Biochimica et Biophysica Acta 1833 (2013) 1511–1526

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Biochimica et Biophysica Acta

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A novel snRNA-like transcript affects amyloidogenesis and cell cycle progression through perturbation of Fe65L1 (APBB2) alternative splicing

Ilaria Penna a, Irene Vassallo a, Mario Nizzari b, Debora Russo c, Delfina Costa a, Paola Menichini c, Alessandro Poggi c, Claudio Russo d, Giorgio Dieci e, Tullio Florio b,f, Ranieri Cancedda a,c, Aldo Pagano a,c,⁎ a Dept. of Experimental Medicine (DIMES), University of Genova, Genova, Italy b Dept. of Internal Medicine (DIMI), University of Genova, Genova, Italy c IRCCS-AOU San Martino-IST, Genova, Italy d Dept. of Health Sciences, University of Molise, Campobasso, Italy e Dept. of Molecular Biology and Biochemistry, University of Parma, Parma, Italy f Center of Excellence for Biomedical Research (CEBR), University of Genova, Italy article info abstract

Article history: FE65 constitute a family of adaptors which modulates the processing of amyloid precursor Received 13 August 2012 and the consequent amyloid β production. Thus, they have been involved in the complex and partially un- Received in revised form 17 January 2013 known cascade of reactions at the base of Alzheimer's disease etiology. However, FE65 and FE65-like proteins Accepted 18 February 2013 may be linked to neurodegeneration through the regulation of cell cycle in post-mitotic neurons. In this work Available online 26 February 2013 we disclose novel molecular mechanisms by which APBB2 can modulate APP processing. We show that APBB2 mRNA splicing, driven by the over-expression of a novel non-coding RNA named 45A, allow the generation of alter- Keywords: β APBB2 alternative splicing native protein forms endowed with differential effects on A production, cell cycle control, and DNA damage re- Cell cycle progression non-coding RNA sponse. 45A overexpression also favors cell transformation and tumorigenesis leading to a marked increase of RNA polymerase III malignancy of neuroblastoma cells. Therefore, our results highlight a novel regulatory pathway of considerable in- Neurodegeneration terest linking APP processing with cell cycle regulation and DNA-surveillance systems, that may represent a molec- β amyloid ular mechanism to induce neurodegeneration in post-mitotic neurons. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Indeed, it was reported that APBB2 co-localizes with the AICD (Amyloid Intracellular C-terminal Domain) [9] and that a transcription- FE65 and FE65-like {FE65L1, also called APBB2 [ ally active APBB1-AICD-Tip60 complex can be formed [10] thus bringing (Aβ) precursor protein-binding, family B, member 2, NP_004298.1] to light an Aβ-independent contribution of APBB proteins to AD. and FE65L2 (BAA78674.1)} proteins constitute a family of adaptors However, specific modulation of transcription for APBB2 has not been which modulates the processing of amyloid precursor protein described yet, suggesting that its influence on global -expression (APP) and the consequent synthesis of amyloid β. Thus, these protein might need to be mediated by other proteins [11]. Notwithstanding, adaptors have been involved in the complex and partially unknown recent experimental observations showing that the AICD overexpression cascade of reactions at the base of Alzheimer's disease (AD) etiology lead to AD-like phenotype in vivo, suggest that the contribute of AICD [1,2]. In particular, the direct involvement of APBB2 in AD may be (and of the modulators of its functional activity) to AD onset might be related to its propensity to vicariate APBB1 (Fe65). In fact, like APBB1, as relevant as that of the secreted portion of APP, Aβ [12]. APBB2 might participate to AD development altering APP processing Besides the role in and APP processing, its involve- through an increased production of amyloid β [2] or interacting with ment in cell cycle regulation is a further aspect of APBB2 biological func- Tip60 [K (lysine) acetyltransferase 5, NC_000011.9] acting in the intracel- tion that might be linked to neurodegeneration. Indeed, it has been lular compartment [3–8]. In both cases we must consider the possible reported that the overexpression of APBB2 in PC12 cells inhibits competition among APBB1 and APBB2 and 3, as APP-interacting proteins. cell cycle progression, via its nuclear translocation and inhibition of thymidilate synthase (TYMS, NC_000018.9) leading, ultimately, to a cell cycle delay [13]. In this context, although the link between TYMS downregulation and AD pathogenesis is not clear yet, it was proposed that this phenomenon might lead to a weakening of the ⁎ Corresponding author at: IRCCS-AOU San Martino-IST, Genova, Italy. Tel.: +39 0105737241; fax: +39 0105737257. DNA repair apparatus with consequences on chromosomal stability E-mail address: [email protected] (A. Pagano). [13]. Thus, the high rate of aneuploidy detected in AD brains constitutes

0167-4889/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbamcr.2013.02.020 1512 I. Penna et al. / Biochimica et Biophysica Acta 1833 (2013) 1511–1526 a further possible link among APBB2 expression, cell cycle control and 2.2. Real time quantitative RT-PCR analysis neurodegeneration, that deserves further investigations. In this work we searched for molecular links among the multiple Total RNAs from samples were extracted using TRIzol reagent roles played by APBB2 in APP processing starting from a novel aspect (Invitrogen) according to the manufacturer's protocol, DNAseI-digested of APBB2 biology: the synthesis of alternative protein forms with pecu- and subjected to reverse transcription by Transcriptor High Fidelity liar biochemical features through mRNA maturation [11]. Among the cDNA Synthesis Kit (Roche 05081955001) following manufacturer's already characterized isoforms, the alternative splicing variants “a”, instructions. The total RNA from samples was measured by real-time “b” and “c” are very similarly spliced except for the lack of exon 8 quantitative RT-PCR using PE ABI PRISM@ 7700 Sequence Detection (63 bp/21 residues-long stretch) in variant “c”. Despite the potential System (Perkin Elmer Corp./Applied Biosystems, Foster City, CA) and relevance in neurophysiopathology of APBB2 alternative splicing, the SybrGreen method as described elsewhere [19].Thesequencesoffor- key factor(s) affecting this process and its tissue- and stage-specificity ward and reverse primers were: 45A: 5′-CATCTATAATGGCTGAATTG are largely unknown. GAA-3′ and 5′-ATGAACTTTCCAACAAATGTTGTT-3′; APBB2 tot.: 5′-GAGC We recently demonstrated that the pol III transcriptome is much CGAGGCGAGCG-3′ and 5′-TTCCATCCAGCAAATGTTTCTG-3′;APBB2ex. larger than expected and that a fraction of these non-coding (nc) RNAs 8: 5′-GTCACTTAGTTCTGTAACGCCATCTC-3′ and 5′-CCCCCATTCAGAACA might act as specific regulators of gene expression of protein-coding GCAA-3′;APP:5′-TGGCCCTGGAGAACTACATCA3′ and 5′-CGCGGACATA target [14–17]. Functional studies of two of these RNAs showed that CTTCTTTAGCATATT-3′. For endogenous control, the expression of they map to an intronic portions of Pol II in antisense configura- Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) was examined. tion and, when expressed, perturb pre-mRNA maturation driving to The sequences for human GAPDH primers were 5′-GAAGGTGAAGGT the synthesis of alternative protein forms with peculiar biological fea- CGGAGTC-3′ and 5′-GAAGATGGTGATGGGATTTC-3′.Thesequencesfor tures [18,19]. Here we report on a novel pol III-transcribed ncRNA human 5S rRNA primers were 5′-TACGGCCATACCACCCTGAA-3′ and (hereafter referred to as 45A) that maps in the intron 1 of APBB2 in an- 5′-GCGGTCTCCCATCCAAGTAC-3 ′ and for human U6 primers were tisense (AS) configuration. In consideration of its sub-chromosomal 5′-AATTGGAACGATACAGAGAAGATTAGC-3′ and 5′-TATGGAACGCTTCA localization and previous studies on other small nuclear (sn) RNA-like CGAATTTG-3′. Relative transcript levels were determined from the transcripts of our collection, we postulated the possible influence of relative standard curve constructed from stock cDNA dilutions, and di- 45A expression on APBB2 pre-mRNA maturation thus linking its geno- vided by the target quantity of the calibrator following manufacturer's mic location to its biological role. instructions. For the determination of APBB2 variants RNA expression Herein, we demonstrate that 45A expression leads APBB2-specific in human post-mortem brain samples, we selected the appropriate pre-mRNA alternative maturation driving to the synthesis of alterna- housekeeping gene [Topoisomerase (DNA) I, NM_003286.2] taking tive protein forms. In turn, these alternative APBB2 molecules perturb advantage of geNorm Housekeeping Gene Selection Kit as described the processing of APP C-terminal fragments (CTFs) and, concomitant- elsewhere [30]. ly, exert deep effects on cell cycle regulation, altering the synthesis of cell cycle control- and DNA repair-related proteins. The phenotype 2.3. Human brain samples driven by 45A overexpression also favors cell transformation and tu- morigenesis leading to a marked increase of malignancy of neuroblasto- Frontal and temporal cortices of 20 human post-mortem samples ma cells both in vitro and in vivo. Therefore, our results highlight a novel were derived from P. Gambetti and Brain Bank (Case Western Reserve pol III-dependent regulatory pathway of considerable interest linking University, Cleveland, OH, USA). APP processing with cell cycle regulation and DNA-surveillance systems as a mechanism of neurodegeneration, and providing a novel possible 2.4. Aβ detection in HEK-293 permanently transfected cells connection among different hypotheses on AD pathology. The amount of secreted Aβx-40 and Aβx-42 in the media of 45A 2. Materials and methods permanently transfected HEK-293 cells was evaluated by sandwich ELISA (IBL, Gumna, Japan) as previously reported [20]. 2.1. Cell culture, transfection and luciferase assay 2.5. Western blot Different cells lines were used: HEK-293 (Human Embryonic Kidney), HeLa (Human epithelial cervical cancer), LoVo (Human Proteins were quantified using a commercial protein quantification Colon Carcinoma) and SH-SY5Y (Human Neuroblastoma) cells were kit (Protein Assay, Bio-Rad 500-0006) following the manufacturer's maintained on Dulbecco's modified Eagles medium (DMEM ECB7501L, instructions. The samples were subsequently analyzed by denaturing EuroClone, Milan, Italy), 10% FBS (LONZA DE14-801F), L-glutamine polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a (2 mM; EuroClone, Milan, Italy), and penicillin–streptomycin (100 U/ nitrocellulose membrane (Whatman, Inc.) as described elsewhere ml/100 μg/ml; EuroClone, Milan, Italy). SKNBE2 (Human Neuroblastoma) [21,22]. The primary antibodies used were: rabbit polyclonal anti-APP, cells were maintained on RPMI 1640 medium (ECB9006L EuroClone, C-terminal (A 8717 Sigma) (1:1000), anti-α-tubulin (T 5168 Sigma) Milan, Italy), 10% FBS (LONZA DE14-801F), L-glutamine (2 mM; (1:2000), anti-APBB2 (ab64749 Abcam). Amyloid C-Terminal Frag- EuroClone, Milan, Italy), penicillin–streptomycin (100 U/ml/100 μg/ml; ments Western blot analysis was performed by running protein sam- Euro Clone, Milan, Italy) (standard medium). HEK-293, SH-SY5Y and ples on precast Tris-tricine gels in gradient 10–20% (Novex® 10–20% HeLa cells were transfected using polyethylenimine (PEI; Sigma P3143), Tris-Glycine Gel 1.0 mm, 12 well). The membranes were blocked by SKNBE2 cells by a Microporator MP-100 (Digital Bio; NanoEnTek, Seoul, an incubation of 1 h in phosphate-buffered saline Tween-20 containing Korea; Labtech France) with pEGFP-N1 as control (hereafter referred 5% non-fat dried milk. The blot was incubated overnight with the pri- to as HEK-M) and pEGFP-N1-45A (hereafter referred to as HEK-45A). mary antibody, rabbit polyclonal anti-APP, C-terminal (A 8717 Sigma)

G418 (Geneticin; Invitrogen) was used in culture medium as mean of (1:1000), diluted in 0.1% NaN3 in PBS. The densitometric analysis of pro- selection up to 1000 μg/ml, until resistant cells were identified. After tein bands was performed using the ImageJ software system. selection, the cells were preserved in 200 μg/ml G-418 in standard culture conditions. The luciferase-based promoter activity assay was 2.6. Immunofluorescence detection (confocal microscopy) performed 48 h after transfection by firefly luciferase activity determina- tion with the dual-luciferase reporter-assay system (Promega, Madison, HEK-M and HEK-45A cells grown on slides, were fixed with meth- WI, USA) according to the manufacturer's protocol. anol, washed 3 times with PBS and incubated with the different I. Penna et al. / Biochimica et Biophysica Acta 1833 (2013) 1511–1526 1513 primary antibodies in PBS plus 1% NGS for 1 h, and finally incubated with enzyme. The cDNA was mixed with RT2 SYBR green/ROX qPCR master antigen-specific secondary antibodies (Alexa 488 or 568-conjugated, and mix (SABiosciences Corporation) and 25 μl aliquots were loaded into AMCA from Invitrogen Corporation, Carlsbad, CA, USA). Cells were then each well of the RT2 Profiler PCR Array (SABiosciences Corporation, mounted by Mowiol and analyzed on BioRad MRC 1024 ES confocal Cat. No. PAHS-020 SABiosciences). The PCR array was designed to microscope, equipped with a Nikon Eclipse TE 300 inverted microscope study the profile of 84 human cell-cycle-related genes. PCR array with a 60× objective lens [23]. Antibodies for rabbit anti-APBB2 (Abcam experiments were performed on an Eppendorf Realplex4 epgradient S 64749) were used at 1:100; Alexa Fluor® 568 and Alexa Fluor® 488 Mastercycler®. Conditions for amplification were as follows: 1 cycle of (Molecular Probes, Invitrogen Corporation, Carlsbad, CA, USA) were 10 min at 95 °C followed by 40 cycles of 15 s at 95 °C and 1 min at used at 1:200. 60 °C. The PCR array data were analyzed by PCR Array Data Analysis web-based software (SABiosciences Corporation). 2.7. Cell proliferation assays

A) We tested proliferation of HEK-M and HEK-45A cells, plating 2.11. Colony forming assay 25 × 104 cells/well in round-bottomed 24-well plates; cells were incu- bating for 24 h and pulsed with [3H]-thymidine (0.3 μ Ci/500 μl/well) HEK-M and HEK-45A cells were seeded at 400 cells in 60 mm dish, (GE Healthcare, New York, NY) for the last 14 h. We calculated the 3 dishes per dose, for the determination of cell survival measured as – fi average proliferation rate by the thymidine uptake assay. B) For cell colony forming ability. After 8 10 days, the dishes were xed and col- 5 onies counted. Adriamycin (Ebewe Italia, Rome, Italy) was diluted in counting studies, HEK-M and HEK-45A cells were seeded at 5 × 10 − cells in 25-cm2 culture flasks, incubated in standard medium and count- PBS , like MMS (Fluka); cells were UV-C irradiated in the absence ed with a hemocytometer after 48 hours. C) Finally cell proliferation of medium with a germicidal lamp emitting 254-nm, as monitored was also assessed by xCELLigence RTCA MP System (Roche, Germany) by a UVX digital radiometer (Ultraviolet Products, San Gabriel, CA). μ μ μ that monitors cellular events in real time by measuring electrical im- Adriamycin was tested at doses 0.01 M, 0.05 M and 0.1 M; MMS pedance across interdigited gold micro-electrodes integrated on the was tested at doses 0.05 mM, 0.1 mM and 0.5 mM; UV doses were 2 2 2 bottom of tissue culture plates. The impedance measurement provides 5 J/m , 10 J/m and 20 J/m . quantitative information about the biological status of the cells, includ- ing cell number, viability, and morphology. Cell-sensor impedance is 2.12. Cytotoxicity assay in real-time expressed as an arbitrary unit called Cell Index (CI). In order to deter- mine the Cell Index, cells were seeded into 100 μL of standard medium Cytotoxicity assay was performed by xCELLigence RTCA MP Sys- in 96× microtiter plates (E-Plate-Roche, Germany). Background imped- tem (Roche, Germany). 104 cells were plated in E-plate 16; for each ance was determined using 50 μl of standard medium. Cell attachment, treatment, cells were seeded in triplicate. 24 h after seeding, cells spreading and proliferation were monitored every 15 min using the were treated with adriamycin (0.5 μMe5μM) and MMS (100 μMe xCELLigence system. Experimental results were performed using RTCA 1 mM) and the CI was monitored every 30 min up to the end of the Software 1.2 that calculated the population doubling by fitting the experiment (140 h). curve to an exponential equation. The number of cells seeded was dif- ferent for every cell type: HEK-293 8 × 103 cells, HeLa 4 × 103 cells, 3 4 SH-SY5Y 7 × 10 cells and SKNBE2 10 cells. 2.13. MN assay

2.8. Cell cycle analysis The day before treatment, cells were seeded in 35-mm dishes with coverslips. Cells were cultured at densities of 3 × 105 cells/coverslip, fi The cell cycle was analyzed after 70/30% ethanol/water xation using conditions ensuring that cells were actively growing after permeabilization with 1% Nonidet P-40 (Sigma) and labeling with 24 h treatment for adriamycin, 1 h treatment for MMS and a time propidium iodide (PI; Sigma). Samples were run on a FACSort variable for UV, depending from the intensity of the lamp. At the ap- fl cyto uorimeter (Becton Dickinson, Palo Alto, CA, USA), and cell cycle propriate harvest time, cells were rinsed and exposed to a hypotonic analysis was performed by the ModFit LT 3.0 computer program (Verity shock in situ using Iskandar solution (0.9% NaCl/75 mM KCl; 9:1). Software House, Topsham, ME, USA). Cells were then fixed with MeOH/HOAc (3:1) and stained with Giemsa. All slides including those of positive and solvent controls were coded 2.9. Apoptosis analysis before analysis and scored blindly for the evaluation of MN by two inde- pendent observers. The criteria used for identifying MN fulfilthose fl Apoptosis was analyzed by ow cytometry, using Annexin V accor- recommended by the HUMNwork [25]:(1)areab1/3 the main nucleus ding to the manufacturer's instructions Annexin V-APC (Apoptosis Detec- area; (2) no overlapping with the nucleus (distinct borders); and – tion Kit I; BD Biosciences, Oxford, UK; DAPI, Sigma Aldrich, Milan, Italy). (3) same aspect as the chromatin. Adriamycin was tested at doses HEK-M and HEK-45A cells were trypsinized, washed in PBS and 0.01 μM and 0.025 μM; MMS was tested at doses 0.05 mM and resuspended in serum free medium. Annexin V-APC and DAPI were 0.1 mM; UV doses were 5 J/m2 and 10 J/m2. added to the cell preparations (105 cells) and incubated for 15 min in the dark at room temperature [24]. Samples were analyzed using a Cyan ADP cytofluorimeter (Beckman-Coulter, Brea CA, USA). For each 2.14. Methylcellulose colony formation assay sample, 20,000 events were acquired. The data were analyzed using Summit 4.3.1 software (Beckman-Coulter, USA). Clonogenic assay was performed using a methylcellulose medium consisting of DMEM for HEK-293 cells and RPMI 1640 for SKNBE2 cells 2.10. cDNA synthesis and real time-PCR array with 0.9% methylcellulose (Methocult H4100; StemCell Technologies, Vancouver, BC, Canada), 10% FBS, 100 U/ml penicillin/streptomycin and 2 The cDNA for each RNA sample was obtained using RT First Strand 2mML-glutamine. Cells were plated at a density of 400 cells in 2 ml kit (SABiosciences Corporation, QIAGEN Company, Frederick, MD) volume of methylcellulose medium in humidified 6-well plates. For according to the manufacturer's instructions. Briefly, after genomic each assay, suspension cells were plated in triplicate. Colonies were DNA elimination, the reverse transcription reaction was performed at counted at 12 days for HEK-293 cells and at 14 days for SKNBE2 cells 42 °C for 15 min and then heated at 95 °C for 5 min to inactivate the after plating. 1514 I. Penna et al. / Biochimica et Biophysica Acta 1833 (2013) 1511–1526

2.15. Cell migration assay mouse had the tumor mass. For SKNBE2-45A mice, we calculated the weighted mean of the daily measures as follows: to mice #1, #2, #3 Cell migration was analyzed by the new technique of real-time migra- and #4, characterized by a similar growth progression, we attributed a tion monitoring using the CIM-Plate 16 and xCELLigence System RTCA DP relative weight value of 22.5%, with respect to the mouse #5 to which Instrument (Roche). The day before migration assay, cells were plated in we attributed a relative weight value of 10%. This method has been starvation medium (medium with FBS 0.5%). After 24 h, 4 × 104 cells, used to reduce the effect of the fifth mouse, whose growth progression resuspended in 100 μl of serum-free medium, were seeded in the upper in terms of time and tumor size was atypical. For each group we also chamber of a CIM-Plate 16. The upper chamber was then placed on derived the averaged progression rate by the linear regression. the lower chamber of the CIM-Plate 16 containing growth medium supplemented with 20% FBS as an attractant, or without FBS (negative 2.20. Statistical analysis control). Time-dependent cell migration was monitored over a period of up to 18 h, and experimental results were analyzed using RTCA Soft- Statistical significance of observed differences among different exper- ware 1.2 considering the interval of 0–6h. imental groups was calculated using a two-tailed t-test. In the figures, * and ** indicate statistical significance at P b 0.05 and 0.01, respectively. 2.16. Cell adhesion assay 3. Results This protocol was adapted from [26]. Cells were plated at 1,5 × 105 cells/well in a 24-well tissue culture plate; 3 h after cell plating, the 3.1. 45A is a novel snRNA-like transcription unit multiwell plate was centrifuged twice in inverted position (well bottom placed up in a swing-out rotor) at 1000 rpm for 30 s. The cells still By bioinformatic search for Proximal Sequence Element (PSE)/TATA- adherent to the wells were then fixed in 3.7% paraformaldehyde in containing snRNA-like elements in the , we identified a PBS, stained with methylene blue and counted (each well was divided novel transcription unit that synthesize a 78 nt long transcript (hereafter in quadrants and 3 quadrants/well were counted). Each value repre- referred to as 45A RNA) [16]. To gain hints onto its transcriptional activity sents the mean ± SD of three independent wells. in different cells and/or conditions we fused 45A pol III type 3 promoter to a luciferase silencer hairpin (pSHAG-45A, hereafter referred to as pS- 2.17. Immunofluorescence 45A). In this condition, if the 45A promoter is active in a specificcell line, the transcription of the hairpin drives the post-transcriptional silenc- For morphological examination, HEK-M and 45A cells were seeded ing of a co-transfected luciferase cDNA and, ultimately, the decrease of in l-Slide eight-well (Ibidi GmbH, Martinsried, Germany) in standard luciferase signal; on the contrary, unaltered luminescent signal would in- medium. After 24 h, the cells were fixed in pre-warmed (37 °C) 4% dicate that luciferase is not silenced and thus the 45A promoter is not ac- paraformaldehyde in phosphate-buffered saline (PBS) for 20 min at tively transcribed in the cell line tested. As shown in Fig. 1A, we detected a 37 °C, permeabilized with 0.1% Triton X-100 in PBS for 5 min, and 42% and a 46% decrease of luciferase emission 48 h after co-transfection blocked for 1 h in 2% bovine serum albumin/PBS. Then the cells were in- of pS-45A with a plasmid expressing luciferase (pGL3), in HEK-293 and cubated with rabbit anti-α tubulin (1:200 dilution; Abcam, Cambridge, SKNBE2 cells respectively, demonstrating efficient transcription of the UK) in PBS–bovine serum albumin 1% for 24 h at room temperature. hairpindrivenby45Apromoterinboththecelltypes.Aspositivecon- Following three rinses in PBS of 10 min each, cells were incubated trols, constructs harboring the same luciferase-silencing hairpin under with TRITC-labelled secondary antibody [anti-rabbit IgG (H + L) 1:75 the control of two well assessed Pol III type 3 promoters (those of dilution; Jackson Immunoresearch Laboratories, Inc. West Grove, PA, the U6 RNA gene, coding for an RNA component of the spliceosome, USA] in PBS–bovine serum albumin 1% for 1 h at room temperature. and the H1 gene, coding for the RNA component of RNAse P) led to a The incubation was stopped by three 10 min rinses with PBS. Cells significant luminescence reduction whereas a negative control (NP, were stained with DAPI (100 μg/ml; Invitrogen, Carlsbad, CA, USA) in a promoter-less hairpin) did not alter luciferase signal (Fig. 1A). Altogeth-

H2O for 5 min. Fluorescent images were captured with a Zeiss Axiovert er, these results demonstrate that 45A transcription unit is active both 200 M inverted microscope (Zeiss, Jena, Germany), equipped with HEK-293 and SKNBE2 cell lines. Apotome technology, at a final magnification of 63×, and processed by using Zeiss AxioVision 4.8 software (Zeiss). 3.2. The expression of 45A promotes APBB2 splicing perturbation

2.18. Mice Since 45A transcription unit maps within intron 1 of APBB2 in anti- sense configuration (see Fig. S1) and considering that this protein is Homozygous NOD-SCID (NOD.CB17-Prkdcscid) mice were pur- subjected to alternative splicing that generates alternative protein forms chased from the Jackson Laboratory (Bar Harbor, MA). Mice were [as reported by AceView database (http://www.ncbi.nlm.nih.gov/IEB/ used between 5 and 9 weeks of age. All animals were bred and Research/Acembly/)] (Fig. 1B), we hypothesized that, similarly to other maintained at the institution's animal facility of the National Institute pol III-transcribed ncRNAs recently characterized by us [17–19], 45A tran- for Cancer Research, Genova, Italy. The care and the use of the animals scription might perturb APBB2 pre-mRNA maturation leading to the were in compliance with laws of the Italian Ministry of Health and the synthesis of alternatively spliced protein forms. guidelines of the European Community. To test this hypothesis we generated a cell line stably overexpressing 45A RNA driven by its endogenous promoter. In order to better visualize 2.19. In vivo tumorigenicity assay theeffectsof45Aexpression,wechoosecellsexpressingthelowestlevel of endogenous 45A [HEK-293 cells (see Fig. 1F)] to be equipped with SKNBE2-Mock (hereafter referred to as SKNBE2-M), SKNBE2-45A, extra-copies of 45A transcription unit together with a GFP reporter HEK-M and HEK-45A cell suspensions (3 × 106 cells in PBS) were sub- cassette (hereafter referred to as HEK-45A cell line). As control, HEK- cutaneously injected into 4 NOD/SCID mice groups composed of 5 mice 293-Mock cell line (hereafter referred to as HEK-M) was obtained inte- each. Mice were observed weekly for the appearance of tumors at injec- grating an empty pEGFP-N1 plasmid vector in HEK-293 genome. After tion sites; tumor size was measured daily with calipers in all groups and clonal selection, the expression level of 45A was measured by Real Time these experimental data have been represented by a progression tumor RT-PCR in both the transgenic cell lines. HEK-45A cells express graph. To calculate the tumor development function by the polynomial 43.14-fold increased level of 45A ncRNA with respect to HEK-M cells interpolation, we used the average of the daily measures when each (Fig. 1C). Similar levels of overexpression of 45A ncRNA were detected I. Penna et al. / Biochimica et Biophysica Acta 1833 (2013) 1511–1526 1515 AB 1.0 1.0 a

0.58 0.54 0.42 0.33 0.25 b 0.25 Luciferase emission

c pS-H1 pS-H1 pS-U6 pS NP pS NP pS-U6 pS-45A pS-45A HEK-293 SKNBE2

C 45 ** 5 73°C 5

44 43.14 4 4 81°C 77°C 3 43 3 ** ** 2 2 2 1.00 1.0 1.00 45A(-fold increase) 5S (-fold increase) 1 1 0 U6 (-fold increase) 1 0.40 0.24 0 0 0 M 45A M 45A M 45A D E ** ** 1.2 1.00 77.6°C 1.2 1.5 1.5 ** 2 ** 1.00 ** 0.88 1.50 1.00 1.00 0.81 86°C 1.5 0.8 0.8 1 0.83 1 1.00 0.55 1 0.4 0.4 0.5 0.5 (-fold increase) 0.5 45A (-fold increase) (-fold increase) APBB2ex.8/TOT Ratio APBB2 ex.8/TOT.

APBB2 EX.8(-fold increase) 0 0 0 0

0 APBB2 TOT (-fold increase) M 45A M45A M45A

F ** ** 5 5 5 5 ** 4.11 3.61 ** 4 4 ** 4 4 ** ** ** 3 ** 3 ** 3 3 ** 2.38 ** ** 1.85 ** 2 ** 1.73 2 1.52 2 1.46 2 1.35 0.83 1.22 1.01 0.92 APBB2 TOT.

0.40 APBB2 ex.8

1 (-fold increase) 1 0.19 (-fold increase) 0.69 0.16 0.14 (-fold increase) 1 1

45A (-fold increase) 0.13 0.07 0.06

0 0 Ratio APBB2 ex.8/TOT 0 0 LoVo LoVo LoVo LoVo HeLa HeLa HeLa HeLa SKNBE2 SKNBE2 SKNBE2 SKNBE2 HEK-293 HEK-293 HEK-293 HEK-293 SH-SY5Y SH-SY5Y SH-SY5Y SH-SY5Y

Fig. 1. The increased expression of 45A ncRNA promotes APBB2 splicing shift. A) Promoter activity transfection assay in HEK-293 and SKNBE2 cells. Transcription of a specific luciferase-silencing hairpin was driven by 45A PSE-dependent promoter. The promoter region encompasses the putative pol III type 3 regulatory regions (TATA box and PSE). pS-NP, pShag-No promoter negative control; pS-U6, pShag-U6 positive control; pS-45A, pSHAG-45A; pS-H1, pSHAG-H1 positive control. B) Schematic view of the three splicing variants (a, b and c) of APBB2 protein [variants a and b have an additive exon (the eighth) with respect to the c; variant a has the sixth intron longer than variant b and variant c (see Aceview annotations at www.ncbi.nlm.nih.gov/IEB/Research/Acembly)]. The location of primers, relative to 45A RNA is reported by darts. C) Expression of 45A, 5S rRNA and U6 snRNA in HEK-M and HEK-45A permanently transfected cell lines. Inset: amplification product dissociation curve unambiguously distinguishes the RNA of interest by peaks at specific temperatures. D) Real-time RT-PCR quantitative determination of APBB2 splicing variants and relative ratio in HEK-M and HEK-45A permanently transfected cell lines (Inset: the amplification product dissociation curve unambiguously distinguishes the RNA of interest by peaks at specific temperatures). E) 45A RNA expression and APBB2 spice variants ratio in HEK293 cells overexpressing Anti-45A RNA. F) 45A expression and determination of APBB2 splicing variants and relative ratio in cells of different origin, as determined by real-time RT-PCR.

also in two independently isolated clones (data not shown). In order to gene-specific and not due to a generic enhancement of RNA polymerase assess the specificity of 45A expression the level of synthesis of two III transcriptional activity (Fig. 1C). Altogether these data demonstrate unrelated pol III-transcribed ncRNAs such as U6 and 5S rRNA was mea- that HEK-45A cells constitute an adequate in vitro model to study the sured in the two cell lines. Results showed that the expression level of effects of 45A overexpression. these transcripts is stable if not slightly diminished in HEK-45A cells, To investigate possible differences in APBB2 splicing occurring in demonstrating that overexpression of 45A RNA in these cells is HEK-45A cells, we measured alternative APBB2 mRNA levels by splice 1516 I. Penna et al. / Biochimica et Biophysica Acta 1833 (2013) 1511–1526 variant-specific Real Time RT-PCR. Indeed, three alternatively spliced was mainly associated to Aβ-x40 release (42%) rather than Aβ-x42, APBB2 protein forms (derived by mRNAs endowed with both 5′ and whose secretion was modestly affected (16%). Therefore, this re- 3′ untranslated regions) have been so far predicted [namely variants sult shows that, besides a general inhibition of amyloid release, “a”, “b”, “c” in AceView (www.ncbi.nlm.nih.gov/IEB/Research/Acembly/ 45A overexpression, driving to a 44% increased Aβ-x42/Aβ-x40 av.cgi?exdb=AceView&db=36a&term=apbb2&submit=Go)]. ratio, may generate conditions favorable to Aβ aggregation These isoforms are not clearly discernible in SDS-PAGE as they only differ (Fig. 2A). by about 2.2 KD. To overcome this limitation we used Real Time RT-PCR In order to mechanistically strengthen the link between the perturba- reactions based on two different pairs of oligos: oligos α (that amplify tion of the relative amount of APBB2 protein variants (triggered by the all the splice variants “a”, “b” and “c”)andoligosβ [(that amplify exclu- overexpression of 45A RNA) and Aβ releasewetransfectedHEK293 sively those splice variants that enclose the 63 bp-long exon 8 (“a” and cells with a plasmid construct encoding for APBB2 protein variant “a” “b”)]. Results, expressed as oligos β vs. oligos α amplified mRNA ratio, thus mimicking the condition driven by 45A-overexpression. We then showed that HEK-45A cells stably produce a 1.5-fold increased β vs. α measured the amount of Aβ−x40 released in the medium 48 h splice variant ratio as a consequence of decreased expression of variant after transfection evidencing a 42% decrease of Aβ released “c” with respect to exon 8-containing variants (“a” and “b”)(Fig. 1D). in cells overexpressing APBB2 variant “a” with respected to Thus, these data demonstrate that 45A-overexpressing cells synthesize pMock-transfected cells (Fig. 2B).Again,thisfinding demonstrates increased amounts of exon 8-containing APBB2 splice forms (var. a, b) that an increase of the relative fraction of APBB2 variant “a” leads to in comparison to the exon 8-missing one (var. c). Although the additional adecreaseofAβ-x40 release. 63 bp-long exon 8 (Q92870) encodes for 21 a.a. most of which are hydro- Next, to assess whether 45A RNA overexpression leads specifically to phobic (52.4%) it has not been possible yet to discern its role in APBB2 adecreaseofAβ release, or rather it inhibits also the non-amyloidogenic function. To demonstrate the effective occurrence the different APBB2 pathway of APP processing we measured the amount of the C-terminal variants in normal human brains, RT-PCR analysis was performed on fragment C83 (CTF83) in HEK-45A and/or in HEK-M control cells. mRNA isolated from 20 human brain post-mortem samples. The re- We found that the level of C83 is markedly reduced by 45A RNA over- sults, expressed as the average of the single samples values, showed expression (−44% as resulting from the average of three independent that, although an expected variability among the different samples determinations) according with a global reduction of APP processing was detected, a significant amount of APBB2 variant “c” was driven by 45A-overexpression (Fig. 2C). detected, reaching a level comparable to HEK-Mock cells (data not To investigate the molecular mechanism mediating these effects, shown). we tested possible variations of APP synthesis in HEK-45A cells at Next, we further demonstrated that the perturbation of the ratio mRNA and at protein level. We show that the overexpression of 45A among different splicing variants was directly triggered by the syn- RNA does not alter the amount of APP suitable for processing, neither thesis of 45A ncRNA measuring the ratio in HEK293-Anti45A cells, a at mRNA nor at protein level (Fig. 2D). The data exclude that the re- transgenic cell line harboring a permanently an additional transcrip- duced release of amyloid β is due to the inhibition of APP synthesis tional unit expressing 45A in antisense (AS) configuration but rather suggest a direct effect on its processing. We thus (Anti-45A). Results showed that in this condition the decrease of addressed the hypothesis that the alternative forms of APBB2 the amount of 45A ncRNA is accompanied by reduction of APBB2 might have a different subcellular localization with marked effects exon8/total variants ratio (Fig. 1E). on their interaction with APP. As previously reported by others To further support the hypothesis that increased synthesis of [27] thiswouldreducetheresidencetimeofthecomplexAPBB2: splice variants a and b is driven by the overexpression of 45A APP that, as a consequence, would less efficiently expose APP to ncRNA, we measured the levels of (1) 45A ncRNA, (2) APBB2 variants γ -secretase cleavage. “a” + “b” + “c”, (3) APBB2 variants “a” + “b”, and (4) the ratio To test this hypothesis we took advantage of an APBB2-specific antise- between exon 8-containing and exon 8-missing protein forms in rum in order to possibly identify changes in the subcellular localization of five different cell lines, namely HEK-293, HeLa, LoVo, SH-SY5Y and APBB2 in 45A overexpressing cells. As shown in Fig. 2E, we found by con- SKNBE2. As shown in Fig. 1F, the synthesis of 45A ncRNA in human focal microscopy analysis, an extensive cellular and perinuclear localiza- cells of different origin is modulated in a cell type-specific manner tion of APBB2 in Mock and 45A-overexpressing cells. Conversely, while and roughly overlaps with the increase of the synthesis of the exon in the nucleus of Mock cells a barely detectable APBB2 signal was 8-containing mRNA variants. observed, a significant APBB2 nuclear localization was detected in 45A Although the comprehensive panel of possible APBB2 splice overexpressing cells (Fig. 2E), suggesting a different sorting within the variants include several other members here not evaluated, our cell of the alternative variants of APBB2. findings support the hypothesis that 45A ncRNA synthesis drives In the light of the above results, we concluded that the alternative a splicing shift of APBB2 leading to the expression of exon forms of APBB2 induced by the overexpression of 45A RNA, are differ- 8-containing protein forms (variants “a” and “b”). Therefore, in entially localized within the cell and that this phenomenon may affect this context we hypothesized that the balance among different APP processing. protein variants with peculiar functional traits might be at the base of cell physiology alterations. 3.4. 45A-driven cascade of reactions affects cell proliferation control

3.3. 45A overexpression inhibits Aβ secretion, increases Aβ-x42/Aβ-x40 The increased nuclear localization of APBB2 and the previously ratio and alters APBB2 subcellular localization reported alteration of cell proliferation rate by FE65 and related proteins overexpression [13], induced us to hypothesize that the message It has been demonstrated that FE65 overexpression increases Aβ brought to the nucleus by APBB2 might be amplified overexpressing secretion, through the enhancement of APP C-terminal fragments 45A RNA. More specifically, we postulated an increase of the cell prolif- processing and the release of AICD induced by a higher γ-secretase eration rate in HEK-45A cells carrying higher relative amounts of APBB2 cleavage. This prompted us to test whether the observed alteration of alternative forms vs. the canonical protein variant. Indeed, it has been the relative amounts of different APBB2 variants may affect Aβ release. previously demonstrated that overexpression of the canonical variants To this aim we measured, by sandwich ELISA, the amount of Aβ-x40 and of FE65 proteins restricts cell proliferation in fibroblasts and that in par- Aβ-x42 released by HEK-M and HEK-45A cells. We found a signi ficant ticular, APBB2 negatively modulates proliferation in PC12 cells through reduction of the total amount of released β−amyloid by 45A- a panel of genes that still remains to be completed [13]. In this context, overexpressing cells (39.22% in comparison to HEK-M). This effect we hypothesized an increased cell proliferation rate in HEK-45A cells in I. Penna et al. / Biochimica et Biophysica Acta 1833 (2013) 1511–1526 1517

A ** B ** ** ** 198.94 0.2 ** 1.00 200 180.77 20 18.17 200 1.0 15.33 0.15 160 16 160 120.92

ratio 0.58 120 105.59 12 120 0.10 0.5 A β tot

A β 1-42 0.1 A β 1-40 80 8 80

40 4 40 A β 42/40 0 A β 40-fold decrease 0 0 0 0 M 45A M 45A M 45A M 45A

C D E 1.2 1 0.95

1.4 M 45A 1.00 0.8

1.2 C83 0.4

1.0 (-fold increase) 0.44 α-tub. APP expression 0.8 0.0 M 45A C83 0.6 1.01 1.2 1 α 0.4 -tub. 0.8 C83 expression/Tubulin

0.2 APP C83 0.4 0.0

expression/Tubulin 0.0 M 45A α-tub. APP α-tub. M 45A

Fig. 2. 45A-driven reduction of APP processing and altered APBB2 subcellular localization. A) Decreased β-amyloid secretion and perturbation of the Aβx-42/Aβx-40 ratio in 45A-overexpressing HEK-293 cells. X axis, cell type; y axis, quantitative determination of Aβ (pg/ml) secreted in the medium as determined by sandwich ELISA. B) Decreased Aβx-40 secretion in APBB2 Var A-overexpressing HEK-293 cells. X axis, cell type; y axis, quantitative determination of Aβ (pg/ml) secreted in the medium as determined by sand- wich ELISA. C) C-terminal fragment 83 (CTF83) western blotting analysis in Mock and 45A cells. D) APP expression levels in Mock and 45A cells either at mRNA or at protein level as resulting by Real Time RT-PCR and western blotting respectively. E) Subcellular localization of APBB2 protein in Mock and 45A cells by confocal microscopy (Fitc: γ-tubulin, Red: anti-APBB2 64749).

which the variant “c” of APBB2 is diminished. To test our hypothesis we we measured by real time RT-PCR the expression of 45A RNA in two measured the proliferation rate of HEK-M and HEK-45A cells by di- SKNBE2-derived cell lines (S1 and S2) engineered in order to exhibit dif- rect determination of population doubling time (PD) and [3H]-thy- ferent levels of malignancy [14] (see Castelnuovo et al.). Results showed midine incorporation. As expected, we found a significant increase that 45A is predominantly expressed in the more malignant S2 cells of HEK-45A proliferation (1.58-fold) in comparison to HEK-M, whereas its amount is 2.7-fold decreased in the more differentiated, with a PD of 24 h in HEK-45A cells and of 30 h in HEK-M cells slightly malignant S1 cells (Fig. 3C). In addition, we compared the expres- (Fig. 3A). sion of 45A RNA in two neuroblastoma cell lines with different tumori- This result was validated analyzing cells of different origin: HEK-293 genic potential, such as the Myc-N-amplified SKNBE2 cells vs. the (epithelial), HeLa (epithelial), SH-SY5Y (non-Myc-N amplified Neuro- non-Myc-N-amplified SH-SY5Y cells evidencing again a direct correlation blastoma) and SKNBE2 (Myc-N amplified Neuroblastoma), permanent- between the expression of 45A and the oncogenic potential of the cell ly transfected with a plasmid harboring 45A transcription unit under (Fig. 3D). the control of its endogenous promoter. As resulting by xCELLigence de- Next, to characterize the mechanism by which 45A affects cell prolif- tection system, the stable overexpression of 45A ncRNA enhanced the eration, we analyzed HEK-45A and HEK-M cell cycle distribution by FACS. proliferation rate in all the cell lines tested, evidencing that 45A RNA Results showed only barely detectable differences between the two cell is a general regulator of intracellular pathways involved in the control lines as far as G0/G1 and S phases, whereas the percentage of cells in of cell proliferation (Fig. 3B). Similar results were obtained in SKNBE2/ G2/M (6.45%) was markedly diminished (4.46%) in HEK-45A cell line 45A-overexpressing cells (data not shown). Altogether these results (Fig. 3E). This scenario suggests that a restriction of G2 and G2/M transi- demonstrate again an inhibitory effect on cell cycle progression exerted tion is induced in these cells. In order to assess whether the augmented by the canonical APBB2 splice variant “a” thus supporting a direct role cell proliferation rate is accompanied by higher apoptotic rate, we on cell proliferation control of 45A ncRNA through an APBB2 splicing var- analyzed these cells by Annexin V-APC FACS and found that the percent- iant shift. age of viable vs. apoptotic cells is not affected by 45A overexpression In order to assess whether the effects of 45A RNA expression on cell (Fig. 3F). Altogether these results demonstrate a direct control of cell pro- proliferation might be also extended to the cell tumorigenic potential, liferation mediated by 45A ncRNA and supporting the direct involvement 1518 I. Penna et al. / Biochimica et Biophysica Acta 1833 (2013) 1511–1526 of APBB2 canonical variants in cell cycle arrest previously documented by 3.5. The alternative APBB2 protein forms regulate cell proliferation others [13]. Indeed, these data demonstrate that the down-regulation of modulating the expression of a set of cell cycle-related genes APBB2 isoforms, driven by 45A ncRNA, generates the opposite effect of the overexpression and that, unexpectedly, this process can be regulated Since the overexpression of 45A ncRNA affects cell proliferation, by an upstream pol III-dependent transcript. and considering that the canonical form of APBB2 is involved in the

A ** 1.8 1.58 40 1.6 30 B HEK293 45A 1.4 30 24 MOCK 1.2 1.00 1 20

(hours) 1132537496173 cpm 0.8 HeLa 45A Normalized 0.6 10 MOCK 0.4 Population Doubling 0.2 0 0 1 13 25 37 49 61 73 MOCK 45A MOCK 45A 45A SH-SY5Y C D Cell Index MOCK 6.0 3.0 2.54 4.71 5.0 42210 63 84 105 126 SKNBE2 4.0 45A 20 MOCK 3.0 1.69 45A RNA 1.0 0.79 45A RNA 2.0 (-fold increase) (-fold increase) 1.0 0 0 S1 S2

E 61.84 35.28 58.27 70 40 33.70 8 MOCK 6.45 200 60 35 30 6 50 4.46 100 25 40 0 20 4 0 50 100 150 200 250 30 % of cells Number % of cells 15 200 45A 20 % of cells 10 2

10 5 100

0 0 0 0 MOCK 45A MOCK 45A MOCK 45A 0 50 100 150 200 250 G0/G1 S G2/M PI

F 89.13 91.38 100 100 MOCK 45A 80 80 60 60 3.04 3.42% 7.82 % 5.19% 40 40 10.86 8.61 DAPI % 20 20 Viable cells (%) 0 Apoptotic cells (%) 0 Annexin V-APC I. Penna et al. / Biochimica et Biophysica Acta 1833 (2013) 1511–1526 1519

Fig. 4. The overexpression of 45A ncRNA modulates the expression of specific cell cycle related genes. A) Expression profile of genes involved in cell cycle progressionandincellcyclecontrolin HEK-29345A/HEK-293-M cells; these genes have been considered arbitrarily imposing a cut-off (+22.50 b up-regulated genes b +30 and −2.29 b down-regulated genes b −1).

modulation of nuclear gene expression [13], we hypothesized that Cycle RT2 Profiler™ PCR Array) the simultaneous expression of 84 the expression of this ncRNA (and the consequent alteration of the genes involved in cell cycle control, in HEK-45A and HEK-M cells. By relative abundance of APBB2 isoforms) might modulate the expression arbitrarily imposing appropriate cut-off values (+22.5 b y b +30 of genes involved in cell cycle control. To test this hypothesis we mea- gene expression for the up-regulated genes and −2.29 b y b −1 sured by cell cycle-specific real time low density array (Human Cell gene expression for down-regulated genes), we found 9 genes whose

Fig. 3. Effects of 45A ncRNA on cellular proliferation. A) [3H]-thymidine incorporation assay and population doubling (PD) time of HEK-293M and HEK-293-45A permanently transfected cell lines. B) Cell Index (CI) curves of cellular proliferation of cells of different origin Mock and 45A permanently transfected obtained by the xCELLigence RTCA DP Instrument (Roche). The peculiar time scale bar (in hour) for SH-SY5Y is due to their different proliferation rate. C) Determination by Real Time RT-PCR of the expression of 45A RNA in two transgenic SKNBE2 cell lines with different tumorigenic potential D) and in two different neuroblastoma cell lines. E) Distribution in cell cycle phases of HEK-293M and HEK29345A cells, as determined by PI-based FACS analysis. F) Diagrams of APC-Annexin V/DAPI flowcytometry of HEK-293M and HEK-293-45A cells. The lower left quadrants of each panel show the viable cells, which exclude DAPI and are negative for APC-Annexin V binding. The upper right quadrants (R3) contain the nonviable, necrotic cells, positive for APC-Annexin V binding and for DAPI uptake. The lower right quadrants (R5) represent the apoptotic cells, APC-Annexin V positive and DAPI negative. 1520 I. Penna et al. / Biochimica et Biophysica Acta 1833 (2013) 1511–1526

Fig. 5. 45A ncRNA affects short-term response to genotoxic stress. A) Percentage of colony survival of HEK-293M and HEK-29345A permanently transfected cell lines to adriamycin (Adr), Methyl methanesulfonate (MMS) and UV light treatments. B) Real Time cytotoxicity of HEK-293-M and HEK-29345A to Adr, MMS and UV monitored by the xCELLigence RTCA DP Instru- ment (Roche). C) Micronuclei (MN) induced in HEK-293-M and HEK-29345A cells before and after Adr, MMS and UV radiation treatments. D) Multinucleated cells (MultiN) HEK-293-M and HEK-293-45A before and after Adr, MMS and UV radiation treatments. E) An example of the microscope evaluation of MN (indicated by arrows) and MultiN cells (arrowhead). expression is significantly modulated by 45A overexpression (Fig. 4). play important roles in differentiation and in cell cycle arrest in Interestingly, in HEK-45A cells, up-regulation is observed for those response to DNA damage (Fig. 4). Therefore, this cell cycle-specific genes that promote cell proliferation, whereas down-regulated genes gene expression analysis could draw a novel scenario where the

Fig. 6. 45A ncRNA affects short-term response to cell cycle progression. PI-based FACS analysis, before and after A) adriamycin, B) methyl methanesulfonate and C) UV radiation treatments. Full bars: Mock cells; striped bars: 45A cells. U: untreated sample; T: treated sample. I. Penna et al. / Biochimica et Biophysica Acta 1833 (2013) 1511–1526 1521 1522 I. Penna et al. / Biochimica et Biophysica Acta 1833 (2013) 1511–1526 induction of cell cycle proliferation exerted by 45A ncRNA is mediated Alterations of cell cycle progression are associated to treatment not only by the promotion of cell cycle progression but also by the con- with genotoxic agents. Thus, we investigated whether the expression comitant inhibition of DNA damage response processes. of 45A ncRNA affected the distribution among cell cycle phases fol- lowing adriamycin, MMS and UV treatments. 3.6. The overexpression of 45A RNA affects short-term response to genotoxic To this aim we focused on the most efficient dosages of treatments stress and cell cycle progression previously observed and measured by FACS analysis potential changes of specific cell cycle phases induced by the agents (Fig. 6). Considering together the increased proliferation rate and the down- We found that if normalized to the reactions induced in untreated regulation of genes involved in DNA repair and cell cycle checkpoint acti- cells, the treatment with 0.025 μM adriamycin of 45A cells induces a vation in response to DNA damage (i.e. GADD45A and HUS1), we strong decrease of the percentage of cells in G2/M and a roughly com- hypothesized that the high proliferation activity of these cells might be plementary increase of the population in S phase whereas in Mock associated to increased susceptibility to genotoxic damage. To test this hy- cells this effect was undetected (Fig. 6A). Similarly, the treatment pothesis we investigated the response of Mock and 45A cells to the treat- with MMS of 45A cells increases the S phase reducing concomitantly ment with chemical and/or physical agents known to induce specificDNA G0/G1. Conversely, a significant decrease of the percentage of cells in damages resulting in the activation of different DNA repair pathways. G2/M was observed both in Mock and in 45A cells (Fig. 6B). Since UV To this aim cells were treated with adriamycin, methyl metha- treatment determine a slow down of the cell cycle evident as a high nesulfonate (MMS) and ultraviolet radiation (UV) and cell viability was fraction of cells in S phase and a reduction of cells in G2/M phase, we mea- assessed by colony forming ability and xCELLigence RTCA. Survival curves sured the cell cycle distribution of Mock and 45A cells following UV treat- obtained through the colony forming ability assay did not reveal signifi- ment. We found that in 45A-overexpressing cells, similarly to the Mock cant differences between the two cell lines (Fig. 5A).However,thereal cells, the S phase fraction is increased but 45A-overexpressing cells time monitoring of cell proliferation following adriamycin and MMS partially escape the block of cell cycle maintaining an increased fraction exposure revealed a higher sensitivity of 45A than Mock cells to these of cells in G2/M not found in Mock cells. This peculiar cell cycle distribu- agents (Fig. 5B). These differences may rely on the ability of the tion is balanced by the concomitant reduction of the G0/G1 cell phase in xCELLigence system to measure short-term effects of the treatment that Mock and even more in 45A cells. may not be detectable with the colony assay. Therefore, to better investi- These results indicate that the expression of 45A significantly affects gate the influence of 45A-overexpression on DNA damage response, the the progression through the cell cycle following exposure to different induction of micronuclei (MN) was determined. MN assay has emerged DNA damaging agents. However, while after adriamycin and MMS as one of the preferred methods for assessing damage treatments, 45A cells show a higher percentage of S-phase cells than [28,29]. MN are known to form when cells enter mitosis with unresolved the Mock cells, following UV irradiation the 45A-overexpressing cells chromosomal damage and can only be expressed in cells that complete overcome the cell cycle blockage observed in Mock cells after UV irradi- nuclear division. ation, reducing the G0/G1 phase (Fig. 6C). A possible explanation of the Cells were treated with 0.01 μM and 0.025 μM adriamycin [a dou- above results is that following UV irradiation the up-regulation of cell ble strand break (DSB) inducer that can activate the repair] and the cycle genes and the down regulation of GADD45A observed in generation of micronuclei in Mock and 45A cell populations was mea- 45A-overexpressing cells would speed up the progression through the sured. In HEK-M cells a dose-dependent increase of MN was detected, cell cycle. Nevertheless, this response did not influence genotoxic dam- whereas at high dosages of adriamycin HEK-45A cells generate a re- age formation (detected as MN and multinucleated cells), suggesting that duced number of micronuclei (Fig. 5C). Accordingly, treatment with the NER pathway was likely not affected by 45A-overexpression. On the MMS, an alkylating agent known to induce single strand breaks and other hand, treatment of 45A cells with adriamycin and MMS likely acti- chromosomal aberrations and efficiently activate the base excision vated other DNA damage response pathways that slow down repair (BER) pathway, increased MN formation in HEK-M but only DNA replication and, consequently, did not allow an exhaustive MN slightly modified their occurrence in HEK-45A cells, although this var- expression. iation was not statistically significant (Fig. 5C). The treatment of the same cells with UV, a physical agent activating the nucleotide exci- 3.7. 45A increases malignant potential of neuroblastoma cells in vitro sion repair (NER), did not affect MN formation both in Mock and in and in vivo 45A cells (Fig. 5C). In order to better clarify the effects of the genotoxic agents on Since 45A RNA induces marked effects on cell proliferation and 45A-overexpressing cells, we also tested the possible differential sus- on the capability of the cells to respond to genotoxic damages, we hy- ceptibility to adriamycin, MMS and UV also in terms of generation of pothesized that it might also affect biochemical pathways involved in multinucleated cells (Fig. 5D). Results showed that although the treat- neoplastic transformation which might accompany a forced increase ment with adriamycin induces an apparent reduction of multinucleated of cell proliferation induced by 45A RNA overexpression. To test this cells in Mock cells, the induction of multinucleated 45A-cells was not hypothesis we first evaluated the colony forming potential in non- affected by the treatment. Mock cells treated with MMS showed a adhesive conditions (methylcellulose) of both normal (HEK-293) and dose-dependent increase of multinucleated cells that was not observed tumoral (SKNBE2) cells. The overexpression of 45A in SKNBE2 neuro- in 45A cells. Last, we analyzed the effects of UV treatment on the two blastoma cells significantly increases the colony forming potential, cell populations. We found that in Mock sample multinucleated supporting a possible enhancement of cancer malignancy (Fig. 7A). cells increase proportionally to the stimulus, whereas 45A cells form On the contrary, the stable overexpression of 45A RNA in HEK-293 multinucleated cells only at the highest dose of UV. A graphical repre- cells does not increase the colony forming capacity suggesting that sentation together with the microscopic evaluation of these results at although 45A ncRNA promotes cell proliferation in both the cell lines, the most critical dosages of the experiments is reported in Figs. 5D it might favor malignant potential in a cell type-specificmannerin and E. vitro (Fig. 7B). Altogether these results suggest that the 45A-dependent increase of Besides the quantitative evaluation of the colony forming potential, cell proliferation rate is not accompanied by a significant increase of MN the qualitative characteristics of 45A-overexpressing cells provided us and multinucleated cells. Since cytotoxicity experiments showed a with indications of their possible acquisition of altered migratory capac- higher sensitivity of 45A cells toward adriamycin and MMS, the lack ity. Indeed, while HEK-M cells form rounded colonies in adherent con- of MN induction in 45A cells can be related to the consequences of ditions, 45A cells show less organized clusters of cells that tend to these exposures on cell cycle progression soon after the treatments. disperse throughout the surrounding space, according with the I. Penna et al. / Biochimica et Biophysica Acta 1833 (2013) 1511–1526 1523

Fig. 7. Increase of adhesiveness and migratory ability in 45A-overexpressing cells. A) Capacity of SKNBE2 and B) HEK-293 cells to form colonies in methylcellulose. C) Morphological analysis of colonies grown in adhesive condition of HEK-293-M and HEK-293-45A cells. Right panels: GFP-positive colonies, as shown in the overlay picture. Left panels: transmitted light picture. D) Migration assay: using the xCELLigence RTCA DP instrument (Roche), migratory capability of HEK-293-M and HEK-29345A cells has been quantified by the slope of the migration curve calculated by the RTCA 1.2 Software. E) Adhesion properties of HEK-293M and HEK-29345A permanently transfected cell lines. Adherent cells were stained by methylene blue staining (lower panel) and quantified by calculating the mean of cells counted in 3 independent wells (upper panel). F) Morphological analysis of HEK-293-M and HEK-29345A permanently transfected cell lines by ApoTome Microscope Technique from Carl Zeiss. Blue = DAPI, green = GFP, red = anti αtubulin.

acquisition of marked migratory capacity (Fig. 7C). To better investigate the methylene blue cell adhesion assays. Both the assays showed that this aspect we measured the changes in the migratory potential of HEK- 45A-overexpression leads to the acquisition of marked migratory activ- 45A cells by two additional approaches, the xCELLigence migratory and ity (Figs. 7D and E). Accordingly, the morphological analysis of these 1524 I. Penna et al. / Biochimica et Biophysica Acta 1833 (2013) 1511–1526 cells support their increased adhesiveness as HEK-45A cells, labeled volume of 3689 mm3 (see supplementary online material S2A). These with α Anti-tubulin IgGs, exhibited, in immunofluorescence analysis, results demonstrate that although our experimental setting using a strongly organized and extended net of tubulin fibers that is not de- HEK-293 cells does not allow to appreciate differences between the two tectable in HEK-M cells (Fig. 7F). Altogether, these experiments suggest cell lines in terms of capacity to initiate tumors, it clearly demonstrates that besides a general increase of cell proliferation the overexpression of that tumor progression potential might be significantly affected by 45A 45A might also favor transformation processes increasing malignant overexpression (Figs. 8AandS2A). potential. Next, we derived the polynomial interpolation by the average of To assess whether the increased expression of 45A RNA may favor daily measurements of tumor volumes in order to obtain a repre- tumor formation in vivo, we injected subcutaneously 3 × 106 45A- sentative function of the tumor progression. As expected, the overexpressing HEK-293 and SKNBE2 cells and their Mock controls in a growth trend of HEK-45A tumor masses is higher than HEK-M con- pool of 20 NOD-SCID mice and evaluated possible differences in tumor trol masses. Indeed, calculating the averaged progression rate of formation capacity and/or in tumor progression rate associated to 45A- the growing nodules starting from their mass ≥5mm3 to the time overexpression. In HEK-293 cells we did not observe a strike difference of sacrifice we found that the averaged progression rate among in terms of capacity to form tumors since, after a prolonged time the HEK-M-injected mice was 98,529 mm3/day whereas that of (39 days after the injection), 5 out of 5 (100%) of the HEK-M-injected the HEK-45A-injected counterparts was 134,43 mm3/day (inset in mice and 5 out of 5 (100%) of the HEK-45A-injected mice developed Fig. 8A). Altogether these results demonstrate that in HEK-293 tumor nodules. This result suggests that the capacity of HEK-293 cells to cells the overexpression of 45A does not affect strongly the capacity initiate tumors is partially independent on 45A-overexpression, as we to initiate tumor but augments significantly the tumor progression previously observed in vitro. Conversely, the tumor progression efficiency rate and the growth speed of nodules. was strongly different in HEK-45A- compared to HEK-M-injected mice. Even clearer was the effect of 45A RNA overexpression on SKNBE2 All the mice in the control group showed a tumor nodule volume cells. Here, we observed a significant difference in tumor initiation ≥5mm3 after 39 days and 4 out of 5 mice already showed a volume capacity between Mock- and 45A-injected mice as 29 days after the in- ≥5mm3 only after 30 days. On the contrary the group of HEK-45A- jection 5/5 (100%) of the SKNBE2-45A-injected mice and only 2/5 (40%) injected mice reached a tumor nodule volume ≥5mm3 after 23 days. of the SKNBE2-M-injected mice developed a tumor nodule ≥5mm3 Moreover, 3 out of 5 of HEK-M-injected mice did not reach a tumor vol- (See Supplementary Material online S2B for details). This is consistent ume of 2000 mm3 atthetimeofsacrifice (58 days), whereas with a marked increase of SKNBE2 tumor formation capacity driven HEK-45A-injected mice at the time of sacrifice had an averaged tumor by 45A RNA expression, according to the data obtained in vitro.Also

Fig. 8. 45A ncRNA expression favors cancer progression in vivo. Tumor development function by the polynomial interpolation of A) HEK-293 tumor nodules and B) SKNBE2 tumor nodules. In the inset of both figures it is represented the linear regression of the averaged progression rate. I. Penna et al. / Biochimica et Biophysica Acta 1833 (2013) 1511–1526 1525 the tumor progression rate showed marked differences between enhanced levels of 45A might disturb a homeostatic control of repairing SKNBE2-45A-injected and SKNBE2-M-injected mice as in the control mechanisms at chromosomal level directly or in competition with Fe65. group mice showed a 5 mm3 tumor nodule after an averaged 25th Although we did not observe an increased formation of micronuclei day whereas after the same lag of time the averaged tumor mass of in HEK-293 cells overexpressing the 45A ncRNA following different the SKNBE2-45A-injected mice was 16.32 mm3. Again, SKNBE2-45A- genotoxic stress, the results on cell survival suggest an impairment of overexpressing tumor nodules grow faster than their Mock counter- the DNA damage response in these cells. It has to be noted that the parts as resulting by the polynomial interpolation of daily averaged overexpression of 45A significantly increases the colony forming poten- measurements (Fig. 8B). Accordingly, the average progression rate tial in SKNBE2 neuroblastoma but not in HEK-293 cells, indicating that among SKNBE2-M-injected mice was 150.84 mm3/day whereas that the it could favor malignant potential in a cell type-specific manner. of their SKNBE2-45A-injected counterparts was 211.88 mm3/day Thus, the impairment of DNA surveillance mechanisms in other neuro- (inset in Fig. 8B). These results demonstrate that the overexpression blastoma cells overexpressing the 45A ncRNA, (i.e. SKNBE2), deserves of 45A RNA affects significantly both the capacity to initiate tumors further investigations. and the tumor progression/growth rate of nodules, providing a novel The highly efficient tumor formation capacity of 45A-overexpressing link between APBB2 maturation, Aβ release and the regulation of cell cells concomitant to a perturbation of Aβ processing toward more cycle. fibrilogenic species might represent the first molecular link between the effects exerted by the same effector on Aβ releaseandontheregulation 4. Discussion of cell cycle. In this context 45A ncRNA biological effects bring to light thepossibleco-regulationofthetwophenomenaandsupportsprevious In the recent past an active role of FE65 protein family (and in partic- observations on neurodegeneration, reconciling alternative hypothesis ular that of APBB2) in neurodegeneration has been repeatedly proposed, for neurodegenerative processes. Indeed, our data are consistent with and its involvement in pathways that cross Alzheimer's disease has been the most recent literature showing that aneuploid neurons characterize experimentally documented [11].Inparticular,aroleforAPBB2inmak- the brain of AD patients and that 3–4% of pyramidal neurons from AD ing APP available to γ-secretase cleavage, favoring its processing and hippocampus are aneuploid [38] and might provide novel hints to join the release of Aβ in the extracellular space has been described [2].In together different hypothesis [39]. light of this notion, if alternative APBB2 protein variants, with reduced In conclusion, here we demonstrate that the perturbation of the capability to interact with APP, are preferentially expressed, it is reason- relative levels of different splice variants of APBB2 is crucial for the able to expect reduced formation of APBB2:APP protein complex, less determination of the cell phenotype although how and which is the efficient γ-secretase cleavage of APP, and, ultimately, decreased Aβ specific contribution of each variant to the phenotype observed is secretion. unclear and deserves further studies. In this scenario, the determina- In this work we propose that the occurrence of this phenomenon tion of the level of synthesis of 45A RNA in neurodegeneration and/or is driven and regulated by the synthesis of a newly identified pol tumor samples deserves further investigations in order to bring to III-transcribed non-coding RNA. This transcript perturbs the matura- light its contribution to cancer and/or neurodegeneration and a possi- tion of APBB2 pre-mRNA altering the ratio among alternative protein ble molecular link between these two pathologies. variants. Although the specific biochemical features of each single Supplementary data to this article can be found online at http:// protein variant (and their affinity for APP) still need to be established dx.doi.org/10.1016/j.bbamcr.2013.02.020. in detail, in this work we show that by affecting APBB2 splicing, the β synthesis of 45A ncRNA ultimately conveys a reduced release of A . Acknowledgements This phenomenon leads to a general increase of Aβ-x42/Aβ-x40 ratio, a condition that might favor amyloid aggregation also in the Prof. Giuseppina Minopoli (University of Naple, Federico II) and absence of a generally increased release of total amyloid. In this Prof. Tommaso Russo (University of Naple, Federico II) are greatly ac- scenario, 45A RNA might, indirectly, contribute to the aggregation of knowledged for providing the APBB2-overexpressing plasmid. A.P. was fi plaques and proto bers [31] delineating its possible involvement in supported by MIUR (2007 PRIN Program prot. 2007945BZN), by the neurodegeneration or in other conditions that imply impaired APP Associazione Italiana Ricerca sul Cancro (2009 AIRC Program n° IG9378) β processing or increased A aggregation. and by the Associazione Italiana per la Lotta al Neuroblastoma (Genoa, fi However, although the nding of a possible upstream regulator of Italy). G.D. was supported by Fondazione Cariparma (2010 grant pro- β A -dependent pathways is novel per se, in our view the most intrigu- gram) and by the Italian Ministry of Education, University and Research fi ing aspect of our ndings is related to independent processes trig- (MIUR, PRIN Program). T.F was supported by the Italian Ministry of Edu- gered by 45A culminating with an unprecedented role of this small cation, University and Research (MIUR, FIRB Accordi di Programma 2011 RNA in the regulation of cell cycle and malignant potential. With RBAP11HSZS). this respect, it is interesting to note that the strongly increased prolif- eration rate demonstrated in 45A-overexpressing cells of different or- References igins is associated to the inhibition of the expression of cell cycle control pathways and possibly to a decreased efficiency of DNA dam- [1] H. Tanahashi, T. Tabira, Characterization of an amyloid precursor protein-binding age response mechanisms. 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