Retinoic acid induced downregulation of MYCN is not mediated through changes in Sp1/Sp3.
BACKGROUND
Use of retinoic acid (RA) has become the standard of care in the treatment of high risk neuroblastoma (NB). In vitro, RA induces growth arrest and differentiation, an effect that likely underlies its activity in the clinical setting. An important event in differentiation is the transcriptional downregulation of the MYCN oncogene, which is frequently activated in aggressive tumors. While it is known that Sp1/Sp3 and E2F are necessary to drive basal MYCN expression, the mechanism for its downregulation by RA remains enigmatic. Changes in E2F binding have been reported, however these occurred after the actual transcriptional response. Here, post-translational modifications of Sp proteins were examined as an alternate mechanism of RA-mediated promoter regulation.


METHODS
Western blot was used to evaluate steady state levels of nuclear/cytoplasmic Sp1/Sp3. Promoter binding and DNA conformation were determined by gel shift, circular permutation, and chromatin immunoprecipitation assays. Immunoprecipitation/western and (32)P-phosphoamino analyses were used to detect glycosylation, acetylation, sumoylation, and phosphorylation.


RESULTS
RA did not affect the cellular level of Sp1/Sp3 proteins, their nuclear/cytoplasmic distribution, ability to bind the MYCN promoter, degree of Sp-induced DNA bending, or post-translational modifications.


CONCLUSIONS
MYCN RA response is not mediated solely though the region controlling basal activity. RA may be exerting its effects via multiple non-adjacent regulatory regions, potentially including basal motifs, either within the MYCN promoter or distally, on the same or even different chromosomes. Such cooperative trans-type DNA-protein interactions could explain the inaccessibility of this mechanism to the locus-specific approaches employed up to this point.
Kanemaru
Kelli K
KK
Natural Products and Cancer Biology Program, Cancer Research Center of Hawaii, Honolulu, Hawaii, USA.
Tuthill
Matthew C
MC
Takeuchi
Kenneth K
KK
Sidell
Neil
N
Wada
Randal K
RK
D000970:Antineoplastic Agents / Q000494:pharmacology*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D015536:Down-Regulation; D024202:Electrophoretic Mobility Shift Assay; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D007136:Immunoglobulins / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D047468:Immunoprecipitation; D000071447:N-Myc Proto-Oncogene Protein; D009447:Neuroblastoma; D009687:Nuclear Proteins / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D015513:Oncogene Proteins / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D051705:Sp3 Transcription Factor / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D014212:Tretinoin / Q000494:pharmacology*
D000970:Antineoplastic Agents; D007136:Immunoglobulins; C459801:MYCN protein, human; D000071447:N-Myc Proto-Oncogene Protein; D009687:Nuclear Proteins; D015513:Oncogene Proteins; C090698:SP1 antigen; C497121:SP3 protein, human; D051705:Sp3 Transcription Factor; D014212:Tretinoin