF siRNA. Cisplatin (CIS), etoposide (ETO) and doxorubicin (DOXO). (B) Viability of neuroblastoma cell lines SH-SY-5Y co-treated for 24 hours with BDNF siRNA and different drugs or with the indicated drugs without siRNA. Quantification of the viability assay for differentiated cells was performed by manually counting the positive cells with respect to total number of cells stained with nuclear marker Hoechst 33258 (each column is the mean ± s.e.m. from n=3 cultures). (C) BDNF quantification in cell lysates and supernatant of SK-N-BE cultures treated with the drug alone, drug + K252a or drug + BDNF siRNA was carried out by ELISA (Promega). (D) BDNF quantification in cell lysates and supernatant of SH-SY-5Y cultures treated with the drug alone, drug + K252a or drug + BDNF siRNA. Only treatment with siRNA shows a significant reduction in BDNF expression and secretion. Statistical analysis was performed with ANOVA (*P<0.05; **P<0.01).

 

Figure 6
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  		Fig. 6. Expression and silencing efficacy of BDNF siRNA isoforms in differentiated SK-N-BE or SH-SY-5Y neuroblastoma cell lines. RNA extraction and cDNA preparation was performed on three different cultures of retinoic-acid-treated cells (5 days with 5 µM retinoic acid) and splice variants were detected by PCR amplification. (A) Differentiated SK-N-BE cultures express the BDNF exons 1, 2A, 2B, 4, 5, 6A, 6B, 7, 8 and 9a whereas differentiated SH-SY-5Y cells express exons 2A, 2B, 4, 5, 6B and 9a. (B) Silencing of the different splice variants through siRNA in differentiated SK-N-BE cells. (C) Quantification of silencing efficacy with respect to basal expression in three different cultures (taken as 100%). Silencing efficacy was ranged from 70% to 80%. The housekeeping gene GAPDH is not silenced by the BDNF siRNA (each column represents the mean ± s.e.m. of three cultures; for quantitative data see supplementary material Table S2).

 

Figure 7
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  		Fig. 7. BDNF mRNA transcripts of exon 4, exon 6 and exon 9a are required for resistance to cytotoxic stress conditions. Viability assay using MTT in SK-N-BE cells treated with each single specific siRNA against BDNF mRNA isoforms at 6 hours of the following treatments. (A) Control medium with 10% FCS. (B) Control medium with cisplatin (CIS). (C) Serum-free medium (SFM) with no drugs. (D) Serum-free medium (SFM) with cisplatin (CIS). Quantification of the viability assay for cells was performed with MTT (each column is the mean ± s.e.m. of n=3 cultures). Statistical analysis was performed with ANOVA (*P<0.05; **P<0.01).

 

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© The Company of Biologists Ltd 2009