Neuroblastoma Breakpoint Figure 7. Induced expression of NBPF1 in human tumor cells impairs colony formation in soft agar. (A) DLD1Tr21/NBPF1 cells express flag-tagged NBPF1 in the cytoplasm, but only when Dox is added to the medium (left panels). DAPI staining was used to visualize nuclei (right panels). (B) DLD1Tr21/NBPF1 and DLD1Tr21/Mock cells were seeded in soft agar in six-well plates in the absence or presence of Dox and then incubated for 2 weeks. NBPF1 induction induces a severe decrease in the ability to form colonies on soft agar. Pictures in the top panel were taken at a 0.86magnification and those in the bottom panel at a 46magnification. Scale bars indicate 200 mm. (C) Quantification of the soft agar colonies (mean6standard deviation for two experiments). Only colonies larger than 100 mm were counted. Induction of NBPF1 expression by adding Dox results in a severe decrease in the number of colonies in comparison to the control setting (Mock) or the non-induced (-Dox) cells. doi:10.1371/journal.pone.0002207.g007 that NBPF1 might act as a TSG, at least in colon cancer. So far, technical difficulties have prevented us from testing this hypothesis in neuroblastoma cell lines. As colorectal cancer is also characterized by frequent deletions or translocations of 1p36 [11], we believe that our present data is a first step in the elucidation of the potential tumor suppressive properties of NBPF1. The underlying mechanism for this NBPF1 activity is currently unknown. ACCN1 encodes an amiloride-sensitive cation channel and can be expressed as two different isoforms differing in their aminoterminal domains [26]. The constitutional translocation in the present patient truncates the longer isoform. Interestingly, restoration of surface expression of this isoform in glioma cells reduces cell growth and migration [40], classifying it as a potential tumor suppressor. Meta-analysis of ACCN1 expression in neuro- blastomas showed a correlation between 1p36 LOH and decreased levels of ACCN1, but no such correlation was observed between 11q23 LOH and the expression level of ACCN1 [34]. LOH of 1p36 and of 11q23 are both associated with high-risk disease, but only 1p36 LOH is associated with MYCN amplifica- tion [6]. Another report showed a decreased expression level of ACCN1 in neuroblastomas with high telomerase activity [41]. Knockout mice defective in both ACCN1 isoforms were viable, fertile, and showed no morphological or behavioral abnormalities, but did indicate that ACCN1 has a role in retinal function [42]. No data have been reported on tumor incidence or tumor aggressiveness in these mice. The most frequent chromosomal abnormality in neuroblastomas is gain of chromosome 17 [43], which appears to conflict with a potential tumor suppressor function for ACCN1 in neuroblastoma. However, most 17q PLoS ONE | www.plosone.org 10 May 2008 | Volume 3 | Issue 5 | e2207