Expression and DNA-binding activity of MYCN/Max and Mnt/Max during induced differentiation of human neuroblastoma cells.
Amplification of MYCN is one of the most important prognostic markers for neuroblastoma and is correlated with rapid tumor progression and poor prognosis. MYCN belongs to the Myc/Max/Mad/Mnt network of proteins that regulate proliferation, apoptosis, and differentiation. It is well established that MYCN is downregulated during induced differentiation of neuroblastoma cells carrying an amplified MYCN gene, but very little is known about other components of the network, i.e., the Max, Mad, and Mnt proteins, during this process. In this study we show that Mad and Mnt expression was only modestly regulated in differentiating SK-N-BE(2) neuroblastoma cells, while MYCN was rapidly downregulated. This downregulation was reflected in a decreased MYCN/Max DNA-binding activity while the Mnt/Max binding did not change during differentiation. In parallel experiments we also analyzed the Myc/Max/Mad expression and DNA binding capacity during induced differentiation in the MYCN single copy neuroblastoma cell line SH-SY5Y. In this cell line only modest changes in expression of the components of the MYCN/Max/Mad/Mnt network was detected, but since the cell line expresses relatively low levels of MYCN and c-Myc, these changes might be of functional significance. Cell cycle analyses of SK-N-BE(2) demonstrated an increase in the G1-phase fraction after RA-treatment. These data show that the decreased MYCN expression and MYCN DNA-binding is correlated with retarded cell cycle progression. Furthermore, when Mad1 or Mnt was overexpressed in SK-N-BE(2) cells they retained the capacity to differentiate, underscoring the notion that MYCN downregulation, and not changes in Mad/Mnt expression, is essential for neuroblastoma cell differentiation.
Smith
Anna Grynfeld
AG
Department of Laboratory Medicine, Molecular Medicine, Lund University, University Hospital MAS, S-205 02 Malmö, Sweden.
Popov
Nikita
N
Imreh
Martha
M
Axelson
Håkan
H
Henriksson
Marie
M
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D050976:Basic-Leucine Zipper Transcription Factors; D015152:Blotting, Northern; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D005434:Flow Cytometry; D006801:Humans; D000071447:N-Myc Proto-Oncogene Protein; D009447:Neuroblastoma / Q000378:metabolism* / Q000473:pathology; D009687:Nuclear Proteins / Q000378:metabolism*; D015513:Oncogene Proteins / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000378:metabolism*
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C499330:MAX protein, human; C106465:MNT protein, human; C459801:MYCN protein, human; C067850:Myc associated factor X; D000071447:N-Myc Proto-Oncogene Protein; D009687:Nuclear Proteins; D015513:Oncogene Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D004247:DNA