Introduction 1 2 3 4 5 4 6 TP53 7 9 p53 ARF 10 11 Trp53 Patched Ptch+/− 12 TP53 TP53 4 5 wild-type TP53 7 MDM2 MDM2 TP53 13 MDM2 14 15 MDM2 16 WIP1 wild-type p53-induced phosphatase 1 or protein phosphatase, magnesium-dependent 1, delta, PPM1D wild-type TP53 17 19 17 21 WIP1 22 23 24 WIP1 WIP1 WIP1 WIP1 Materials and methods Medulloblastoma tissue and cell lines n 1 Ten patients were female and 23 were male. The mean age at diagnosis was 82 months (median 79 months) with four patients less than 36 months old (range: 12–216 months). The median and mean time of follow-up was approximately 42 months from diagnosis. The majority of patients were M0 (non-metastatic), while one was M2 and eight were M3 stage at diagnosis. All studies were performed according to the institutional guidelines of Texas Children’s Hospital, and with the approval of the Institutional Review Board of Baylor College of Medicine. WIP1 l 2 Fluorescence in situ hybridization (FISH) analysis 25 WIP1 26 WIP1 27 WIP1 Quantitative real-time RT-PCR (qRT-RTPCR) analysis 12 WIP1 TP53 GAPDH GAPDH Western immunoblot analysis 2 Transfections and luciferase reporter assays 28 29 For flow cytometry, the EGFP-expressing pMaxGFP plasmid (Amaxa, Gaithersburg, MD) provided transfection controls. For functional assessment of in vitro p53 activation, cells transfected with luciferase-encoding reporter plasmids were harvested and processed for dual color luciferase assay according to manufacturer’s recommendations (Promega, Madison, WI), and quantitated using a BenchMark Plus plate spectrophotometer (Bio-Rad). Flow cytometric and terminal deoxynucleotidyl transferase-mediated biotinylated-dUTP nick end-labeling (TUNEL) analysis 0 1 n 0 1 t 0 1 . Results WIP1 DNA copy number is increased in medulloblastomas with 17q gain WIP1 1 WIP1 WIP1 1 WIP1 1 WIP1 1 Fig. 1 WIP1 WIP1 green red A WIP1 B WIP1 C D L D H I L WIP1  WIP1 24 1 WIP1 Table 1 WIP1 Specimen Copy number (FISH) Amplification* CNA (CGH) CEP 17 WIP1 WIP1 Chr 17 status D283 2–4 4–9 Y i17q Daoy 4 3 N +17 MCF-7 4 >50 Y ** MB13 2 6–7 Y −17p12-p13, +17p11.2-q25 MB1 2–4 5–8 Y i17q MB3 2–4 3–7 Y i17q MB14 2–4 2–7 Y +17 MB15 2–4 2–6 Y +17 MB5 2 6 Y i17q MB2 3–4 6–11 Y i17q MB6 2 4 N i17q MB28 2 2 N N/C MB27 2 2 N N/C MB4 2–3 2–3 N i17q WIP1 WIP1 WIP1 P *Amplification is defined as >4 copies as determined by FISH **Reportedly amplified, but not determined in the present study Abbreviations 1 WIP1 1 4 WIP1 P t WIP1 WIP1 WIP1 WIP1 WIP1 WIP1 WIP1 WIP1 2 WIP1 2 WIP1 Fig. 2 WIP1 A WIP1 +17q/i17q MB n non-17q gain MB n WIP1- Columns error bars Y-axis GAPDH B WIP1 n C WIP1 WIP1 open diamond open circle Abbreviations  WIP1 2 2 WIP1 n n WIP1 P 2 2 WIP1 2 2 n WIP1 n WIP1 2 2 WIP1 WIP1 P 2 Table 2 WIP1 Tumors grouped by CNA n WIP1 P +17q MB 3 28.3 0.02* i17q MB 6 25.4 <0.0001* +17q or i17q MB 9 26.3 <0.00001* Non-17q gain MB 2 6.5 – WIP1 7 27.7 0.05** WIP1 4 14.1 – Tumors analyzed by FISH (subtotal) 11   +17q MB 5 20.2 0.0533* i17q MB 11 20.3 <0.0001* +17q or i17q MB 16 20.3 <0.00001* Non-17q gain MB 17 5.9 – All tumors analyzed by CGH (total) 33 13.0 – WIP1 WIP1 P WIP1 P P t P t WIP1 Abbreviations Medulloblastomas with gain of 17q overexpress WIP1 protein 3 3 3 30 3 WIP1 3 Fig. 3 A 2 WIP1- B n Abbreviations n  3 WIP1 n n 3 3 Genotoxic stress induces p53 phosphorylation and WIP1 expression in medulloblastoma cell lines WIP1 TP53 4 WIP1 wild-type TP53 4 Fig. 4 A 2 wild-type WIP1 B TP53 WIP1 TP53 WIP1 C TP53 error bars D  TP53 WIP1 WIP1 TP53 4 WIP1 TP53 WIP1 wild-type TP53 4 VP-16 and constitutively expressed p53 induce apoptosis 0 1 4 0 1 P 5 wild-type Fig. 5 A n 0 1 n 2 0 1 B C D E Y-axis X-axis F G 0 1 Y-axis X-axis 0 1 t P P P P P  0 1 P 5 0 1 P 5 TP53 wild-type TP53 0 1 wild-type 5 wild-type TP53- 0 1 P 5 TP53 WIP1 overexpression limits VP-16- and p53-induced apoptosis wild-type TP53 5 WIP1 TP53 0 1 P 5 WIP1 TP53 5 0 1 5 . WIP1 wild-type WIP1 WIP1 5 WIP1 wild-type WIP1 WIP TP53 0 1 5 WIP1 TP53 Discussion TP53 WIP1. WIP1 WIP1 22 WIP1 23 WIP1 WIP1 WIP1 20 31 21 Wip1 Wip1 TP53 20 32 WIP1 17 18 WIP1 TP53 WIP1 17 WIP1 33 34 TP53 WIP1 WIP1 WIP1 WIP1 WIP1 WIP1 WIP1 WIP1 22 23 WIP1 21 30 33 WIP1 wild-type TP53- wild-type 35 37 WIP1 WIP1 WIP1 wild-type TP53 38 39