BMC Genomics 2007, 8:139 http://www.biomedcentral.com/1471-2164/8/139 activated signaling pathways, such as Ras/MAPK [81]. Activation of the low affinity NGF receptor p75NTR by NGF results in apoptosis in Schwann cells [82] through the activity of p53 [83]. TrkA high affinity and p75NTR low affinity NGF receptors may represent a useful model through which the divergent 'choices' between cell cycle arrest/differentiation and apoptosis by p53 protein func- tion can be studied. Notably, NGF-induced PC12 cell dif- ferentiation results in little or no detectable apoptosis, despite markedly increased p53 levels and transcriptional activity over the extended time course of several days. It is thus likely that both the inducing agent (i.e. stimulus) and cell type lend specificity to the p53-controlled transcrip- tional response observed for other p53-responsive condi- tions [84]. In addition, the continued transcription of MDM2 and cyclin G1 described here is in apparent oppo- sition to elevated p53 protein levels following NGF treat- ment. MDM2 functions as the major negative regulator of p53 protein within the cell [85], while cyclin G1 exerts its negative regulatory role through MDM2 dephosphoryla- tion and subsequent stabilization [86]. However, contin- ued stabilization of p53 protein during NGF-induced differentiation occurs despite the increased MDM2 and cyclin G1 RNA levels shown here and maintained MDM2 protein levels (data not shown). This suggests the pres- ence of an intermediate event either inhibiting MDM2 function, or protecting p53 protein from MDM2 function, in differentiating PC12 cells. The incongruent time frames of activation for NGF-induced p53 compared to the rela- tively rapid stabilization via genotoxic agents further sug- gests the presence of a mechanism distinct from DNA damage through which p53 stabilization and activation may occur during NGF-mediated signaling. The approach used here in the identification of p53-regu- lated elements in neuronal differentiation represents a useful strategy amenable to the analyses of DNA binding by other transcription factors. The recognized roles of NGF-induced p53 transcriptional targets identified in this report raise new prospects for a role of p53 in differentia- tion and development. Further, we provide evidence dem- onstrating stimulus-specific selectivity by activated p53 protein in transactivation. Conclusion Nerve growth factor-mediated activation of p53-depedent transcription represents an unusual example of receptor- mediated, non-genotoxic p53 activation. Using a global chromatin immunoprecipitation cloning technique, we have identified and validated 14 novel transcriptional tar- gets of NGF-activated p53 protein during neuronal PC12 differentiation. This study provides evidence of a func- tional role for p53 in differentiation. Methods Cell culture and treatments PC12 cells (ATCC, Manassas, VA) were propagated in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supple- mented with 2 mM L-glutamine, 10% horse serum (Invit- rogen), 5% fetal bovine serum (Invitrogen) and appropriate antibiotics in a humidified incubator main- tained at 37° with 5% CO2. PC12 cells (passage 10–25) were differentiated on rat tail collagen-coated culture ves- sels (Sigma, St. Louis, MO) in RPMI 1640 medium con- taining 1% horse serum, 2 mM L-glutamine and antibiotics with 50 ng/mL mouse nerve growth factor, (NGF 2.5S) (Chemicon, Temecula, CA) for indicated time intervals. Differentiation medium was replaced and fresh NGF 2.5S was added every third day to differentiating cells. PC12 cells were incubated with genotoxic agents at the following concentrations, at the indicated time points: bleomycin, 10 µg/mL (Sigma) and 5-fluorouracil, 375 µM (Sigma). Reporter assay was performed by transient trans- fection of p53TA-luc vector (BD Pharmingen, San Diego, CA) into naïve PC12 cells plated onto collagen-coated dishes using Lipofectamine 2000 (Invitrogen). Cells were treated with NGF the following day and luciferase activity was quantified using the Luciferase Assay System (Promega, Madison, WI) at the indicated time intervals on a Tropix (Bedford, MA) TR717 luminometer. Statistical analysis of reporter assay data was performed using one- way ANOVA followed by Tukey's HSD post-hoc test for significance. Generation of stable shRNA expressing cells Stable anti-p53 shRNA expressing PC12 cells were gener- ated via lentiviral-mediated infection, integration and sta- ble selection (BlockIt Lentiviral Expression System, Invitrogen). Hairpin sequences were designed and synthe- sized (IDT, Coralville, IA) with appropriate overhangs for cloning into the pENTR/U6 entry vector. The informative p53-targeting sense hairpin sequences are: p53-2mid – 5'- GCATACGGATTTCCTTCCACC-3', p53-3low – 5'-ATATC- CGACTGTGAATCCTCC-3', p53-4mid – 5'-AAAATTAG- GTGACCCTGTCGC-3'. shRNAs were designed using the 4-base loop sequence CGAA. Hairpin sequences were cloned into the pENTR/U6 vector and selected clones were sequence-verified. The U6 promoter-shRNA cassettes were transferred into the pLenti6-BLOCK-iT-DEST vector. The resulting destination clones were used to generate len- tiviral particles by transfection of HEK293FT cells along with the pLP1, pLP2 and pLP/VSVG plasmids in equal amounts using FuGene (Roche Diagnostics, Indianapolis, IN) lipid reagent. Transfection efficiencies were moni- tored by concomitant tranfection of the pEYFP-C1 vector (BD Clontech, Mountain View, CA), demonstrating effi- ciency ≥ 90% in all cases. Lentiviral-laden media was col- lected 48 hours following transfection, clarified by centrifugation and used with 4 µg/mL polybrene (Sigma) Page 13 of 17 (page number not for citation purposes)