Identification of the tissue of origin of a tumor is vital to its management. Previous studies showed tissuespecific expression patterns of microRNA and suggested that microRNA profiling would be useful in addressing this diagnostic challenge. MicroRNAs are well preserved in formalin-fixed, paraffin-embedded (FFPE) samples, further supporting this approach. To develop a standardized assay for identification of the tissue origin of FFPE tumor samples, we used microarray data from 504 tumor samples to select a shortlist of 104 microRNA biomarker candidates. These 104 microRNAs were profiled by proprietary quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) on 356 FFPE tumor samples. A total of 48 microRNAs were chosen from this list of candidates and used to train a classifier. We developed a clinical test for the identification of the tumor tissue of origin based on a standardized protocol and defined the classification criteria. The test measures expression levels of 48 microRNAs by qRT-PCR, and predicts the tissue of origin among 25 possible classes, corresponding to 17 distinct tissues and organs. The biologically motivated classifier combines the predictions generated by a binary decision tree and K-nearest neighbors (KNN). The classifier was validated on an independent, blinded set of 204 FFPE tumor samples, including nearly 100 metastatic tumor samples. The test predictions correctly identified the reference diagnosis in 85% of the cases. In 66% of the cases the two algorithm predictions (tree and KNN) agreed on a single-tissue origin, which was identical to the reference diagnosis in 90% of cases. Thus, a qRT-PCR test based on the expression profile of 48 tissue-specific microRNAs allows accurate identification of the tumor tissue of origin.

The kinesin-related protein HsEg5 plays essential roles in mitotic spindle dynamics. Although inhibition of HsEg5 has been suggested as an aid in cancer treatment, the effects of such inhibition on human cells have not been characterized. Here we studied the effects of monastrol, an allosteric HsEg5 inhibitor, on AGS and HT29 cell lines and compared them to those of taxol. While both cell lines were similarly sensitive to taxol, AGS cells were more sensitive to monastrol. The differences in sensitivity were determined by the degree of inhibitory effect on cell proliferation, reversibility of monastrol-induced G2/M arrest, intracellular phenotypes and induction of apoptosis. In both cell lines, monastrol-induced apoptosis was accompanied by mitochondrial membrane depolarization and poly-ADP-ribose polymerase 1 cleavage. In AGS, but not HT29 cells, monastrol-induced apoptosis involved a prominent cleavage of procaspases 8 and 3. While in AGS cells, monastrol induced the formation of symmetric microtubule asters only, in HT29 cells, asymmetric asters were also formed, which may be related to specific HsEg5 functions in HT29 cells.

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objective:To evaluate the accumulation pattern of intramyocellular lipids (IMCLs) in striated muscle during the development and progression of diabetes, using a novel scanning electron microscopic method. Methods and Procedures: Hyperglycemia was induced by feeding diabetes-prone (DP) Psammomys obesus a high-energy (HE) diet. Lipid accumulation within gastrocnemius muscle fibers was assessed in formalin-fixed muscle samples during the development of hyperglycemia using high resolution imaging in a scanning electron microscope. We evaluated the temporal relationship between changes in IMCL quantity and morphology and the altered glucose metabolism and assessed the effect of reversal of hyperglycemia on IMCL level and morphology. Diabetes-resistant (DR) P. obesus served as controls.Results: Lipid accumulation in the muscle fibers of DP animals was increased with the development of hyperglycemia. This was characterized by increased lipid density as well as by an abundance of large lipid droplets. Reversal of the phenotype resulted in the disappearance of large lipid droplets. The IMCL level and the distribution of lipid droplet size were similar in muscles of both the normoglycemic DR and DP animals, with an abundance of small lipid droplets. This profile was changed following a HE diet only in the DP animals. Discussion: Lipid accumulation in the muscle of P. obesus during the development of hyperglycemia is characterized by increased quantity and accumulation of large lipid droplets. These changes were reversible upon normalization of blood glucose. The evaluated methodology is a useful tool for the study of the dynamics of lipid accumulation in different metabolic conditions.