Summary Background The frequent recurrence of early-stage non-small-cell lung cancer (NSCLC) is generally attributable to metastatic disease undetected at complete resection. Management of such patients depends on prognostic staging to identify the individuals most likely to have occult disease. We aimed to develop and validate a practical, reliable assay that improves risk stratification compared with conventional staging. Methods A 14-gene expression assay that uses quantitative PCR, runs on formalin-fixed paraffin-embedded tissue samples, and differentiates patients with heterogeneous statistical prognoses was developed in a cohort of 361 patients with non-squamous NSCLC resected at the University of California, San Francisco. The assay was then independently validated by the Kaiser Permanente Division of Research in a masked cohort of 433 patients with stage I non-squamous NSCLC resected at Kaiser Permanente Northern California hospitals, and on a cohort of 1006 patients with stage I–III non-squamous NSCLC resected in several leading Chinese cancer centres that are part of the China Clinical Trials Consortium (CCTC). Findings Kaplan-Meier analysis of the Kaiser validation cohort showed 5 year overall survival of 71·4% (95% CI 60·5–80·0) in low-risk, 58·3% (48·9–66·6) in intermediate-risk, and 49·2% (42·2–55·8) in high-risk patients (ptrend=0·0003). Similar analysis of the CCTC cohort indicated 5 year overall survivals of 74·1% (66·0–80·6) in low-risk, 57·4% (48·3–65·5) in intermediate-risk, and 44·6% (40·2–48·9) in high-risk patients (ptrend<0·0001). Multivariate analysis in both cohorts indicated that no standard clinical risk factors could account for, or provide, the prognostic information derived from tumour gene expression. The assay improved prognostic accuracy beyond National Comprehensive Cancer Network criteria for stage I high-risk tumours (p<0·0001), and differentiated low-risk, intermediate-risk, and high-risk patients within all disease stages. Interpretation Our practical, quantitative-PCR-based assay reliably identified patients with early-stage non-squamous NSCLC at high risk for mortality after surgical resection. Funding UCSF Thoracic Oncology Laboratory and Pinpoint Genomics.

Tumor necrosis factor-alpha (TNF) and interleukin-1beta (IL-1) are cytokines that induce expression of various genes through activation of the redox-sensitive transcription factor nuclear factor-kappaB (NF-kappaB). We have previously cloned the entire human MnSOD (SOD2) gene and found several NF-kappaB-binding sites in the 5' and 3' flanking and intronic regions. To test whether these putative NF-kappaB-binding sites are able to respond to TNF and IL-1, we performed induction analysis using various deletion constructs ligated to a luciferase reporter gene. We found that the 5' and 3' flanking regions containing several NF-kappaB-binding sites do not mediate MnSOD induction by TNF or IL-1. When a 342-bp intron 2 fragment containing NF-kappaB, C/EBP, and NF-1 binding sites was linked to the basal promoter of the SOD2 gene, transcriptional activities were significantly increased in response to TNF and IL-1 in an orientation- and position-independent manner. To accurately identify the element that is most critical for the enhancer activity, deletions and specific mutations of each individual site were studied. The results indicated that the NF-kappaB binding site is essential but not sufficient for TNF- or IL-1-mediated induction. Furthermore, NF-kappaB elements in the 5' and 3' flanking regions could be made to function in TNF or IL-1 induction when they were transposed to the intronic fragment. Taken together, these results suggest that an NF-kappaB element and its location in the SOD2 gene is critical for TNF/IL-1-mediated induction. However, a complex interaction between NF-kappaB and other transcription elements is needed for a high-level induction.

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The purpose of this study was to determine the effects of dietary fat, vitamin E and iron on oxidative damage and antioxidant status. Male Swiss-Webster mice (1 mo old) were fed a basal vitamin E-deficient diet that contained either 8% fish oil + 2% corn oil or 10% lard with or without 1 g dl-alpha-tocopheryl acetate. The diets without vitamin E contained either 0.21 or 0.95 g ferric citrate/kg. Diets were fed for 4 wk/kg diet. Compared with the vitamin E-supplemented groups, mice fed diets without vitamin E (with or without supplemental iron) had significantly (P < 0.05) higher hepatic levels of thiobarbituric acid-reactive substances (TBARS), conjugated dienes and protein carbonyls when they were fed fish oil, but not lard. The levels of TBARS were further increased by iron supplementation in the mice fed fish oil. Significantly lower concentrations of alpha-tocopherol and higher glutathione (GSH) were found in the liver of mice fed fish oil and vitamin E than in those fed lard and vitamin E (P < 0.05). The activities of superoxide dismutase and glucose-6-phosphate dehydrogenase were lower in the fish oil-fed mice than in those fed lard (P < 0.05). The activities of Se-GSH peroxidase, non-Se-GSH peroxidase, catalase, and glutathione reductase were not altered by dietary fat or vitamin E/iron. The results obtained provide experimental evidence of the prooxidative effects of high dietary fish oil and iron, and suggest that vitamin E protects not only lipid-soluble compounds, but also water-soluble constituents, against oxidative damage. Further, dietary lipid plays a key role in determining cellular susceptibility to oxidative stress.

The transfection of p14(ARF) into mesothelioma cells led to the overexpression of p14(ARF), which resulted in G(1)-phase arrest and apoptotic cell death. These results suggest that this gene therapy-based approach may be of use in the treatment of mesothelioma.

In a study by researchers from San Francisco and Taiwan, a ganglial culture system is used to screen various growth factors. The aim was to assess these growth factors as possible therapeutic agents for pelvic floors nerve injuries. They found that vascular endothelial growth factor causes a marked neurotrophic effect. Other researchers from Ann Arbor describe a method of immobilising extracellular matrix proteins to potential growth surfaces, and found that this enhances the attachment of cultured cells. They felt that this might lead to the development of other methods of tissue engineering. The authors from Japan describe a study wherein they investigated whether prenatal stress affected the pituitary‐testicular axis and thus testicular descent in rat fetuses; they report that this may indeed be the mechanism. OBJECTIVE To investigate the feasibility of using a ganglial culture system to screen various growth factors as potential therapeutic agents for pelvic nerve injuries. MATERIALS AND METHODS The major pelvic ganglia (MPG) were isolated from male rats and attached to culture dishes with the aid of MatrigelTM (Becton Dickinson, Mountain View, CA, USA). Alternatively, the dorso‐caudal region (DCR) of MPG, from which the cavernous nerves originate, was dissected and then attached to a Matrigel‐coated coverslip. The MPG or DCR was cultured in serum‐free medium supplemented with phosphate‐buffered saline (PBS, control), 50 ng/mL of vascular endothelial growth factor (VEGF), 20 ng/mL of a neurotrophin (BDNF, NT3, or NT4), or combinations of these growth factors. After 2 days of incubation, the ganglial tissues with their outgrowing nerve fibres were stained for the expression of NADPH‐diaphorase, tyrosine hydroxylase (TH) and acetylcholinesterase (AChE). The length and staining intensity of nerve fibres were analysed. RESULTS The outgrowing fibres were significantly longer in MPG treated with any of the four tested growth factors than in PBS‐treated MPG. The combination of VEGF and NT3 induced the best fibre growth. Improvements to the culturing conditions allowed a histological examination of the outgrowing fibres for the expression of nitric oxide synthase (NOS), TH and AChE. VEGF and BDNF were equally capable of inducing NOS‐ and TH‐expressing fibres. BDNF was much weaker than VEGF for inducing AChE‐expressing fibres. CONCLUSIONS This improved culturing system is potentially useful for screening nerve‐regenerating factors; VEGF had neurotrophic effects comparable with BDNF, NT3, or NT4.

Nucleophosmin (NPM) is an important nucleolar phosphoprotein with pleiotropic functions in various cellular processes. In this study, we have further examined the largely uncharacterized role of NPM in transcriptional regulation by uncovering novel NPMbinding transcriptional factors. Among potential interactors, we found that activating protein transcription factor 2 (AP2)a forms a complex with NPM during retinoic-acid-induced cell differentiation. We show that this complex is recruited to the promoters of certain retinoic-acid-responsive genes, including NPM itself. Such binding of AP2a, and consequent recruitment of NPM, is selective and dependent on a consensus AP2a-binding sequence. Remarkably, suppression of NPM by RNA interference alleviates the repression of gene expression mediated by retinoic acid and AP2a. Our findings further show that, on promoter binding, NPM probably exerts its repressive effect by inducing a change in local chromatin structure that also engages histone deacetylases. This study unveils a hitherto unrecognized transcriptional corepressor function of the NPM protein, and highlights a novel mechanism by which NPM regulates cell growth and differentiation.

Although natural biological matrices have demonstrated modest improvement in the survival of cells transplanted into the infarcted myocardium, these materials have not been amenable to systematic optimization and therefore have limited potential to treat post infarct cardiac injuries. Here we have developed tunable bioactive semi-interpenetrating polymer network (sIPN) hydrogels with matrix metalloproteinase (MMP) labile crosslinkers to be used as an assistive microenvironment for transplantation of bone marrow derived mesenchymal stem cells (BMSCs) into the infarcted myocardium. Injectable sIPN hydrogels were designed with a range of mechanical and biological properties that yielded material-dependent BMSC proliferation in vitro. Five groups were evaluated to treat myocardial infarction (MI) in adult mice: saline injection; green fluorescent protein (GFP)(+)-BMSCs delivered in saline; a sIPN matrix; a sIPN + GFP(+)-BMSCs; and Matrigel™ + GFP(+)-BMSCs. Injection of cells alone created a transient improvement in LV function that declined over time, and the synthetic hydrogel without cells resulted in the highest LV function at 6 weeks. Donor GFP-positive cells were detected after matrix-enhanced transplantation, but not without matrix support. Biomimetic sIPN hydrogel matrices succeeded both in mechanically supporting the injured myocardium and modestly enhancing donor cell survival. These matrices provide a foundation for systematic development of "pro-survival" microenvironments, and improvement in the long-term results of cardiac stem cell transplantation therapies.