Use of RNA interference to elucidate the effect of MYCN on cell cycle in neuroblastoma.
BACKGROUND
MYCN amplification marks poor prognosis in neuroblastoma (NB) tumors. In evaluating the mechanisms by which retinoic acid (RA) or nerve growth factor (NGF) decrease cell number in MYCN amplified NB cells, we have identified a number of proteins whose expression either decreases (E2F, CDC2, CDK6, cyclin dependent kinase activity) or increases (p27) in association with a decrease in MYCN expression. However, it was still unclear which were MYCN dependent effects or not.


METHODS
This study aimed to determine which changes in cell cycle gene expression are modulated as a consequence of the decrease in MYCN. We silenced MYCN expression using siRNA targeted to the coding region of MYCN. Then, by using siRNA transient transfections, we analyzed the change of cell cycle related genes and cell cycle in MYCN amplified NB cell lines.


RESULTS
We demonstrate that expression of MYCN can be suppressed by almost 60% in MYCN amplified NB cell using siRNAs targeted to MYCN. Functionally, the decrease in MYCN leads to a decrease in cells in the S-phase of the cell cycle. Decreases in MYCN are associated with decreases in E2F1-2 and ID2 along with increases in p27 protein levels by post-transcriptional modification. Moreover, we find that a decrease in MYCN is accompanied by a decrease in cdk6 mRNA and protein expression.


CONCLUSIONS
These results show that E2F and ID2 expression is associated with MYCN regulation and that cdk6 is a possible new transcriptional target of MYCN.
Woo
Chan-Wook
CW
Department of Pediatrics, College of Medicine, Korea University, Seoul 152-703, South Korea. wooc@korea.ac.kr
Tan
Fei
F
Cassano
Hope
H
Lee
Junghwa
J
Lee
Kwang Chul
KC
Thiele
Carol J
CJ
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D002453:Cell Cycle / Q000235:genetics; D018797:Cell Cycle Proteins / Q000096:biosynthesis / Q000235:genetics; D048708:Cell Growth Processes / Q000235:genetics; D045744:Cell Line, Tumor; D020868:Gene Silencing; D006801:Humans; D051379:Mice; D000071447:N-Myc Proto-Oncogene Protein; D041681:NIH 3T3 Cells; D009447:Neuroblastoma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology*; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000172:deficiency / Q000235:genetics*; D015513:Oncogene Proteins / Q000096:biosynthesis / Q000172:deficiency / Q000235:genetics*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection; D014212:Tretinoin / Q000494:pharmacology
D000970:Antineoplastic Agents; D018797:Cell Cycle Proteins; C459801:MYCN protein, human; D000071447:N-Myc Proto-Oncogene Protein; D009687:Nuclear Proteins; D015513:Oncogene Proteins; D034741:RNA, Small Interfering; D014212:Tretinoin