BMC Cancer 2008, 8:30 http://www.biomedcentral.com/1471-2407/8/30 cFDeiCglluXlirnaeen3d phospho-DCX (P-DCX) expression in SK-N-SH DCX and phospho-DCX (P-DCX) expression in SK-N-SH cell line. Lysates (50 μg/lane) of SK-N-SH control and 6 days RA treated cells were processed with or without calf intestine alkaline phosphatase (CIP) and analyzed by WB. DCX is a phosphoprotein and appears as a doublet that resolves to a single band after CIP treatment (+CIP) in both C and RA samples, indicating that the slower-migrating species is phosphorylated DCX (P-DCX). utilized an anti-DCX antibody which reacts with both the phosphorylated and the unphosphorylated DCX forms [25]. Protein analysis reveals that the exposure of SK-N-SH cells to RA for 6 days significantly decreases both DCX (- 87.4%) and LIS1 (-38.4%) levels, whereas NF-68 protein content (+86.3%) increases and VIM remains unchanged (Fig. 4). A time-course WB analysis shows that 2- or 4 days treat- ment with RA significantly reduces DCX levels (Fig. 5) without modifying those of LIS1, VIM and NF-68 (data not shown). The DCX low level, observed after 6 days treatment, is not further diminished by extending RA exposure up to 12 days. Moreover, the down regulation exerted by 6 days RA exposure on DCX persists after an additional 6 days period following treatment withdrawal (Fig 5). Only DCX expressing cells show spontaneous motility after RA treatment To evaluate the motility of SK-N-SH cells we have then analyzed their response to wound healing assay, either in control conditions or after RA treatment. As shown in Fig. 6, untreated SK-N-SH cells are able to invade the scratched area that is fully re-colonised 24 hr later. IF analysis shows that all the cells moving in the scratched area are DCX+ (Fig. 6). Six days RA treatment strongly reduces the migration rate of the SK-N-SH cells. In fact, 24 hr after the scratch very few cells are in the scratched area and the distance between the borders of the wound is not significantly dif- ferent from that measured immediately after the scratch (Fig. 6 panel A). The few cells that move into the scratched area are DCX+, most of them have short or no neurites, and small and refractile cell bodies (Fig. 6 panel B and C). Quantitative analysis clearly indicates a significant (about -40%) decrease of the cell migration rate following RA treatment (Fig. 7). Neuroblastoma cells with invasive activity are DCX+ NB cell invasiveness has been evaluated by means of a modified microchemotaxis assay. Cells cultured in nor- mal conditions or treated with RA for 6 days were seeded into culture well containing polycarbonate membranes inserts coated with a film of Matrigel. After 48 hr, invasive cells migrated through the matrix-membrane unit and attached to the bottom of the membrane were dislodged and their number was estimated using a nuclear fluores- cent dye. The results obtained show that RA treatment sig- nificantly reduces the amount of NB cell able to invade the Matrigel (chart of Fig. 8). Some of the insert membranes have been processed for IF. Invading cells attached to the bottom of the membrane were fixed and immunostained for DCX. We found that, in both control and RA treated samples, all the cells migrated through the matrix/membrane unit are DCX+, even with different intensity (Fig. 8, right panels). Discussion In the present study we demonstrate a differential expres- sion of DCX in the cell subtypes composing human NB cell line SK-N-SH. DCX appears to be expressed exclu- sively in the cells that show high motility and invasive- ness; these parameters are reduced by RA treatment that also down regulates DCX expression. It is noteworthy to say that after RA treatment all the cells that maintain motility and invasiveness express DCX. DCX is a microtubule-associated protein (MAP) mainly involved in cell migration [10,12]. Modifications of DCX expression in neuron-like cell and its possible correlation with motility/invasion has been studied in mouse or in rat experimental models. For instance, it has been demon- strated that overexpression of DCX increases mouse neu- ronal migration rates in vitro [17]. On the contrary, targeted mutation or the shutdown of DCX disrupts the migration of neuronal cells [26,27]. Even though by the present data a direct involvement of DCX in these mechanisms cannot be assessed, to our knowledge this is the first evidence that in human NB cell line SK-N-SH DCX is specifically expressed in neuroblastic (N-type) cells, but it is absent in substrate adherent (S- type) cell type. Moreover, DCX is expressed in some, but not all, cells possessing an intermediate phenotype (I- type). Since it is sometime quite difficult to clearly distin- guish the I-type from the N-type cells by their morphol- ogy, we found easier to identify the cells composing SK-N- Page 7 of 12 (page number not for citation purposes)