Neuroblastoma Breakpoint median expression level that was lower in cell lines with NBPF1 loss, and this difference was significant for amplicons A, B and D (p,0.05), but not for amplicon C (p = 0.13). (C) Meta-analysis of the NBPF expression level in neuroblastoma tumors using the Oncomine database [32]. The reporter used in the array experiment was derived from NBPF1, but due to the high sequence identity among the NBPF paralogs, this analysis also represents the expression level of all NBPF genes. Significantly lower levels of NBPF transcripts are observed in tumors that relapse within five years. doi:10.1371/journal.pone.0002207.g004 gene, so we also determined the expression profile of this gene specifically. We designed primers located on the 59 UTR of the NBPF1 gene, where it has slightly lower sequence homology with the other NBPF paralogs. Specific NBPF1 amplification was determined by melting curve analysis, which showed a single peak, and by sequence analysis of the amplicon. The expression level of NBPF1, determined for a panel of 32 neuroblastoma cell lines, varied widely between the different samples (Figure 5), like it did in the expression analysis of the total NBPF family. Interestingly, we did not observe complete loss of NBPF1 expression in any cell line, as has been described for other genes located in the 1p36 region [16,17]. Statistical analysis of these data with the Wilcoxon rank sum test revealed a significantly lower expression level of NBPF1 in cell lines with a heterozygous deletion of the NBPF1 locus compared to cell lines without NBPF1 loss (p,0.05). Our analysis of ACCN1 (Figure 6) was limited to meta-analysis using the Oncomine database [32]. We observed a significantly weaker expression of ACCN1 in neuroblastomas with MYCN amplification (p,0.01) [34] or with 1p36 loss of heterozygosity (p,0.01) [34] compared to tumors without these alterations. Additionally, weaker ACCN1 expression was correlated with higher stages of disease (p,0.01) [33]. On the other hand, no difference was observed between neuroblastomas with or without 11q23 loss of heterozygosity (LOH) (p = 0.10) [34]. NBPF1 expression suppresses anchorage-independent growth The DLD1Tr21 cell line is a human colon cancer cell line expressing high levels of the tetracycline repressor protein, resulting in a tight control of the expression of the gene of interest under control of a responsive tetracycline operator element [21]. This reliable cell system was stably transfected with an expression vector harboring an inducible flag-tagged full length NBPF1. As shown in Figure 7A, the addition of doxycyclin (Dox) to the cell cultures for 48 h resulted in the expression of NBPF1 in the cytoplasm of the cells. To determine whether overexpression of NBPF1 influences anchorage-independent growth, we compared the ability of DLD1Tr21/mock and DLD1Tr21/NBPF1 cells to form colonies in soft agar in the absence and presence of Dox. NBPF1- expressing cells formed significantly fewer colonies than the control cell lines or the non-induced DLD1Tr21/NBPF1 (figures 7B and 7C). These data indicate that overexpression of NBPF1 strongly inhibits the ability of this human cancer cell line to form colonies in soft agar. Figure 5. Expression analysis of NBPF1 in a panel of 32 neuroblastoma cell lines. Expression levels were normalized to two reference genes [19], and the lowest value was set to 1. Inset: relative expression levels of NBPF1 in neuroblastoma cell lines with normal NBPF1 status (grey; NO) or with a heterozygous deletion of the NBPF1 locus (white; YES). Significantly lower NBPF1 expression is observed in cell lines with NBPF1 loss (p,0.05). doi:10.1371/journal.pone.0002207.g005 PLoS ONE | www.plosone.org 8 May 2008 | Volume 3 | Issue 5 | e2207