Expression of doublecortin in human neuroblastoma cell line SK-N-SH and its regulation by retinoic acid.

Doublecortin (DCX), a microtubule-associated protein which is required for normal migration of neurons into the cerebral cortex, is encoded by the gene DCX, located on chromosome Xq22.3-q23. The product of the autosomal gene LIS1 located on 17p13.3 (LIS1), regulates microtubule function and dynein motor activity. DCX and LIS1 display overlapping localization and cross-talk in vitro and in vivo during normal neuronal migration. Recently, Oltra S. (Oltra et al., Diagn. Mol. Pathol. Vol. 14, number 1; pp. 53-57, March 2005) has proposed the use of DCX gene as a molecular marker of neuroblastoma cells in vivo. Human neuroblastoma, the most common extracranial solid tumor in the pediatric population, arises from the developing neural crest along its migratory pathway. Consistent with an origin from multipotent neural crest cells, neuroblastoma often comprises multiple cell phenotypes. Retinoic acid (RA) treatment can interfere with neuroblastoma cell growth, reducing in vitro cell migration, inducing differentiation and lowering tumorigenicity and aggressivness. We were interested in elucidating whether DCX and LIS1 are expressed by neuroblastoma cell line SK-N-SH and whether their expression is affected by RA. Immunofluorescence analysis (IF) showed the presence of LIS1 in all cell types, whereas DCX is expressed only in most but not all of the neuroblastic type (N-type) cells. The treatment with RA (10 mM for 6 days) reduced the number of N-type cells immunoreactive for DCX, without affecting LIS1 expression. Western blot analysis confirmed quantitative DCX reduction after RA treatment, as observed in IF. Since we didn’t observe any quantitative DCX mRNA difference between control and treated cells, we postulated that RA might exert its effects on the translation or the post-transcriptional maturation of DCX mRNA. Moreover, after RA treatment few N-type cells (10% of the total) maintain DCX expression and seem to be unaffected by the treatment. This subset of cells might be the “side-population” of neuroblastoma cells, which shows high resistance to chemotherapeutic agents due to the strong expression of drug-transporter proteins. Reduction of DCX protein expression might represent one of the mechanisms through which RA exerts its differentiating and anti-invasive effects on neuroblastoma cells. (Supported by FIRB: RBAU01FXRT)