Downregulation of histone H3 lysine 9 methyltransferase G9a induces centrosome disruption and chromosome instability in cancer cells

Background

Modifications of the histone amino-terminal tails affect access of regulatory factors and complexes to chromatin and thereby influence biological processes. Cancer cells are characterized by prominent epigenetic dysregulation, including histone modifications. However, the functional roles of the histone methyltransferases (HMT) in cancer remain unclear.

Methodology/Principal Findings

We studied RNAi-based inhibition (knockdown, KD) of 2 different H3K9 HMTs, SUV39H1 and G9a. Knockdown of the 2 HMTs in PC3 cancer cell line markedly inhibited cell growth and caused profound morphological changes with loss of telomerase activity and shortened telomeres. SUV39H1 KD cells showed substantial increase in G2/M fraction. G9a KD cells showed increased DNA content (1.7-fold in 2 independent clones) compared with FACS analyses to control. Karyotype analyses showed that this was due to an increased number of chromosomes (from 61 to 102) in G9a KD cells compared to parental PC3. Intriguingly, we found abnormal centrosome morphology and number in about 25% of the G9a KD cells, while centrosomes were morphologically normal in control cells. Microarray analyses after KD of SUV39H1 or G9a showed very few genes up-regulated among the 39,000 genes. The silenced tumor-suppressor genes p16 and RASSF1A were not activated in KD cells.

Conclusions/Significance

These data suggest that the 2 HMTs, SUV39H1 and G9a are required to perpetuate the malignant phenotype. Furthermore, G9a plays a critical role in regulating centrosome duplication presumably through chromatin structure rather than through affecting gene expression in cancer cells. Targeting these histone methyltransferases may be of therapeutic benefit in cancers.     

Induction and regulation of matrix metalloproteinase-12in human airway smooth muscle cells

Background

The elastolytic enzyme matrix metalloproteinase (MMP)-12 has been implicated in the development of airway inflammation and remodeling. We investigated whether human airway smooth muscle cells could express and secrete MMP-12, thereby participating in the pathogenesis of airway inflammatory diseases.

Methods

Laser capture microdissection was used to collect smooth muscle cells from human bronchial biopsy sections. MMP-12 mRNA expression was analysed by quantitative real-time RT-PCR. MMP-12 protein expression and secretion from cultured primary airway smooth muscle cells was further analysed by Western blot. MMP-12 protein localization in bronchial tissue sections was detected by immunohistochemistry. MMP-12 activity was determined by zymography. The TransAM AP-1 family kit was used to measure c-Jun activation and nuclear binding. Analysis of variance was used to determine statistical significance.

Results

We provide evidence that MMP-12 mRNA and protein are expressed by in-situ human airway smooth muscle cells obtained from bronchial biopsies of normal volunteers, and of patients with asthma, COPD and chronic cough. The pro-inflammatory cytokine, interleukin (IL)-1β, induced a >100-fold increase in MMP-12 gene expression and a >10-fold enhancement in MMP-12 activity of primary airway smooth muscle cell cultures. Selective inhibitors of extracellular signal-regulated kinase, c-Jun N-terminal kinase and phosphatidylinositol 3-kinase reduced the activity of IL-1β on MMP-12, indicating a role for these kinases in IL-1β-induced induction and release of MMP-12. IL-1β-induced MMP-12 activity and gene expression was down-regulated by the corticosteroid dexamethasone but up-regulated by the inflammatory cytokine tumour necrosis factor (TNF)-α through enhancing activator protein-1 activation by IL-1β. Transforming growth factor-β had no significant effect on MMP-12 induction.

Conclusion

Our findings indicate that human airway smooth muscle cells express and secrete MMP-12 that is up-regulated by IL-1β and TNF-α. Bronchial smooth muscle cells may be an important source of elastolytic activity, thereby participating in remodeling in airway diseases such as COPD and chronic asthma.     

Ultrastructural, tomographic and confocal imaging of the chondrocyte primary cilium in situ

Hyaline cartilage chondrocytes express one primary cilium per cell, but its function remains unknown. We examined the ultrastructure of chick embryo sternal chondrocyte cilia and their interaction with extracellular matrix molecules by transmission electron microscopy (TEM) and, for the first time, double-tilt electron tomography. Ciliary bending was also examined by confocal immunohistochemistry. Tomography and TEM showed the ciliary axoneme to interdigitate amongst collagen fibres and condensed proteoglycans. TEM also revealed the presence of electron-opaque particles in the proximal axoneme which may represent intraciliary-transport (ICT) particles. We observed a wide range of ciliary bending patterns. Some conformed to a heavy elastica model associated with shear stress. Others were acutely deformed, suggesting ciliary deflection by collagen fibres and proteoglycans with which the cilia make contact. We conclude that mechanical forces transmitted through these matrix macromolecules bend the primary cilium, identifying it as a potential mechanosensor involved in skeletal patterning and growth.     

DSCR2, a Down syndrome critical region protein, is localized to the endoplasmic reticulum of mammalian cells

We used immunocytochemical and fluorescence assays to investigate the subcellular location of the protein encoded by Down syndrome critical region gene 2 (DSCR2) in transfected cells. It was previously suggested that DSCR2 is located in the plasma membrane as an integral membrane protein. Interestingly, we observed this protein in the endoplasmic reticulum (ER) of cells. We also studied whether the truncated forms of DSCR2 showed different subcellular distributions. Our observations indicate that DSCR2 probably is not inserted into the membrane of the endoplasmic reticulum since the fragments lacking the predicted transmembrane (TM) helices remained associated with the ER. Our analyses suggest that, although DSCR2 is associated with the endoplasmic reticulum, it is not an integral membrane protein and it is maintained on the cytoplasmic side of the ER by indirect interaction with the ER membrane or with another protein.     

Effect of manganese and calcium deficiency on the growth and oxygen exchange ofScenedesmus intermedius cultured for successive generations

The green algaScenedesmus intermedius was grown in synchronous culture under manganese or calcium deficiency for six successive generations. The growth rate, pigment and protein contents gradually decreased in comparison with the control. In Mn-deficient cells, the rate of oxygen evolution was sharply decreased. This inhibition was restored to normal in less than 1 h (40–60 min) by adding Mn salt to the suspension medium. In Ca-deficient cells, the inhibition of photosynthesis appears to be irreversible.     

Expression and secretion of human alpha1(I) procollagen fragment by Hansenula polymorpha as compared to Pichia pastoris

Secretion of a human collagen alpha1(I) chain fragment was achieved in Hansenula polymorpha using the native alpha1(I) procollagen secretory signal sequence. The N-terminal propeptide in the fragment was cleaved off during secretion, yielding the N-terminus of mature alpha1(I) collagen. In Pichia pastoris transformants, the expression of the fragment could be detected on RNA-level, but the product could not be determined extracellularly. After fusion of the fragment with a myc-HIS6 epitope, the intact product was found intracellularly. The difference in the extracellular level of the protein between the two expression hosts is most likely caused by difference in secretion efficiency.     

Neuregulin 3 promotes excitatory synapse formation on hippocampal interneurons

Hippocampal GABAergic interneurons are crucial for cortical network function and have been implicated in psychiatric disorders. We show here that Neuregulin 3 (Nrg3), a relatively little investigated low‐affinity ligand, is a functionally dominant interaction partner of ErbB4 in parvalbumin‐positive (PV) interneurons. Nrg3 and ErbB4 are located pre‐ and postsynaptically, respectively, in excitatory synapses on PV interneurons in vivo. Additionally, we show that ablation of Nrg3 results in a similar phenotype as the one described for ErbB4 ablation, including reduced excitatory synapse numbers on PV interneurons, altered short‐term plasticity, and disinhibition of the hippocampal network. In culture, presynaptic Nrg3 increases excitatory synapse numbers on ErbB4⁺ interneurons and affects short‐term plasticity. Nrg3 mutant neurons are poor donors of presynaptic terminals in the presence of competing neurons that produce recombinant Nrg3, and this bias requires postsynaptic ErbB4 but not ErbB4 kinase activity. Furthermore, when presented by non‐neuronal cells, Nrg3 induces postsynaptic membrane specialization. Our data indicate that Nrg3 provides adhesive cues that facilitate excitatory neurons to synapse onto ErbB4⁺ interneurons.     

Conditional and unconditional QTL mapping of drought-tolerance-related traits of wheat seedling using two related RIL populations

For discovering the quantitative trait loci (QTLs) contributing to early seedling growth and drought tolerance during germination, conditional and unconditional analyses of 12 traits of wheat seedlings: coleoptile length, seedling height, longest root length, root number, seedling fresh weight, stem and leaves fresh weight, root fresh weight, seedling dry weight, stem and leaves dry weight, root dry weight, root to shoot fresh weight ratio, root-to-shoot dry weight ratio, were conducted under two water conditions using two F_8:9 recombinant inbred line (RIL) populations. The results of unconditional analysis are as follows: 88 QTLs accounting for 3.33–77.01% of the phenotypic variations were detected on chromosomes 1A, 1B, 1D, 2A, 2B, 2D, 3A, 3B, 4A, 4B, 4D, 5A, 5B, 5D, 6A, 6B, 6D, 7A, 7B and 7D. Among these QTLs, 19 were main-effect QTLs with a contribution rate greater than 10%. The results of the conditional QTL analysis of 12 traits under osmotic stress on normal water conditions were as follows: altogether 22 QTLs concerned with drought tolerance were detected on chromosomes 1B, 2A, 2B, 3B, 4A, 5D, 6A, 6D, 7B, and 7D. Of these QTLs, six were main-effect QTLs. These 22 QTLs were all special loci directly concerned with drought tolerance and most of them could not be detected by unconditional analysis. The finding of these QTLs has an important significance for fine-mapping technique, map-based cloning, and molecular marker-assisted selection of early seedling traits, such as growth and drought tolerance.