Disassembly and reassembly of the nuclear pore complexes (NPCs) is one of the major events during open mitosis in higher eukaryotes. However, how this process is controlled by the mitotic machinery is not clear. To investigate this we developed a novel in vivo model system based on syncytial Drosophila embryos. We microinjected different mitotic effectors into the embryonic cytoplasm and monitored the dynamics of disassembly/reassembly of NPCs in live embryos using fluorescently labeled wheat germ agglutinin (WGA) or in fixed embryos using electron microscopy and immunostaining techniques. We found that in live embryos Cdk1 activity was necessary and sufficient to induce disassembly of NPCs as well as their cytoplasmic mimics: annulate lamellae pore complexes (ALPCs). Cdk1 activity was also required for keeping NPCs and ALPCs disassembled during mitosis. In agreement recombinant Cdk1/cyclin B was able to induce phosphorylation and dissociation of nucleoporins from the NPCs in vitro. Conversely, reassembly of NPCs and ALPCs was dependent on the activity of protein phosphatases, sensitive to okadaic acid (OA). Our findings suggest a model where mitotic disassembly/reassembly of the NPCs is regulated by a dynamic equilibrium of Cdk1 and OA-sensitive phosphatase activities and provide evidence that mitotic phosphorylation mediates disassembly of the NPC.

Drosophila Merlin, the homolog of the human Neurofibromatosis 2 (NF2) gene, is important for the regulation of cell proliferation and receptor endocytosis. Male flies carrying a Mer3 allele, a missense mutation (Met177→Ile) in the Merlin gene, are viable but sterile; however, the cause of sterility is unknown.

D-glucuronyl C5-epimerase (GLCE) is a potential tumor-suppressor gene involved in heparan sulfate biosynthesis. GLCE expression is significantly decreased in breast tumors; however, the underlying molecular mechanisms remain unclear. This study examined the possible epigenetic mechanisms for GLCE inactivation in breast cancer. Very little methylation of the GLCE promoter region was detected in breast tumors in vivo and in breast cancer cells (MCF7 and T47D) in vitro and GLCE expression in breast cancer cells was not altered by 5-deoxyazacytidine (5-aza-dC) treatment, suggesting that promoter methylation is not involved in regulating GLCE expression. Chromatin activation by Trichostatin A (TSA) or 5-aza-dC/TSA treatment increased GLCE expression by two to 3-fold due to an increased interaction between the GLCE promoter and the TCF4/β-catenin transactivation complex, or H3K9ac and H3K4Me3 histone modifications. However, ectopic expression of TCF4/β-catenin was not sufficient to activate GLCE expression in MCF7 cells, suggesting that chromatin structure plays a key role in GLCE regulation. Although TSA treatment significantly repressed canonical WNT signaling in MCF7 cells, it did not influence endogenous TCF4/β-catenin mRNA levels and activated TCF4/β-catenin-driven transcription from the GLCE promoter, indicating GLCE as a novel target for TCF4/β-catenin complex in breast cancer cells. A correlation was observed between GLCE, TCF4 and β-catenin expression in breast cancer cells and primary tumors, suggesting an important role for TCF4/β-catenin in regulating GLCE expression both in vitro and in vivo. Taken together, the results indicate that GLCE expression in breast cancer is regulated by a combination of chromatin structure and TCF4/β-catenin complex activity.

Here we present the analysis of alternative splicing events on an example of glioblastoma cell culture samples using a set of computer tools in combination with database integration. The gene expression profiles of glioblastoma were obtained from cell culture samples of primary glioblastoma which were isolated and processed for RNA extraction. Transcriptome profiling of normal brain samples and glioblastoma were done by Illumina sequencing. The significant differentially expressed exon-level probes and their corresponding genes were identified using a combination of the splicing index method. Previous studies indicated that tumor-specific alternative splicing is important in the regulation of gene expression and corresponding protein functions during cancer development. Multiple alternative splicing transcripts have been identified as progression markers, including generalized splicing abnormalities and tumor- and stage-specific events. We used a set of computer tools which were recently applied to analysis of gene expression in laboratory animals to study differential splicing events. We found 69 transcripts that are differentially alternatively spliced. Three cancer-associated genes were considered in detail, in particular: APP (amyloid beta precursor protein), CASC4 (cancer susceptibility candidate 4) and TP53. Such alternative splicing opens new perspectives for cancer research.