BMC Cancer 2008, 8:30 http://www.biomedcentral.com/1471-2407/8/30 FQiugaunrteific7ation of RA effect on neuroblastoma cell motility Quantification of RA effect on neuroblastoma cell motility. Graph shows that NB cell migration rate of 6 days RA treated cells (RA) is signif- icantly reduced in comparison with untreated cells (C). Data are obtained from a set of scratch test analysis and are expressed as means ± standard error; N = 4;double star = p < 0.01. VIM (DCX-/VIM-). There is not, at the moment, a clear explanation for the meaning of these three I-cell subtypes, however further studies are now in progress in the authors' lab. RA treatment is known to cause differentiation of NB N- type cells into a more mature phenotype and to induce changes in the expression of specific markers [30,31]. Our data, while confirming previous evidences, show a progressive decrease of DCX expression during the RA- induced differentiation of N-type cells. As a consequence, the number of DCX+ cells is greatly reduced by the treat- ment. On the contrary, S-type cells are slightly affected by the treatment, and we did not observe any of these cells becoming DCX+. This observation confirms once more that RA acts specifically upon N-type cells [30,31]. In N- type cells, we observe that DCX level starts to decrease after 2 days of RA exposure and reaches the lowest value after 6 days of treatment. It should be noted that DCX level remains very low also after RA treatment withdrawal. Altogether, the results here reported indicate that DCX nRFeAigssutrreeat8ment reduces SK-N-SH neuroblastoma cell invasive- RA treatment reduces SK-N-SH neuroblastoma cell invasive- ness. Invasiveness of NB cells is significantly reduced after 6 days RA treatment. The amount of invasive cells has been evaluated by means of a fluorescence assay (see Materials and Methods). Data are expressed as rel- ative fluorescence units (RFU). Columns are means ± standard error; N = 4; double star = p < 0.01. Representative images of the cells on the bot- tom side of the insert filter show that all NB cells invading the matrix are DCX+ (red) in both control and RA treated samples. Nuclei are counter- stained with DAPI (blue). down-regulation starts very early after the onset of RA treatment, it is progressive, time-dependent, and irrevers- ible. Also LIS1 expression is reduced after RA treatment, but there are no cells completely lacking LIS1. Therefore, it can be argued that this protein is not directly correlated with cell differentiation. In the present paper, we also report that SK-N-SH cell motility is greatly reduced by RA. The only cells able to move from the scratched borderlines into the wounded area are the few DCX+ ones still present after the treat- ment. In accordance to scratch test results, we also demon- strate that RA exposure is able to reduce NB cell invasiveness; the only cells that invade the Matrigel- coated membrane are DCX+. This last observation under- lines the importance of DCX as a marker for invasive NB cells. The results here reported indicate that NB cell motility and invasiveness are characteristics of DCX+ cells, and that, also after RA treatment, both of these capabilities are retained only by the cells expressing this protein. Joshi and coworkers reported that 24 hr of RA exposure stimulates SH-SY5Y cell migration and invasiveness, while long term treatment (5 days) is less effective in stim- ulating migration but significantly reduces invasiveness [32]. Voight and co-workers [24] demonstrated that 3 days RA treatment does not modify cell migration of SK- N-FI, SH-SY5Y, SK-N-MC and SK-N-LO NB cell lines. In Page 10 of 12 (page number not for citation purposes)