Rac1 activation driven by 14-3-3ζ dimerization promotes prostate cancer cell-matrix interactions, motility and transendothelial migration

14-3-3 proteins are ubiquitously expressed dimeric adaptor proteins that have emerged as key mediators of many cell signaling pathways in multiple cell types. Its effects are mainly mediated by binding to selective phosphoserine/threonine proteins. The importance of 14-3-3 proteins in cancer have only started to become apparent and its exact role in cancer progression as well as the mechanisms by which 14-3-3 proteins mediate cancer cell function remain unknown. While protein 14-3-3σ is widely accepted as a tumor suppressor, 14-3-3ζ, β and γ isoforms have been shown to have tumor promoting effects. Despite the importance of 14-3-3 family in mediating various cell processes, the exact role and mechanism of 14-3-3ζ remain unexplored. In the current study, we investigated the role of protein 14-3-3ζ in prostate cancer cell motility and transendothelial migration using biochemical, molecular biology and electric cell-substrate impedance sensing approaches as well as cell based functional assays. Our study indicated that expression with wild-type protein 14-3-3ζ significantly enhanced Rac activity in PC3 cells. In contrast, expression of dimer-resistant mutant of protein 14-3-3ζ (DM-14-3-3) inhibited Rac activity and associated phosphorylation of p21 activated kinase-1 and 2. Expression with wild-type 14-3-3ζ or constitutively active Rac1 enhanced extracellular matrix recognition, lamellipodia formation, cell migration and trans-endothelial migration by PC3 cells. In contrast, expression with DM 14-3-3ζ or DN-Rac1 in PC3 cells significantly inhibited these cell functions. Our results demonstrate for the first time that 14-3-3ζ enhances prostate cancer cell-matrix interactions, motility and transendothelial migration in vitro via activation of Rac1-GTPase and is an important target for therapeutic interventions for prostate cancer.     

Mapping candidate hotspots of meiotic recombination in segments of human DNA cloned in the yeast<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

The hotspots of meiotic recombination in the human genome can be localized by genetic techniques. The resolution of these techniques is in the range of kilobases and depends on the density of the physical markers identifying allelic variants of the chromosomal loci. We thought it would be interesting to localize these sites with higher resolution. Assuming that some human chromosomal sites conserve their propensity for recombination when cloned in yeast, we localized the hotspots of recombination in several yeast artificial chromosomes (YACs) carrying human DNA. A number of potential recombination hotspots could be identified in the clones studied. Among them there are two classes of sites that are particularly recombination prone also in human meiotic cells: sites associated with CpG islands and sites located in the vicinity of long minisatellite sequences.Communicated by G. P. Georgiev     

Excitation Energy Transfer in Cyanobacteria Synechocystis Embedded in Polymer Film and Partially Bleached by Polarized Irradiation

The polarized absorption, photoacoustic, fluorescence emission, and fluorescence excitation spectra of whole cells of cyanobacteria Synechocystis sp. embedded in a polymer film were measured. The bacteria cells, as it follows from anisotropy of absorption and fluorescence spectra, were even in a non-stretched polyvinyl alcohol film oriented to a certain extent. The measurements were done for such film in order to avoid the deformation of cyanobacteria shapes. Part of the samples was bleached by irradiation with strong polarized radiation with electric vector parallel to the orientation axis of cells. The anisotropy of photoacoustic spectra was higher than that of absorption spectra and it was stronger changed by the irradiation. Polarized fluorescence was excited in four wavelength regions characterised by different contribution to absorption from various bacteria pigments. The shapes of emission spectra were different depending on wavelength of excitation, polarization of radiation, and previous irradiation of the sample. The fluorescence spectra were analysed on Gaussian components belonging to various forms of pigments from photosystems (PS) 1 and 2. The results inform about excitation energy transfer between pools of pigments, differently oriented in the cells. Energy of photons absorbed by phycobilisomes was transferred predominantly to the chlorophyll of PS2, whereas photons absorbed by carotenoids to chlorophylls of PS1.     

Sodium orthovanadate exerts influence on liver Golgi complexes from control and streptozotocin-diabetic rats

The paper presents the effect of one-week 3mM sodium orthovanadate (Na3VO4) oral treatment of control and streptozotocin[STZ]-diabetic rats. The body weight decreased as compared with untreated control (C group) in both vanadate treated groups (C + V and D + V) and in diabetic untreated rats (D group)--in all cases p < 0.01. A similar tendency was demonstrated by the weight of the livers, which was statistically significant lower than in the controls (p < 0.01). The fluid and food intake were lower in comparison with control vanadium treated groups, in D + V as compared with D it was limited, however, not achieved control level. A high mortality rate, approx. 67%, after the administration of streptozotocin and vanadate (D + V group) was noted; such result had never been previously found within all study groups of rats. But the surviving rats show very good decreased (60%) free blood sugar levels, however euglycaemia was not achieved. The activity of galactosyltransferase, the Golgi complex marker enzyme in group D, was statistically lower than the controls (p < 0.001). Treatment of STZ-diabetic rats with orthovanadate did not increase the enzyme activity toward control level, in both diabetic groups (treated and untreated with Na3VO4) similar dispersion of individual results was found. Morphological study demonstrated, for the first time, no larger cellular lesion in C + V group. The Golgi complex was well developed; showed several cisterns at the trans side, which were grossly distended and contained electron-lucid floccular material. In D + V group typical, cylindrical forms of Golgi complexes predominated. These structures consisted of 3-4 almost practically non-distended cisterns. Also in this case, large, electron-dense vesicles were noted in the vicinity. In this group, small in size, myelin-like structures were also found. These structures might indicate a relatively small, but nevertheless clear damage of the internal membrane system. The external cistern of the cylindrical forms of Golgi complexes, which corresponded the trans side, was often markedly distended and formed a vacuole-like structure filled with electron lucent material; the structure itself sometimes looked empty. Multi-vesicular structures were observed also in this case, but they were seen much more rarely.     

P21 Activated Kinase-1 (Pak1) Promotes Prostate Tumor Growth and Microinvasion via Inhibition of Transforming Growth Factor β Expression and Enhanced Matrix Metalloproteinase 9 Secretion

P21-activated kinases (Paks) are major effectors downstream of the small Rho family of GTPases. Among the six isoforms, Pak1 is the most ubiquitous and the best characterized member. Previous studies have shown that inhibition of Pak6, which is predominantly present in the prostate compared with other tissues, inhibits prostate tumor growth in vivo. Even though Pak1 has been identified in normal prostatic epithelial cells and cancer cells, its specific role in the development of prostate cancer remains unclear. We report here that highly invasive prostate cancer cells express significantly higher levels of Pak1 protein compared with non-invasive prostate cancer cells. Furthermore, prostate tumor tissues and prostate cancer metastasized to lungs showed a higher expression of Pak1 compared with normal tissues. Interestingly, Pak6 protein expression levels did not change with the invasive/metastatic potential of the cancer cells or tumors. Although inhibition of Pak1, and not Pak6, resulted in impaired PC3 cell migration, the effects of Pak1 knockdown on transendothelial migration (microinvasion), tumor growth, and tumor angiogenesis was higher compared with Pak6 knockdown. Finally, gene array data revealed reduced expression of matrix metalloproteinase 9 with the ablation of either Pak1 or Pak6 gene expression in PC3 cells, whereas protein levels of TGFβ was elevated significantly with specific modulation of Pak1 activity or ablation of the Pak1 gene. Our observations suggest that although some level of functional redundancy exists between Pak1 and Pak6 in prostate cancer cells, targeting Pak1 is a potential option for the management of prostate tumor growth, microinvasion, and metastasis.