6 research outputs found
Decreased activity and enhanced nuclear export of CCAAT-enhancer-binding protein beta during inhibition of adipogenesis by ceramide.
To identify novel molecular mechanisms by which ceramide regulates cell differentiation, we examined its effect on adipogenesis of 3T3-L1 preadipocytes. Hormonal stimulation of 3T3-L1 preadipocytes induced formation of triacylglycerol-laden adipocytes over 7 days; in part, via the co-ordinated action of CCAAT-enhancer-binding proteins alpha, beta and delta (C/EBP-alpha, -beta and -delta) and peroxisome-proliferator-activated receptor gamma (PPARgamma). The addition of exogenous N-acetylsphingosine (C2-ceramide) or increasing endogenous ceramide levels inhibited the expression of C/EBPalpha and PPARgamma, and blocked adipocyte development. C2-ceramide did not decrease the cellular expression of C/EBPbeta, which is required for expression of C/EBPalpha and PPARgamma, but significantly blocked its transcriptional activity from a promoter construct after 24 h. The ceramide-induced decrease in the transcriptional activity of C/EBPbeta correlated with a strong decrease in its phosphorylation, DNA-binding ability and nuclear localization at 24 h. However, ceramide did not change the nuclear level of C/EBPbeta after a period of 4 or 16 h, suggesting that it was not affecting nuclear import. CRM1 (more recently named 'exportin-1') is a nuclear membrane protein that regulates protein export from the nucleus by binding to a specific nuclear export sequence. Leptomycin B is an inhibitor of CRM1/exportin-1, and reversed the ceramide-induced decrease in nuclear C/EBPbeta at 24 h. Taken together, these data support the hypothesis that ceramide may inhibit adipogenesis, at least in part, by enhancing dephosphorylation and premature nuclear export of C/EBPbeta at a time when its maximal transcriptional activity is required to drive adipogenesis
Detecting and classifying circulating tumor cell subclasses from On-Q-ity microfluidic chips using a customized imaging platform.
Strategies for H-score Normalization of Preanalytical Technical Variables with Potential Utility to Immunohistochemical-Based Biomarker Quantitation in Therapeutic Reponse Diagnostics
Digital quantitative immunohistochemical analysis of protein biomarker expression offers a broad dynamic range against which clinical outcomes may be measured. Semi-quantitative expression data represented as an H-score is produced by computer generated average intensity of positive staining given weight by the percentage of cells showing positive staining. While patient H-scores vary for biological reasons, variation may also arise from preanalytic technical issues, such as differences in fixation protocols. In this study, we present data on two candidate calibrator nuclear-localized proteins, SNRPA and SnRNP70, with robust and consistent expression levels across breast cancers. Quantitative expression measurement of these two candidate biomarkers may potentially be used to eliminate the effect of differences in preanalytic processing of specimens by normalizing H-scores derived from test biomarkers of interest. To examine the effects of preanalytical fixation variation on biomarker quantitation and potential utility of candidate calibrators to address such issues, 6 surgically-resected human breast cancer patient specimens were divided into 6 portions and fixed under distinct conditions (fixation following resection in formalin for 2 hr, 8 hr or 48 hr, or held overnight at 4°C in buffered saline prior to formalin fixation for 2 hr, 8 hr, or 48 hr). We find H-score variation between fixation conditions within individual patient's tumors that were stained for XPF, ATM, BRCA1, pMK2 and PARP1. Most interestingly, detectable expression of SNRPA and SnRNP70 is covariant to test biomarkers under distinct fixation conditions and so these hold the potential for serving as calibration standards for general antigen preservation and reactivity