Purpose: Most neuroblastomas initially respond to therapy but many relapse with chemoresistant disease. p53 mutations are rare in diagnostic neuroblastomas, but we have previously reported inactivation of the p53/MDM2/p14 ARF pathway in 9 of 17 (53%) neuroblastoma cell lines established at relapse. Hypothesis: Inactivation of the p53/MDM2/p14 ARF pathway develops during treatment and contributes to neuroblastoma relapse. Methods: Eighty-four neuroblastomas were studied from 41 patients with relapsed neuroblastoma including 38 paired neuroblastomas at different stages of therapy. p53 mutations were detected by automated sequencing, p14 ARF methylation and deletion by methylation-specific PCR and duplex PCR, respectively, and MDM2 amplification by fluorescent in situ hybridization.Results: Abnormalities in the p53 pathway were identified in 20 of 41 (49%) cases. Downstream defects due to inactivating missense p53 mutations were identified in 6 of 41 (15%) cases, 5 following chemotherapy and/or at relapse and 1 at diagnosis, postchemotherapy, and relapse. The presence of a p53 mutation was independently prognostic for overall survival (hazard ratio, 3.4; 95% confidence interval, 1.2-9.9; P = 0.02). Upstream defects were present in 35% of cases: MDM2 amplification in 3 cases, all at diagnosis and relapse and p14 ARF inactivation in 12 of 41 (29%) cases: 3 had p14 ARF methylation, 2 after chemotherapy, and 9 had homozygous deletions, 8 at diagnosis and relapse. Conclusions: These results show that a high proportion of neuroblastomas which relapse have an abnormality in the p53 pathway. The majority have upstream defects suggesting that agents which reactivate wild-type p53 would be beneficial, in contrast to those with downstream defects in which p53-independent therapies are indicated. Clin Cancer Res; 16(4); 1108-18. ©2010 AACR.Neuroblastoma is the most common extracranial pediatric solid tumor. It remains one of the most difficult cancers to cure, with <40% of patients with high-risk disease (stage 4 over 18 months of age or MYCN-amplified disease) becoming long-term survivors. Most high-risk neuroblastomas initially respond to cytotoxic therapy, however, over half relapse with chemoresistant disease and this often correlates with the intensity of therapy (1).The p53 gene is inactivated by mutation in >50% of human malignancies (2). p53 is a key regulator of cell cycle checkpoints and apoptosis, which upon activation by cellular stress, particularly DNA damage, binds DNA in a sequence-specific manner to activate the transcription of a large number of downstream genes, including p21 and MDM2, which results in apoptosis, cell cycle arrest, differentiation, and DNA repair (reviewed in ref.3). MDM2 functions upstream of p53 as a ubiquitin ligase that targets p53 for proteosome-mediated degradation, forming an autoregulatory feedback loop which tightly regulates p53 cellular levels (4). MDM2 amplification has been shown in some tumors and could suppress the activity of p53 by increasing its degradation.The INK4...

In gene delivery, non-viral vectors have become the preferred carrier system for DNA delivery. They can overcome major viral issues such as immunogenicity and mutagenicity. Cationic lipid-mediated gene transfer is one of the most commonly used non-viral vectors, which have been shown to be a safe and effective carrier. However, their use in gene delivery often exhibits low transfection efficiency and stability. The aim of this study was to examine the effectiveness of novel non-viral gene delivery systems. This study has investigated the encapsulation and transfection efficiency of cationic liposomes prepared from DOTAP and carboxymethyl-β-cyclodextrin (CD). The encapsulation efficiency of the CD-lipoplex complexes were also studied with and without the addition of Pluronic-F127, using both microfluidic and thin film hydration methods. In vitro transfection efficiencies of these complexes were determined in COS7 and SH-SY5Y cell lines. Formulation stability was evaluated using liposomes size, zeta potential and polydispersity index. In addition, the external morphology was studied using transmission electron microcopy (TEM). Results revealed that formulations produced by microfluidic method had smaller, more uniform and homogenious size and zeta-potential as well as higher encapsulation efficiency when compared with liposomes manufactured by thin film hydration method. Overall, the results of this study show that carboxymethyl-β-cyclodextrin increased lipoplexes’ encapsulation efficiency using both NanoAssemblr and rotary evaporator manufacturing processes. However, this increase was reduced slightly following the addition of Pluronic-F127. The addition of carboxymethyl-β-cyclodextrin to cationic liposomes resulted in an increase in transfection efficiency in mammalian cell lines. However, this increase appeared to be cell line specific, COS7 showed higher transfection efficiency compared to SH-SY5Y.

BackgroundPrimary culture and animal and cell-line models of prostate and bladder development have limitations in describing human biology, and novel strategies that describe the full spectrum of differentiation from foetal through to ageing tissue are required. Recent advances in biology demonstrate that direct reprogramming of somatic cells into pluripotent embryonic stem cell (ESC)-like cells is possible. These cells, termed induced pluripotent stem cells (iPSCs), could theoretically generate adult prostate and bladder tissue, providing an alternative strategy to study differentiation.ObjectiveTo generate human iPSCs derived from normal, ageing, human prostate (Pro-iPSC), and urinary tract (UT-iPSC) tissue and to assess their capacity for lineage-directed differentiation.Design, setting, and participantsProstate and urinary tract stroma were transduced with POU class 5 homeobox 1 (POU5F1; formerly OCT4), SRY (sex determining region Y)-box 2 (SOX2), Kruppel-like factor 4 (gut) (KLF4), and v-myc myelocytomatosis viral oncogene homolog (avian) (MYC, formerly C-MYC) genes to generate iPSCs.Outcome measurements and statistical analysisThe potential for differentiation into prostate and bladder lineages was compared with classical skin-derived iPSCs. The student t test was used.Results and limitationsSuccessful reprogramming of prostate tissue into Pro-iPSCs and bladder and ureter into UT-iPSCs was demonstrated by characteristic ESC morphology, marker expression, and functional pluripotency in generating all three germ-layer lineages. In contrast to conventional skin-derived iPSCs, Pro-iPSCs showed a vastly increased ability to generate prostate epithelial-specific differentiation, as characterised by androgen receptor and prostate-specific antigen induction. Similarly, UT-iPSCs were shown to be more efficient than skin-derived iPSCs in undergoing bladder differentiation as demonstrated by expression of urothelial-specific markers: uroplakins, claudins, and cytokeratin; and stromal smooth muscle markers: α-smooth-muscle actin, calponin, and desmin. These disparities are likely to represent epigenetic differences between individual iPSC lines and highlight the importance of organ-specific iPSCs for tissue-specific studies.ConclusionsIPSCs provide an exciting new model to characterise mechanisms regulating prostate and bladder differentiation and to develop novel approaches to disease modelling. Regeneration of bladder cells also provides an exceptional opportunity for translational tissue engineering.