differentiation genes. HOXC9 expression is upregulated by RA and knockdown of HOXC9 expression confers resistance to RA- induced growth arrest and differentiation. These findings identify HOXC9 as a key regulator of neuroblastoma differentiation [25]. In addition to HOXC9, it has been reported that RA treatment of neuroblastoma cells leads to a significant induction of several HOXD genes [13,14,15]. In this report, we present a comprehen- sive analysis of RA induction of HOXD genes in the regulation of neuroblastoma cell proliferation and differentiation. Results RA induction of HOXD genes in neuroblastoma cells We conducted this study in BE(2)-C cells, an RA-sensitive human neuroblastoma cell line enriched for cells capable of self- renewal and multi-lineage differentiation [26,27]. BE(2)-C cells treated with RA display morphologic changes characteristic of neuronal differentiation and G1 arrest [25,26,27]. Previous studies with the human neuroblastoma cell lines SK-N-BE, CHP-134, SK-N-SH, LA-N-1 and LA-N-5 using standard RT-PCR have revealed marked induction of HOXD1 and HOXD8 following RA treatment in all of the neuroblastoma cell lines, as well as low levels of HOXD4 and HOXD9 induction in some neuroblastoma cell lines [14,15]. The human HOXD complex contains nine genes HOXD1, HOXD3, HOXD4, HOXD8, HOXD9, HOXD10, HOXD11, HOXD12 and HOXD13, which are clustered from 39 to 59 in an approximately 100-kb stretch on chromosome 2q31.1, with HOXD1 at the 3’ end and HOXD13 the 59. end (Figure 1 A). We performed two independent time-course studies of RA- induction of HOXD genes in BE(2)-C cells that were treated continuously with RA. Quantitative RT-PCR showed a marked increase (,10- to 50-fold) in mRNA levels for all HOXD genes following RA treatment, relative to their levels in untreated cells (Figure 1 B). The transcriptional activation of HOXD genes by RA occurred in an apparent two-stage order colinear with their 39 to 59 arrangement in the cluster: marked induction (.10 fold) of the six 39 HOXD genes (HOXD1, HOXD3, HOXD4, HOXD8, HOXD9 and HOXD10) occurred essentially at the same time, as early as 6 h following RA treatment, whereas similar levels of induction (.10 fold) of HOXD11, HOXD12 and HOXD13 was not observed until 48 h after RA treatment (Figure 1 B). The transcriptional upregulation of the six 39 HOXD genes was transient and their mRNA levels declined substantially by 48 h. By contrast, the mRNA levels of HOXD11, HOXD12 and HOXD13 continued to increase for at least 6 days (Figure 1 B). Overall, these findings are consistent with the phenomenon of ‘‘RA sensitivity colinearity’’ that was first observed in human embryonal carcinoma cells and later confirmed in the mouse embryo: HOX genes located at the 39 end are activated generally earlier by RA than those positioned more 59 within the cluster [28,29]. These observations suggest that RA induction of HOXD genes in neuroblastoma cells is of physiological relevance. Distinct functions of HOXD proteins in regulation of neuroblastoma cell proliferation To assess the functional consequence of HOXD induction by RA in neuroblastoma cells, we generated BE(2)-C-derived cell lines with inducible expression of individual HOXD genes in the absence of doxycycline. Quantitative RT-PCR analysis revealed that the mRNA levels of most HOXD genes were increased between 40- to 60-fold by day 3 following doxycycline removal (Figure S1 A). The lowest induction was observed with HOXD9 (,25-fold) and the highest with HOXD13 (,70-fold) (Figure S1 A). The induction HOXD Regulation of Neuroblastoma Differentiation of HOXD proteins was confirmed by immunoblot analysis using an antibody against the Myc-tag (Figure S1 B). We first examined the effect of individual HOXD genes on the proliferation of BE(2)-C cells. Induction of HOXD3, HOXD8, HOXD9, HOXD10 or HOXD12 led to a marked decrease in the number of cells that expressed KI67 (Figure 2 A-B), a nuclear protein specifically expressed in proliferating cells [30]. By contrast, no such effect was observed following the induction of HOXD1, HOXD4, HOXD11 or HOXD13 (Figure 2 A-B). Consis- tent with the finding, fluorescence-activated cell sorting (FACS) showed that individual induction of HOXD3, HOXD8, HOXD9, HOXD10 or HOXD12 caused G1 arrest, whereas induction of HOXD1, HOXD4, HOXD11 or HOXD13 had no significant effect on cell cycle progression (Figure 2 C). Together, these findings demonstrate distinct functions of HOXD proteins in regulation of neuroblastoma cell proliferation, with only HOXD3, HOXD8, HOXD9, HOXD10 and HOXD12 proteins being able to recapitulate the G1 arrest phenotype induced by RA. Anti-growth HOXD proteins downregulate the expression of cell cycle-promoting genes Cell cycle progression is driven by cyclins and cyclin-dependent kinases (CDKs) [31,32]. RA-induced G1 arrest is associated with downregulation of cyclin A2, cyclin B1 and CDK1 [25,33,34]. This effect of RA could be fully recapitulated by individual induction of HOXD3, HOXD8, HOXD9, HOXD10 or HOXD12, which all led to significant downregulation of cyclin A2, cyclin B1 and CDK1 (Figure 3). By contrast, the HOXD proteins that failed to induce G1 arrest (HOXD1, HOXD4, HOXD11 and HOXD13) had no apparent effects on the protein levels of cyclin A2, cyclin B1 and CDK1 (Figure 3). We further examined the specificity of these anti-growth HOXD genes in downregulation of cyclins and CDKs. Induction of HOXD3, HOXD8, HOXD9, HOXD10 or HOXD12 had no significant effect on the expression levels of cyclin D1, CDK4 and CDK6, which are essential for maintaining cells in the G1 phase of the cell cycle (Figure S2). Together, these findings suggest that HOXD3, HOXD8, HOXD9, HOXD10 and HOXD12 inhibit cell proliferation by downregulating cyclins and CDKs that are required for cell cycle progression through the S, G2 and M phases. Distinct functions of HOXD proteins in regulation of neuroblastoma cell differentiation BE(2)-C cells treated with RA display morphologic changes characteristic of neuronal differentiation with extensive outgrowth of neurites [25,26,27]. At the molecular level, RA-induced neuronal differentiation is characterized by downregulation of progenitor cell markers, for example, paired-like homeobox 2B (PHOX2B) [25,35], and upregulation of neuronal differentiation markers, such as neurofilament medium polypeptide (NEFM) [25,36]. Individual induction of HOXD8, HOXD9, HOXD10 or HOXD12 in BE(2)-C cells also led to morphologic changes of neuronal differentiation with marked neurite outgrowth (Figure 4 A). Consistent with the morphologic change, these cells displayed a significant reduction in PHOX2B expression and a marked increase in NEFM expression (Figure 4 B–D). These observations indicate that induction of HOXD8, HOXD9, HOXD10 or HOXD12 can recapitulate the neuronal differentiation phenotype induced by RA. By contrast, induction of HOXD1 in BE(2)-C cells resulted in no apparent changes in both morphology and expression levels of PHOX2B and NEFM (Figure 4). The remaining HOXD proteins PLoS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e40728