Keshan disease (KD) is an endemic dilated cardiomyopathy with unclear etiology. In this study, we compared mitochondrial-related gene expression profiles of peripheral blood mononuclear cells (PBMCs) derived from 16 KD patients and 16 normal controls in KD areas. Total RNA was isolated, amplified, labeled and hybridized to Agilent human 4 × 44k whole genome microarrays. Mitochondrial-related genes were screened out by the Third-Generation Human Mitochondria-Focused cDNA Microarray (hMitChip3). Quantitative real-time PCR, immunohistochemical and biochemical parameters related mitochondrial metabolism were conducted to validate our microarray results. In KD samples, 34 up-regulated genes (ratios ≥ 2.0) were detected by significance analysis of microarrays and ingenuity systems pathway analysis (IPA). The highest ranked molecular and cellular functions of the differentially regulated genes were closely related to amino acid metabolism, free radical scavenging, carbohydrate metabolism, and energy production. Using IPA, 40 significant pathways and four significant networks, involved mainly in apoptosis, mitochondrion dysfunction, and nuclear receptor signaling were identified. Based on our results, we suggest that PGC-1alpha regulated energy metabolism and anti-apoptosis might play an important role in the compensatory mechanism of KD. Our results may lead to the identification of potential diagnostic biomarkers for KD in PBMCs, and may help to understand the pathogenesis of KD.
We have investigated a potential relationship between expression of beta 1-integrins and adhesiveness of the beta 2-integrin LFA-1 (alpha L beta 2, CD11a/CD18). By an approach of random mutagenesis and selection we established clones from the human acute lymphatic leukemia cell line HPB-ALL with (i) constitutively active LFA-1 and (ii) with no apparent integrin-beta 1 cell surface expression. Thirty seven of 42 clones selected for activated LFA-1 were found to have lost apparent integrin-beta 1 expression. Conversely, 7 of 21 clones selected for lack of beta 1 expression were found to have activated LFA-1. Since this pointed toward a possible coupling between beta 1 expression and LFA-1 activity, we further analyzed at which level beta 1 expression was blocked. We focused on one clone, HAP4, with activated LFA-I and no detectable beta 1 cell surface expression and found, surprisingly, that it expressed wild-type levels of beta 1 mRNA and, in Western blots of whole cell lysates, apparently normal levels of beta 1 protein. However, in addition to beta 1 of the expected molecular weight, HAP4 expressed a unique 48-kDa band recognized by the polyclonal anti-beta 1 antiserum. Immunoprecipitation experiments revealed that the epitope recognized by the anti-beta 1 antibody 4B4 was hidden or lost. The alpha 4-chain was found in its precursor form but it did not associate with any beta-chain, and it was not processed to its mature form. Instead alpha 4-chains were eventually degraded. Taken together this showed that beta 1-chains were produced but not properly processed in HAP4. From this we propose that HAP4 is deficient in a gene product required both for proper beta 1 folding and for repression of LFA-1 adhesiveness.
Op18/stathmin (Op18) is a microtubule-destabilizing protein that is phosphorylation-inactivated during mitosis and its normal function is to govern tubulin subunit partitioning during interphase. Human tumors frequently overexpress Op18 and a tumor-associated Q18-->E mutation has been identified that confers hyperactivity, destabilizes spindle microtubules, and causes mitotic aberrancies, polyploidization, and chromosome loss in K562 leukemia cells. Here we determined whether wild-type and mutant Op18 have the potential to cause chromosomal instability by some means other than interference with spindle assembly, and thereby bypassing the spindle assembly checkpoint. Our approach was based on Op18 derivatives with distinct temporal order of activity during mitosis, conferred either by differential phosphorylation inactivation or by anaphase-specific degradation through fusion with the destruction box of cyclin B1. We present evidence that excessive Op18 activity generates chromosomal instability through interference occurring subsequent to the metaphase-to-anaphase transition, which reduces the fidelity of chromosome segregation to spindle poles during anaphase. Similar to uncorrected merotelic attachment, this mechanism evades detection by the spindle assembly checkpoint and thus provides an additional route to chromosomal instability.
The genomes of immortalized cell lines (and cancer cells) are characterized by multiple types of aberrations, ranging from single nucleotide polymorphisms (SNPs) to structural rearrangements that have accumulated over time. Consequently, it is difficult to estimate the relative impact of different aberrations, the order of events, and which gene functions were under selective pressure at the early stage towards cellular immortalization. Here, we have established novel cell cultures derived from Drosophila melanogaster embryos that were sampled at multiple time points over a one-year period. Using short-read DNA sequencing, we show that copy-number gain in preferentially stress-related genes were acquired in a dominant fraction of cells in 300-days old cultures. Furthermore, transposable elements were active in cells of all cultures. Only a few (<1%) SNPs could be followed over time, and these showed no trend to increase or decrease. We conclude that the early cellular responses of a novel culture comprise sequence duplication and transposable element activity. During immortalization, positive selection first occurs on genes that are related to stress response before shifting to genes that are related to growth.
Adult mesenchymal stem cells have self-renewal and multiple differentiation potentials, and play important roles in regenerative medicine. However, their use may be limited by senescence or age of the donor, leading to changes in stem cell functionality. We investigated morphological, molecular and functional differences between bone marrow-derived (MSC) and adipose-derived (ASC) stem cells isolated from neonatal, young and old rats compared to Schwann cells from the same animals. Immunocytochemistry, RT-PCR, proliferation assays, western blotting and transmission electron microscopy were used to investigate expression of senescence markers. Undifferentiated and differentiated ASC and MSC from animals of different ages expressed Notch-2 at similar levels; protein-38 and protein-53 were present in all groups of cells with a trend towards increased levels in cells from older animals compared to those from neonatal and young rats. Following co-culture with adult neuronal cells, dMSC and dASC from animals of all ages elicited robust neurite outgrowth. Mitotracker (R) staining was consistent with ultrastructural changes seen in the mitochondria of cells from old rats, indicative of senescence. In conclusion, this study showed that although the cells from aged animals expressed markers of senescence, aged MSC and ASC differentiated into SC-like cells still retain potential to support axon regeneration.
To investigate the pathogenesis of Kashin-Beck disease (KBD), we compared the common signaling pathways in peripheral blood mononuclear cells (PBMCs) obtained from healthy juvenile and adults and KBD patients, and also from osteoarthritis (OA) patients. The PBMCs from 12 KBD and 12 healthy juvenile, and those from 20 adult KBD patients and 12 healthy donors were separately collected among the people living in the KBD endemic area. The patients were distinguished according to the national diagnosis criteria. Total RNAs were extracted for the determination of gene expressions by microarray analysis. Ingenuity Pathways Analysis (IPA) was employed to identify the signaling pathways significantly affected by juveniles' and adults' KBD, and OA. The expressions of NFκB-p65, cIAP2 and RANKL in the articular cartilage from both juvenile and adults were detected by immunohistochemistry. NF-κB signaling, apoptosis signaling, death receptor signaling and IL-6 signaling pathways were revealed to be the common affected signaling pathways in the juvenile and adult KBD and the OA. BIRC3 and EGR1 were identified as two common differentially expressed genes. The percentages of positive staining of NFκB-p65, cIAP2 and RANKL were reduced in adult KBD patients but significantly increased in juvenile KBD patients. NF-κB, one of the common signaling pathways between adult and juvenile KBD, was less prominent in the adult KBD patients.
Physiological and pathological turnover of basement membranes liberates biologically active cryptic molecules. Several collagen-derived fragments possess anti-angiogenic activity. Arresten is the 26-kDa non-collagenous domain of type IV collagen alpha1 chain. It functions as an efficient inhibitor of angiogenesis and tumor growth in mouse models, but its anti-angiogenic mechanism is not completely known. Here we show that arresten significantly increases apoptosis of endothelial cells in vitro by decreasing the amount of anti-apoptotic molecules of the Bcl-family; Bcl-2 and Bcl-xL. Although the pro-apoptotic effect of arresten is endothelial cell specific in vitro, in mouse tumors arresten induced apoptosis both in endothelial and tumor cells. The tumor cell apoptosis is likely an indirect effect due to the inhibition of blood vessel growth into the tumor. The active site of arresten was localized by deletion mutagenesis within the C-terminal half of the molecule. We have previously shown that arresten binds to alpha1beta1 integrin on human umbilical vein endothelial cells. However, the microvascular endothelial cells (MLECs) are more important in the context of tumor vasculature. We show here that arresten binds also to the microvascular endothelial cells via alpha1beta1 integrin. Furthermore, it has no effect on Matrigel neovascularization or the viability of integrin alpha1 null MLECs. Tumors implanted on integrin alpha1 deficient mice show no integrin alpha1 expression in the host-derived vascular endothelium, and thus arresten does not inhibit the tumor growth. Collectively, this data sheds more light into the anti-angiogenic mechanism of arresten.
Arginine methylation is a post-translational modification of proteins implicated in RNA processing, protein compartmentalization, signal transduction, transcriptional regulation and DNA repair. In a screen for proteins associated with the nuclear envelope in the yeast Saccharomyces cerevisiae, we have identified the arginine methyltransferase Rmt2, previously shown to methylate the ribosomal protein L12. By indirect immunofluorescence and subcellular fractionations we demonstrate here that Rmt2 has nuclear and cytoplasmic localizations. Biochemical analysis of a fraction enriched in nuclei reveals that nuclear Rmt2 is resistant to extractions with salt and detergent, indicating an association with structural components. This was supported by affinity purification experiments with TAP-tagged Rmt2. Rmt2 was found to co-purify with the nucleoporins Nup49, Nup57 and Nup100, revealing a novel link between arginine methyltransferases and the nuclear pore complex. In addition, a genome-wide transcription study of the rmt2Delta mutant shows significant downregulation of the transcription of MYO1, encoding the Type II myosin heavy chain required for cytokinesis and cell separation.
Although embryonic patterning and early development of the nervous system have been studied for decades, our understanding of how signals instruct ectodermal derivatives to acquire specific identities has only recently started to form a coherent picture. In this mini-review, we summarize recent findings and models of how a handful of well-known secreted signals influence progenitor cells in successive binary decisions to adopt various cell type specific differentiation programs.
Op18/stathmin (Op18) is a phosphorylation-regulated and differentially expressed microtubule-destabilizing protein in animal cells. Op18 regulates tubulin monomer-polymer partitioning of the interphase microtubule system and forms complexes with tubulin heterodimers. Recent reports have shown that specific tubulin-folding cofactors and related proteins may disrupt tubulin heterodimers. We therefore investigated whether Op18 protects unpolymerized tubulin from such disruptive activities. Our approach was based on inducible overexpression of two tubulin-disrupting proteins, namely TBCE, which is required for tubulin biogenesis, and E-like, which has been proposed to regulate tubulin turnover and microtubule stability. Expression of either of these proteins was found to cause a rapid degradation of both alpha-tubulin and beta-tubulin subunits of unpolymerized, but not polymeric, tubulin heterodimers. We found that depletion of Op18 by means of RNA interference increased the susceptibility of tubulin to TBCE or E-like mediated disruption, while overexpressed Op18 exerted a tubulin-protective effect. Tubulin protection was shown to depend on Op18 levels, binding affinity, and the partitioning between tubulin monomers and polymers. Hence, the present study reveals that Op18 at physiologically relevant levels functions to preserve the integrity of tubulin heterodimers, which may serve to regulate tubulin turnover rates.
Nijmegen breakage syndrome (NBS) is a rare genetic instability syndrome associated with a high incidence of lymphoid malignancies. The NBS1 protein has been implicated in telomere biology suggesting that cells from NBS patients might have deficient telomere maintenance capacity. In this study we characterized spontaneously immortalized T-cell lines derived from three NBS patients regarding growth characteristics, telomere biology, expression of cell-cycle regulators, and response to DNA damage to understand the role of NBS1 in the immortalization process. In all the NBS T-cell lines the acquisition of an immortal phenotype was associated with telomere length stabilization, high telomerase activity, and increased mRNA expression of the catalytic subunit of telomerase (hTERT), together with c-myc up-regulation. Our findings provide evidence that telomere length maintenance was intact in the T lymphocytes in the absence of a full-length NBS protein, presumably due to the presence of an alternatively transcribed NBS protein of 70 kDa. Normal protein expression patterns for pRb and p53 in all the immortal lines coincided with altered expression of some cell-cycle proteins as well as with an impaired G1/S arrest after gamma irradiation, despite a seemingly normal p53/p21 pathway. The here described, spontaneously immortalized NBS derived T-cell lines can be useful in future analysis of the biologic effects in the NBS.
Adipose derived stem cells (ADSC) can be differentiated into Schwann cell-like cells which enhance nerve function and regeneration. However, the signalling mechanisms underlying the neurotrophic potential of ADSC remain largely unknown. In this study, we hypothesised that ADSC, upon stimulation with a combination of growth factors, could rapidly produce brain derived neurotrophic factor (BDNF) with a similar molecular mechanism to that functioning in the nervous system. Within 48h of stimulation, ADSC demonstrated potent neurotrophic effects on dorsal root ganglion neurons, at a magnitude equivalent to that of the longer term differentiated Schwann cell-like cells. Stimulated ADSC showed rapid up-regulation of the neuronal activity dependent promoter BDNF exon IV along with an augmented expression of full length protein encoding BDNF exon IX. BDNF protein was secreted at a concentration similar to that produced by differentiated Schwann cell-like cells. Stimulation also activated the BDNF expression gating transcription factor, cAMP responsive element binding (CREB) protein. However, blocking phosphorylation of CREB with the protein kinase A small molecule inhibitor H89 did not suppress secretion of BDNF protein. These results suggest rapid BDNF production in ADSC is mediated through multiple compensatory pathways independent of, or in addition to, the CREB neuronal activation cascade.
One of the most important ways in which animal species vary is in their size. Individuals of the largest animal ever thought to have lived, the blue whale (Balaenoptera musculus), can reach a weight of 190 t and a length of over 30 m. At the other extreme, among the smallest multicellular animals are males of the parasitic wasp, Dicopomorpha echmepterygis, which even as adults are just 140 mu m in length. In terms of volume, these species differ by more than 14 orders of magnitude. Since size has such profound effects on an organism's ecology, anatomy and physiology, an important task for evolutionary biology and ecology is to account for why organisms grow to their characteristic sizes. Equally, a full description of an organism's development must include an explanation of how its growth and body size are regulated. Here I review research on how these processes are controlled in the nematode, Caenorhabditis elegans. Analyses of small and long mutants have revealed that in the worm, DBL-1, a ligand in the TGF beta superfamily family, promotes growth in a dose-dependent manner. DBL-1 signaling affects body size by stimulating the growth of syncytial hypodermal cells rather than controlling cell division. Signals from chemosensory neurons and from the gonad also modulate body size, in part, independently of DBL-1-mediated signaling. Organismal size and morphology is heavily influenced by the cuticle, which acts as the exoskeleton. Finally, I summarize research on several genes that appear to regulate body size by cell autonomously regulating cell growth throughout the worm.
BACKGROUND: Acquired resistance to cisplatin treatment is a caveat when treating patients with non-small cell lung cancer (NSCLC) and malignant pleural mesothelioma (MPM). Ceramide increases in response to chemotherapy, leading to proliferation arrest and apoptosis. However, a tumour stress activation of glucosylceramide synthase (GCS) follows to eliminate ceramide by formation of glycosphingolipids (GSLs) such as globotriaosylceramide (Gb3), the functional receptor of verotoxin-1. Ceramide elimination enhances cell proliferation and apoptosis blockade, thus stimulating tumor progression. GSLs transactivate multidrug resistance 1/P-glycoprotein (MDR1) and multidrug resistance-associated protein 1 (MRP1) expression which further prevents ceramide accumulation and stimulates drug efflux. We investigated the expression of Gb3, MDR1 and MRP1 in NSCLC and MPM cells with acquired cisplatin resistance, and if GCS activity or MDR1 pump inhibitors would reduce their expression and reverse cisplatin-resistance.
METHODS: Cell surface expression of Gb3, MDR1 and MRP1 and intracellular expression of MDR1 and MRP1 was analysed by flow cytometry and confocal microscopy on P31 MPM and H1299 NSCLC cells and subline cells with acquired cisplatin resistance. The effect of GCS inhibitor PPMP and MDR1 pump inhibitor cyclosporin A for 72h on expression and cisplatin cytotoxicity was tested.
RESULTS: The cisplatin-resistant cells expressed increased cell surface Gb3. Cell surface Gb3 expression of resistant cells was annihilated by PPMP whereas cyclosporin A decreased Gb3 and MDR1 expression in H1299 cells. No decrease of MDR1 by PPMP was noted in using flow cytometry, whereas a decrease of MDR1 in H1299 and H1299res was indicated with confocal microscopy. No certain co-localization of Gb3 and MDR1 was noted. PPMP, but not cyclosporin A, potentiated cisplatin cytotoxicity in all cells.
CONCLUSIONS: Cell surface Gb3 expression is a likely tumour biomarker for acquired cisplatin resistance of NSCLC and MPM cells. Tumour cell resistance to MDR1 inhibitors of cell surface MDR1 and Gb3 could explain the aggressiveness of NSCLC and MPM. Therapy with GCS activity inhibitors or toxin targeting of the Gb3 receptor may substantially reduce acquired cisplatin drug resistance of NSCLC and MPM cells.
Stem cells generally reside in a stem cell micro environment, where cues for self-renewal and differentiation are present. However, the genetic program underlying stem cell proliferation and multipotency is poorly understood. Transcriptome analysis of stem cells and their in vivo microenvironment is one way of uncovering the unique sternness properties and provides a framework for the elucidation of stem cell function. Here, we characterize the gene expression profile of the in vivo neural stem cell microenvironment in the lateral ventricle wall of adult mouse brain and of in vitro proliferating neural stem cells. We have also analyzed an Lhx2-expressing hematopoietic-stem-cell-like cell line in order to define the transcriptome of a well-characterized and pure cell population with stem cell characteristics. We report the generation, assembly and annotation of 50,792 high-quality 5'-end expressed sequence tag sequences. We further describe a shared expression of 1065 transcripts by all three stem cell libraries and a large overlap with previously published gene expression signatures for neural stem/progenitor cells and other multipotent stem cells. The sequences and cDNA clones obtained within this framework provide a comprehensive resource for the analysis of genes in adult stem cells that can accelerate future stem cell research.
Leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) is a recently discovered negative regulator of growth factor signaling. The LRIG1 integral membrane protein has been demonstrated to regulate various oncogenic receptor tyrosine kinases, including epidermal growth factor (EGF) receptor (EGFR), by cell-autonomous mechanisms. Here, we investigated whether LRIG1 ectodomains were shed, and if LRIG1 could regulate cell proliferation and EGF signaling in a paracrine manner. Cells constitutively shed LRIG1 ectodomains in vitro, and shedding was modulated by known regulators of metalloproteases, including the ADAM17 specific inhibitor TAPI-2. Furthermore, shedding was enhanced by ectopic expression of Adam17. LRIG1 ectodomains appeared to be shed in vivo, as well, as demonstrated by immunoblotting of mouse and human tissue lysates. Ectopic expression of LRIG1 in lymphocytes suppressed EGF signaling in co-cultured fibroblastoid cells, demonstrating that shed LRIG1 ectodomains can function in a paracrine fashion. Purified LRIG1 ectodomains suppressed EGF signaling without any apparent downregulation of EGFR levels. Taken together, the results show that the LRIG1 ectodomain can be proteolytically shed and can function as a non-cell-autonomous regulator of growth factor signaling. Thus, LRIG1 or its ectodomain could have therapeutic potential in the treatment of growth factor receptor-dependent cancers.