Patched heterozygous mice (Goodrich et al., 1997) were obtained from Matthew Scott’s lab at Stanford, and maintained by breeding with 129X1/SvJ mice from Jackson Laboratories. Math1-GFP transgenic mice (Lumpkin et al., 2003) were provided by Jane Johnson at UT Southwestern Medical Center. Math1-GFP/patched+/- mice were generated by crossing patched heterozygotes with Math1-GFP mice, and then backcrossing to Math1-GFP mice three times before further analysis. All mice were maintained in the Cancer Center Isolation Facility at Duke University Medical Center.
ISOLATION OF CELLS
Granule cell precursors (GCPs) were isolated from 7-day-old (P7) patched+/- mice; preneoplastic cells were obtained from 6 week-old patched mutants; and tumor cells were obtained from 10-25-week old patched mutants displaying physical and behavioral signs of medulloblastoma. Cells were isolated from each source using a protocol described in (Wechsler- Reya and Scott, 1999). Briefly, cerebella were digested in solution containing 10 U/ml papain Oliver et al. Pre-Neoplastic Stage of Medulloblastoma - 8 - (Worthington, Lakewood, NJ) and 250 U/ml DNase (Sigma) and triturated to obtain a cell suspension. This suspension was centrifuged through a step gradient of 35% and 65% Percoll (Amersham Biosciences, Piscataway, NJ), and cells were harvested from the 35%-65% interface. Cells were resuspended in serum-free culture medium consisting of Neurobasal containing B27 supplement, sodium pyruvate, L-glutamine, and penicillin/streptomycin (all from Invitrogen, Carlsbad, California) and counted on a hemacytometer. Cells used for RNA isolation were centrifuged and flash frozen in liquid nitrogen. For proliferation assays or immunostaining, cells were plated on poly-D-lysine (PDL)-coated tissue culture vessels and incubated in serum-free culture medium.
MICROARRAY HYBRIDIZATION AND ANALYSIS
RNA from GCPs, pre-neoplastic cells and tumor cells (isolated as described above, but not FACS-sorted) and from normal adult cerebellum (not dissociated) was converted to cDNA using the Superscript Choice cDNA kit (Invitrogen) and a T7-dT(24) primer (Genset/Proligo, Boulder, CO). cRNA was generated using a T7-transcription/labeling kit from Enzo Life Sciences and hybridized to Affymetrix U74Av2 chips (Affymetrix, Santa Clara, CA). Chips were scanned, and hybridization data were acquired using Affymetrix Suite 5.0 software. Affymetrix CEL files were normalized and quantified using Bioconductor software with the gcRMA model to quantify gene expression levels (Gentleman and Carey, 2002). Unsupervised principal components analysis (PCA) was used to identify the relationships among normal adult cerebellum, GCPs, pre-neoplastic cells and tumor cells based on expression profiles. To identify genes that were differentially expressed among GCPs, pre-neoplastic cells and tumor cells, supervised analysis was carried out. A gene-by-gene ANOVA model with three groups (GCP, pre-neoplastic, tumor) was used to fit the log2-transformed intensities. To correct for multiple comparisons, the nominal p-value was adjusted using the false discovery rate (Benjamini and Hochberg, 1995). Genes were considered to be differentially expressed if they satisfied all of the following criteria: a difference in expression greater than 1.9-fold between any two groups, a maximum absolute intensity difference larger than 32 units, and an adjusted pvalue < 0.01. There were 118 genes that met these criteria. The identities of differentially expressed genes were verified by integrating data from the Affymetrix and Unigene databases. Gene functions were determined using information from Gene Ontology, Unigene, LocusLink and PubMed databases. Clustering was performed with Cluster and Treeview (Eisen et al., 1998). All statistical analysis was performed using R-1.7 software (Dalgaard, 2002). Results were visualized with Spotfire 6.0 (Somerville, MA).
CORRESPONDING AUTHOR
Robert J. Wechsler-Reya* Dept. of Pharmacology & Cancer Biology, Duke University Medical Center Box 3813, Durham, NC 27710 Phone: 919-613-8754. Fax: 919-668-3556. Email: [email protected] Keywords = medulloblastoma Keywords = brain tumor
