Identification of phosphoproteins as possible differentiation markers in all-trans-retinoic acid-treated neuroblastoma cells

PLoS One. 2011 May 5;6(5):e18254. doi: 10.1371/journal.pone.0018254.

Abstract

Background: Neuroblastic tumors account for 9-10% of pediatric tumors and neuroblastoma (NB) is the first cause of death in pre-school age children. NB is classified in four stages, depending on the extent of spreading. A fifth type of NB, so-called stage 4S (S for special), includes patients with metastatic tumors but with an overall survival that approximates 75% at five years. In most of these cases, the tumor regresses spontaneously and regression is probably associated with delayed neuroblast cell differentiation.

Methodology/principal findings: In order to identify new early markers to follow and predict this process for diagnostic and therapeutics intents, we mimicked the differentiation process treating NB cell line SJ-NK-P with all-trans-retinoic acid (ATRA) at different times; therefore the cell proteomic pattern by mass spectrometry and the phosphoproteomic pattern by a 2-DE approach coupled with anti-phosphoserine and anti-phosphotyrosine western blotting were studied.

Conclusions/significance: Proteomic analysis identified only two proteins whose expression was significantly different in treated cells versus control cells: nucleoside diphosphate kinase A (NDKA) and reticulocalbin-1 (RCN1), which were both downregulated after 9 days of ATRA treatment. However, phosphoproteomic analysis identified 8 proteins that were differentially serine-phosphorylated and 3 that were differentially tyrosine-phosphorylated after ATRA treatment. All proteins were significantly regulated (at least 0.5-fold down-regulated). Our results suggest that differentially phosphorylated proteins could be considered as more promising markers of differentiation for NB than differentially expressed proteins.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blotting, Western
  • Calcium-Binding Proteins / metabolism
  • Cell Differentiation / drug effects*
  • Cell Line, Tumor
  • Electrophoresis, Gel, Two-Dimensional
  • Humans
  • Mass Spectrometry
  • Microscopy, Phase-Contrast
  • NM23 Nucleoside Diphosphate Kinases / metabolism
  • Neuroblastoma / metabolism*
  • Phosphoproteins / metabolism*
  • Tretinoin / pharmacology*

Substances

  • Calcium-Binding Proteins
  • NM23 Nucleoside Diphosphate Kinases
  • Phosphoproteins
  • reticulocalbin
  • Tretinoin