Cell cycle regulation targets of MYCN identified by gene expression microarrays.
BACKGROUND
We have previously shown that MYCN knockdown causes a G1 arrest in MYCN amplified (MNA), p53 wild type (wt) and p53 mutant MNA neuroblastoma cell lines, with increases in p21(WAF1) and hypo RB in p53 wt cell lines.


OBJECTIVE
MYCN acts by inhibiting p21(WAF1), and also by p21(WAF1) independent mechanisms to override the G1 checkpoint in exponentially growing cells.


METHODS
Genes potentially regulated by MYCN were identified using gene expression microarrays in p53 wt MNA IMR-32 and p53 mutant MNA SKNBE(2c) neuroblastoma cell lines treated with MYCN or scrambled siRNA. Results were validated using qRT-PCR and confirmed using the regulatable MYCN expression system (SHEP Tet21N).


RESULTS
MYCN knockdown altered the expression of several cell cycle related genes. SKP2 was down regulated in both cell lines, and up regulated in MYCN+ Tet21N cells. Expression of the WNT antagonist DKK3 increased in both cell lines and decreased in MYCN+ Tet21N cells. Expression of CDKN1C (p57(cip2)) and TP53INP1 also increased after MYCN knockdown.


CONCLUSIONS
MYCN may override the G1 checkpoint through down-regulation of SKP2 and TP53INP1 resulting in reduced p21(WAF1) expression in p53 wt cell lines, and in addition may act through the WNT signaling pathway in a p53 independent manner.
Bell
Emma
E
Northern Institute for Cancer Research, University of Newcastle upon Tyne, Newcastle upon Tyne, United Kingdom.
Lunec
John
J
Tweddle
Deborah A
DA
D048868:Adaptor Proteins, Signal Transducing; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism; D002453:Cell Cycle / Q000502:physiology*; D045744:Cell Line, Tumor; D018925:Chemokines; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism*; D005720:Gamma Rays; D005786:Gene Expression Regulation; D006360:Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D000071447:N-Myc Proto-Oncogene Protein; D009687:Nuclear Proteins / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D015513:Oncogene Proteins / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D044786:S-Phase Kinase-Associated Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics*
D048868:Adaptor Proteins, Signal Transducing; C493956:CDKN1A protein, human; D002352:Carrier Proteins; D018925:Chemokines; D050759:Cyclin-Dependent Kinase Inhibitor p21; C498624:DKK3 protein, human; D006360:Heat-Shock Proteins; D036341:Intercellular Signaling Peptides and Proteins; C459801:MYCN protein, human; D000071447:N-Myc Proto-Oncogene Protein; D009687:Nuclear Proteins; D015513:Oncogene Proteins; D044786:S-Phase Kinase-Associated Proteins; C429767:TP53INP1 protein, human