Identification of differentially expressed and developmentally regulated genes in medulloblastoma using suppression subtraction hybridization.
To increase our understanding of the molecular pathogenesis of medulloblastoma (MB), we utilized the technique of suppression subtractive hybridization (SSH) to identify genes that are dysregulated in MB when compared to cerebellum. SSH-enriched cDNA libraries from both human and Ptch+/- heterozygous murine MBs were generated by subtracting common cDNAs from corresponding non-neoplastic cerebellum. For the human classic MB library, total human cerebellar RNA was used as control tissue; for the Ptch+/- heterozygous MB, non-neoplastic cerebellum from an unaffected Ptch+/- littermate was used as the control. Through differential screening of these libraries, over 100 upregulated tumor cDNA fragments were isolated, sequenced and identified with the NCBI BLAST program. From these, we selected genes involved in cellular proliferation, antiapoptosis, and cerebellar differentiation for further analysis. Upregulated genes identified in the human MB library included Unc33-like protein (ULIP), SOX4, Neuronatin (NNAT), the mammalian homologue of Drosophila BarH-like 1(BARHL1), the nuclear matix protein NRP/B (ENC1), and the homeobox OTX2 gene. Genes found to be upregulated in the murine MB library included cyclin D2 (Ccnd2), thymopoietin (Tmpo), Musashi-1 (Msh1), protein phosphatase 2A inhibitor-2 (I-2pp2a), and Unc5h4(D). Using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), the mRNA expression levels for these genes were markedly higher in human MBs than in cerebellum. Western blot analysis was used to further confirm the overexpression of a subset of these genes at the protein level. Notch pathway overactivity was demonstrated in the TE671 MB cell line expressing high levels of MSH1 through HES1-Luciferase transfections. This study has revealed a panel of developmentally regulated genes that may be involved in the pathogenesis of MB.
Yokota
Naoki
N
The Arthur and Sonia Labatt Brain Tumor Research Centre, The University of Toronto, Toronto, Ontario, Canada.
Mainprize
Todd G
TG
Taylor
Michael D
MD
Kohata
Tomohiko
T
Loreto
Michael
M
Ueda
Shigeo
S
Dura
Wieslaw
W
Grajkowska
Wiesia
W
Kuo
John S
JS
Rutka
James T
JT
D045744:Cell Line, Tumor; D002528:Cerebellar Neoplasms / Q000235:genetics*; D056742:Cyclin D2; D016213:Cyclins / Q000235:genetics; D018507:Gene Expression Regulation, Developmental; D006609:High Mobility Group Proteins / Q000235:genetics; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D008527:Medulloblastoma / Q000235:genetics*; D008565:Membrane Proteins / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000235:genetics; D009693:Nucleic Acid Hybridization; D011518:Proto-Oncogene Proteins / Q000502:physiology; D051880:Receptors, Notch; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055751:SOXC Transcription Factors; D015534:Trans-Activators / Q000235:genetics; D051153:Wnt Proteins
C412478:BARHL1 protein, human; C080336:CCND2 protein, human; D056742:Cyclin D2; D016213:Cyclins; D006609:High Mobility Group Proteins; D018398:Homeodomain Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011518:Proto-Oncogene Proteins; D051880:Receptors, Notch; C083431:SOX4 protein, human; D055751:SOXC Transcription Factors; D015534:Trans-Activators; D051153:Wnt Proteins