BackgroundThe mechanical properties of the extracellular matrix have an important role in cell growth and differentiation. However, it is unclear as to what extent cancer cells respond to changes in the mechanical properties (rigidity/stiffness) of the microenvironment and how this response varies among cancer cell lines.Methodology/Principal FindingsIn this study we used a recently developed 96-well plate system that arrays extracellular matrix-conjugated polyacrylamide gels that increase in stiffness by at least 50-fold across the plate. This plate was used to determine how changes in the rigidity of the extracellular matrix modulate the biological properties of tumor cells. The cell lines tested fall into one of two categories based on their proliferation on substrates of differing stiffness: “rigidity dependent” (those which show an increase in cell growth as extracellular rigidity is increased), and “rigidity independent” (those which grow equally on both soft and stiff substrates). Cells which grew poorly on soft gels also showed decreased spreading and migration under these conditions. More importantly, seeding the cell lines into the lungs of nude mice revealed that the ability of cells to grow on soft gels in vitro correlated with their ability to grow in a soft tissue environment in vivo. The lung carcinoma line A549 responded to culture on soft gels by expressing the differentiated epithelial marker E-cadherin and decreasing the expression of the mesenchymal transcription factor Slug.Conclusions/SignificanceThese observations suggest that the mechanical properties of the matrix environment play a significant role in regulating the proliferation and the morphological properties of cancer cells. Further, the multiwell format of the soft-plate assay is a useful and effective adjunct to established 3-dimensional cell culture models.

The conserved oligomeric Golgi (COG) complex is an evolutionarily conserved peripheral membrane oligomeric protein complex that is involved in intra-Golgi protein trafficking. The COG complex is composed of eight subunits that are located in two lobes; Lobe A contains COG1-4, and Lobe B is composed of COG5-8. Both in vivo and in vitro protein-protein interaction techniques were applied to characterize interactions between individual COG subunits. In vitro assays revealed binary interactions between Cog2p and Cog3p, Cog2p and Cog4p, and Cog6p and Cog8p and a strong interaction between Cog5p and Cog7p. The two-hybrid assay confirmed these findings and revealed that Cog1p interacted with subunits from both lobes of the complex. Antibodies to COG subunits were utilized to determine the protein levels and membrane association of COG subunits in yeast ⌬cog1-8 mutants. As a result, we created a model of the protein-protein interactions within the yeast COG complex and proposed that Cog1p is a bridging subunit between the two COG lobes. In support of this hypothesis, we have demonstrated that Cog1p is required for the stable association between two COG subcomplexes.

The androgen receptor (AR) is a ligand-regulated transcription factor that belongs to the family of nuclear receptors. In addition to regulation by steroid, the AR is also regulated by post-translational modifications generated by signal transduction pathways. Thus, the AR functions not only as transcription factor, but also as a node that integrates multiple extracellular signals. The importance of the AR in disease cannot be understated as it plays a role in many diseases ranging from complete androgen insensitivity syndrome to spinal bulbar muscular atrophy to prostate and breast cancer. In the case of prostate cancer, dependence on AR signaling has been exploited for therapeutic intervention for decades. However, the effectiveness of these therapies is limited in advanced disease due to restoration of AR signaling. Fully understanding the molecular mechanisms of AR action will enable the development of future therapeutics for the wide range of AR dependent diseases. The AR is subject to regulation by a number of kinases through post-translational modifications on serine, threonine and tyrosine residues. Here we review the AR phosphorylation sites, the kinases responsible for these phosphorylations, as well as the biological context and the functional consequences of these phosphorylations. Finally, what is known about the state of AR phosphorylation in clinical samples is discussed.

In this work, sub-terahertz (THz) spectroscopy is applied to characterize lyophilized and in vitro cultured bacterial cells of non-pathogenic species of Escherichia coli (E. coli) and Bacillus subtilis (BG), spores of BG and DNA fro m E. coli. One of the goals of this research is to demonstrate that Fourier Transform (FT) spectroscopy in the frequency region of 10-25 cm -1 is sensitive enough to reveal characteristic spectral features fro m bio-cells and spores in different environment, to verify the d ifferences between species, and to show the response of spores to vacuum and response of cultured cells to heat. The experimental technique was significantly improved for sensitivity and reliab ility. Observed spectra taken with a spectral resolution of 0.25 cm -1 using FT spectrometer with a detector operating at 1.7 K are rich in well resolved features having spectral widths of ~0.5-1 cm -1 . The reproducibility of experimental results was verified and confirmed. Measured spectra from E. coli DNA and from the entire cell have many similarit ies, thus demonstrating that the cellu lar co mponents might contribute to the v ibrational spectrum of the cell. The results of this work confirm that observed spectroscopic features are caused by fundamental physical mechanism of interaction between THz rad iation and biological macro-mo lecules. Particu larly, the analysis of results indicates that the spectroscopic signatures of microorganis ms originate fro m the co mbination of low frequency vibrational modes or group of modes at close frequencies (vibrational bands) within mo lecular co mponents of bacterial cells/spores, with the significant contribution from the DNA. The significance of this study is justified by necessity for a fast and effective, label free and reagent free optical technology to protect against environmental and other biological threats, as well as for general medical research. The obtained results show that THz v ibrational spectroscopy promises to add quantitative genetic information to the characteristic signatures of biological objects, increasing the detection accuracy and selectivity when appropriate spectral resolution is used.

The Androgen Receptor (AR) is a critical oncogene in prostate cancer (PCa) development and progression. Here we demonstrate cell cycle dependent regulation of AR activity, localization, and phosphorylation. We show that on three AR target genes, androgen-stimulated AR transactivation is highest in G1, decreased in S-phase, and abrogated in G2/M. This change in AR transactivation parallels changes in AR localization and phosphorylation. A combination of imaging techniques and quantitative analysis shows nuclear AR localization during interphase and the exclusion of the majority, but not all, AR from chromatin in mitosis. Flow cytometry analyses using a phospho-S308 AR specific antibody in asynchronous and chemically enriched G2/M PCa cells revealed ligand-independent induction of S308 phosphorylation in mitosis when CDK1 is activated. Consistent with our flow cytometry data, IP-Western blotting showed an increase in S308 phosphorylation in G2/M and an in vitro kinase assay demonstrated that CDK1 was able to phosphorylate the AR on S308. Pharmacological inhibition of CDK1 activity resulted in decreased S308 phosphorylation in PCa cells. Importantly, using a combination of anti-total AR and phospho-S308 specific antibodies in immunofluorescence experiments, we show that the AR is excluded from condensed chromatin in mitotic cells when phosphorylated on S308. In summary, we show that the phosphorylation of the AR on S308 by CDK1 in mitosis regulates AR localization and correlates with changes in AR transcriptional activity. These findings have important implications for understanding AR function as an oncogene.