BMC Cancer 2008, 8:30 http://www.biomedcentral.com/1471-2407/8/30 Background Neuroblastoma (NB), the most common extracranial solid paediatric tumor, is responsible for approximately 15% of cancer deaths occurring in childhood. In older patients the prognosis is still poor although the clinical course may be protracted [1]. Consistent with their origin from neural crest-derived multipotent precursors, NBs are often composed of multiple cellular phenotypes. This het- erogeneity is retained in some cell lines derived from these tumors. In SK-N-SH cell line, derived from a bone marrow metas- tasis, at least three cell types are present: the sympathoad- renal neuroblasts (N), the substrate-adherent non- neuronal (S), and the intermediate (I) cells. [2,3]. The N- type are small rounded cells with short neuritic processes, whereas the S-type are large, flat and strongly attached to the substrate. The I-type have a morphology intermediate between that of N and S, are moderately adherent to the substrate and show small cell body with or without neu- ritic elongations. In addition, the I-type cells have the abil- ity of forming aggregates in culture [3]. The three cell types differ also in the capability to induce the tumor; it has been reported that N-type cells are malignant while the S- type are not, but surprisingly, the I-type cells have the greatest malignancy [2]. Moreover, it has been hypothe- sized that the I-type, may be recognized as tumor stem cells, capable of both self-renewal and bidirectional differ- entiation, being the progenitor of both N- and S-type [4]. The current treatment of NB may involve surgery, chemo- therapy, radiation therapy, megatherapy with stem cell rescue and biological approaches [5]. Arrest of cell growth and morphological differentiation can be observed after retinoic acid (RA) treatment at particular stages of progres- sion of the disease, but the precise mechanism of action of RA remains uncharted; moreover, resistance to RA repre- sents a significant drawback to its clinical utility. In recent years, a number of genetic and biological fea- tures have been investigated in the effort to identify tumor markers that would improve cure rates. Doublecortin (DCX), a microtubule associated protein, has been proposed as a new molecular marker to detect minimal residual disease in human NB, since it has been detected in all the tumors analyzed and it appears to be more efficient than tyrosine hydroxylase [6]. Moreover, DCX, semaphorin3B and SPARC (Osteonec- tin), three genes that play key roles in cellular migration processes, have been indicated as prognosticators of poor prognosis in human glioblastoma [7]. Recently, it has also been reported that DCX is present in tumors of the human central and peripheral nervous sys- tem [8] and that is preferentially expressed in invasive human brain tumors [9]. DCX is the product of the X-linked gene doublecortin (Xq22.3-Xq23) and is required for neuroblastic migration during development of the cerebral cortex [10]. Mutations in this gene result in a deficient neuronal migration that gives rise to the X-linked lissencephaly in males or to the subcortical laminar heterotopia (doublecortex) in female patients [11,12]. DCX is expressed in migrating neurons throughout the central and peripheral nervous system during embryonic and postnatal development, regulates the microtubule cytoskeleton and it is involved in dynamic morphological changes [13-15] and in nuclear translocation occurring during neural cell migration. In this process it acts synergistically with lissencephaly-1 (LIS1), another microtubule associated protein codified by the gene Lis1, located on chromosome 17p.13.3 [16,17]. LIS1 localizes predominantly to the centrosome and acts with DCX for the correct nucleus-centrosome coupling required for normal neuronal migration [17]. To date, the presence of DCX in human NB has been eval- uated only by real-time RT-PCR in tumor samples [6], and by Western blot (WB) analysis in SH-SY5Y cell line [18]. However it is not known whether DCX is expressed in all the different cell-types (S, N and I) composing the tumor or it is a characteristic of some of them, and whether its interacting protein LIS1 is also expressed in the human NB. In the present study we have analysed by immunofluores- cence (IF) the presence of DCX and LIS1 in the different cell subtypes that constitute the SK-N-SH neuroblastoma cell line. We have also correlated the intracellular localiza- tion of these two proteins with tubulin and with two inter- mediate filaments: vimentin (VIM) and neurofilament-68 (NF-68) that have been utilized as possible cell-subtype specific markers. Furthermore, we have investigated by IF and WB analysis whether a treatment with the differenti- ating agent RA might induce modifications on DCX, LIS1, VIM and NF-68 expression. In addition, motility and inva- siveness of SK-N-SH cells have been studied by wound scratch test [19] and by a modified microchemotaxis assay [20], so as to evaluate the capability of RA to influence these parameters. Methods Cell cultures Human SK-N-SH cells were grown as a monolayer in Eagle's Minimum Essential Medium (MEM) supple- mented with 10% of heat-inactivated Foetal Bovine Serum (FBS from Gibco, Invitrogen), 1% non essential Page 2 of 12 (page number not for citation purposes)