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Control Cell Lines (control + cell_line)
Selected AbstractsBrain-Derived Neurotrophic Factor, Neurotrophin-3, and Neurotrophin-4/5 Prevent the Death of Striatal Projection Neurons in a Rodent Model of Huntington's DiseaseJOURNAL OF NEUROCHEMISTRY, Issue 5 2000Esther Pérez-Navarro Abstract: Intrastriatal injection of quinolinate has been proven to be a very useful animal model to study the pathogenesis and treatment of Huntington's disease. To determine whether growth factors of the neurotrophin family are able to prevent the degeneration of striatal projection neurons, cell lines expressing brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), or neurotrophin-4/5 (NT-4/5) were grafted in the adult rat striatum before quinolinate injection. Three days after lesioning, ongoing cell death was assessed by in situ detection of DNA fragmentation. In animals grafted with the control cell line, quinolinate injection induced a gradual cell loss that was differentially prevented by intrastriatal grafting of BDNF-, NT-3-, or NT-4/5-secreting cells. Seven days after lesioning, we characterized striatal projection neurons that were protected by neurotrophins. Quinolinate injection, alone or in combination with the control cell line, induced a selective loss of striatal projection neurons. Grafting of a BDNF-secreting cell line prevented the loss of all types of striatal projection neurons analyzed. Glutamic acid decarboxylase 67-, preproenkephalin-, and preprotachykinin A- but not prodynorphin-expressing neurons were protected by grafting of NT-3- or NT-4/5-secreting cells but with less efficiency than the BDNF-secreting cells. Our findings show that neurotrophins are able to promote the survival of striatal projection neurons in vivo and suggest that BDNF might be beneficial for the treatment of striatonigral degenerative disorders, including Huntington's disease. [source] Profiling of differentially expressed genes induced by high linear energy transfer radiation in breast epithelial cellsMOLECULAR CARCINOGENESIS, Issue 4 2001Debasish Roy Abstract Methods to define patterns of gene expression have applications in a wide range of biological systems. Several molecular biological techniques are used to study expression patterns during the neoplastic progression of breast epithelial cells. In the present study, differential expression of human oncogenes/tumor suppressor genes in human breast epithelial cell lines irradiated with low doses of high linear energy transfer radiation and treated with estrogen was assessed with cDNA expression arrays. Transformed and tumorigenic cell lines were compared with the control cell line to identify differentially expressed genes during tumorigenic progression. Autoradiographic analysis showed that of the 190 genes analyzed, 49 genes showed a high level of altered expression, and 12 genes had minor differences in expression levels. Among these 49 genes, 17 genes were altered at all stages of transformation, 21 were altered only at the early stage, and the remaining 11 were at the late stage of transformation to the tumorigenic stage of progression. Among the 11 late stage,associated genes, seven genes were altered exclusively in the tumorigenic cell lines and in Tumor-T. Of the 17 all-stage genes, six were randomly selected, and we confirmed their altered expression by gene-specific semiquantitative reverse transcription polymerase chain reaction, followed by Northern blot analysis. The results showed that the mRNA expression patterns of all these genes were consistent with the expression pattern seen on the array. Among these six genes, five genes, including c- myc, puf, MNDA, c- yes, and Fra-1 showed upregulation, and the other gene, RBA/p48, showed downregulation in the transformed and tumorigenic cell lines compared with the control MCF-10F cell line. Investigation of these genes should help establish the molecular mechanisms of progression that are altered by radiation and estrogen treatment. A number of candidates reported here should be useful as biomarkers involved in breast carcinogenesis. © 2001 Wiley-Liss, Inc. [source] RNA interference of sialidase improves glycoprotein sialic acid content consistencyBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2006Frederyk A. Ngantung Abstract An important challenge facing therapeutic protein production in mammalian cell culture is the cleavage of terminal sialic acids on recombinant protein glycans by the glycosidase enzymes released by lysed cells into the supernatant. This undesired phenomenon results in a protein product which is rapidly cleared from the plasma by asialoglycoprotein receptors in the liver. In this study, RNA interference was utilized as a genetic approach to silence the activity of sialidase, a glycosidase responsible for cleaving terminal sialic acids on IFN-, produced by Chinese Hamster Ovary (CHO) cells. We first identified a 21-nt double stranded siRNA that reduced endogenous sialidase mRNA and protein activity levels. Potency of each siRNA sequences was compared using real time RT-PCR and a sialidase activity assay. We next integrated the siRNA sequence into CHO cells, allowing production and selection of stable cell lines. We isolated stable clones with sialidase activity reduced by over 60% as compared to the control cell line. Micellar electrokinetic chromatography (MEKC), thiobarbituric acid assay (TAA), and high performance anion exchange chromatography (HPAEC) coupled to amperometric detection were performed to analyze glycan site occupancy, sialic acid content, and distribution of asialo-/sialylated-glycan structures, respectively. Two of the stable clones successfully retained the full sialic acid content of the recombinant IFN-,, even upon cells' death. This was comparable to the case where a chemically synthesized sialidase inhibitor was used. These results demonstrated that RNA interference of sialidase can prevent the desialylation problem in glycoprotein production, resulting improved protein quality during the entire cell culture process. © 2006 Wiley Periodicals, Inc. [source] Role of ataxia telangiectasia mutated in insulin signalling of muscle-derived cell lines and mouse soleusACTA PHYSIOLOGICA, Issue 4 2010I. Jeong Abstract Aim:, Ataxia telangiectasia mutated (ATM) reportedly plays a role in insulin-stimulated activation of Akt in some cell types but not in others. The role of ATM in insulin signalling has not been firmly resolved for skeletal muscle cells, for which Akt phosphorylation is a pivotal step in stimulation of glucose transport. Accordingly, our aim was to determine the role of ATM in insulin effects for cell lines derived from skeletal muscle and for skeletal muscle. Methods:, We examined insulin effects in L6 myotubes, mouse soleus, C2C12 myotubes and differentiated rhabdomyosarcoma (RD) cells in the presence and absence of a low concentration (1 ,m) of the ATM inhibitor KU55933. We also compared insulin signalling in C2C12 cells expressing shRNA against ATM and control cell lines (empty vector; cells expressing non-targeting shRNA). Results:, In L6 myotubes and mouse soleus muscle, KU55933 inhibited insulin-stimulated phosphorylation of the 160 kDa substrate of Akt (AS160) despite no effect on Akt. In contrast, KU55933 prevented insulin-stimulated Akt phosphorylation in C2C12 myotubes. Furthermore, C2C12 myotubes expressing shRNA against ATM displayed reduced insulin-stimulated Akt phosphorylation compared to controls. KU55933 also decreased insulin-stimulated Akt phosphorylation in differentiated RD cells. Conclusion:, These model-dependent differences in the role of ATM in insulin action demonstrate a role of ATM in insulin-stimulated phosphorylation of Akt (in C2C12 and RD cells) but also allow the elucidation of a novel, Akt-independent role of ATM (in L6 myotubes and mouse soleus, at the level of AS160) in insulin signalling. [source] A designed curved DNA segment that is a remarkable activator of eukaryotic transcriptionFEBS JOURNAL, Issue 24 2006Noriyuki Sumida To identify artificial DNA segments that can stably express transgenes in the genome of host cells, we built a series of curved DNA segments that mimic a left-handed superhelical structure. Curved DNA segments of 288 bp (T32) and 180 bp (T20) were able to activate transcription from the herpes simplex virus thymidine kinase (tk) promoter by approximately 150-fold and 70-fold, respectively, compared to a control in a transient transfection assay in COS-7 cells. The T20 segment was also able to activate transcription from the human adenovirus type 2 E1A promoter with an 18-fold increase in the same assay system, and also activated transcription from the tk promoter on episomes in COS-7 cells. We also established five HeLa cell lines with genomes containing T20 upstream of the transgene promoter and control cell lines with T20 deleted from the transgene locus. Interestingly, T20 was found to activate transcription in all the stable transformants, irrespective of the locus. This suggests that the T20 segment may allow stable expression of transgenes, which is of importance in many fields, and may also be useful for the construction of nonviral vectors for gene therapy. [source] Caspase cleavage of exon 9 deleted presenilin-1 is an early event in apoptosis induced by calcium ionophore A 23187 in SH-SY5Y neuroblastoma cellsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 1 2001Bogdan O. Popescu Abstract Presenilins (PSs) are mutated in a majority of familial Alzheimer disease (FAD) cases. Mutated PSs may cause FAD by a number of pro-apoptotic mechanisms, or by regulating ,-secretase activity, a protease involved in ,-amyloid precursor protein processing to the neurotoxic ,-amyloid peptide. Besides their normal endoproteolytic processing, PSs are substrates for caspases, being cleaved to alternative N-terminal and C-terminal fragments. So far little is known about the role of PSs cleavage in the apoptotic machinery. Here, we used SH-SY5Y neuroblastoma cells stably transfected with wild-type or exon 9 deleted presenilin 1 (PS1) in a time-course study after the exposure to the calcium ionophore A23187. During and after exposure to A 23187, intracellular calcium levels were higher in exon 9 deleted PS1 cells as compared with non-transfected and wild-type PS1 transfected cells. Cell death and the enrichment of apoptotic cells after A23187 exposure were increased by overexpression of exon 9 deleted PS1 as compared with the control cell lines. Wild-type PS1 cells were compared with exon 9 deleted PS1 cells and the temporal relationship between PS1 and other caspase substrates cleavages was analyzed. Exon 9 deleted PS1 cells exhibited a higher caspase-3 activation and a greater cleavage of PS1 and poly(ADP-ribose) polymerase (PARP) compared with wild-type PS1 cells. Exon 9 deleted PS1 cleavage occurred earlier than other caspase substrate cleavages (i.e., PARP and gelsolin), simultaneous with minimum detectable caspase-3 activation. Therefore, alternative cleavage of PS1 may play an important role for the regulation of the proteolytic cascade activated during apoptosis. J. Neurosci. Res. 66:122,134, 2001. © 2001 Wiley-Liss, Inc. [source] Mcl-1 overexpression leads to higher viabilities and increased production of humanized monoclonal antibody in Chinese hamster ovary cellsBIOTECHNOLOGY PROGRESS, Issue 4 2009Brian S. Majors Abstract Bioreactor stresses, including nutrient deprivation, shear stress, and byproduct accumulation can cause apoptosis, leading to lower recombinant protein yields and increased costs in downstream processing. Although cell engineering strategies utilizing the overexpression of antiapoptotic Bcl-2 family proteins such as Bcl-2 and Bcl-xL potently inhibit apoptosis, no studies have examined the use of the Bcl-2 family protein, Mcl-1, in commercial mammalian cell culture processes. Here, we overexpress both the wild type Mcl-1 protein and a Mcl-1 mutant protein that is not degraded by the proteasome in a serum-free Chinese hamster ovary (CHO) cell line producing a therapeutic antibody. The expression of Mcl-1 led to increased viabilities in fed-batch culture, with cell lines expressing the Mcl-1 mutant maintaining ,90% viability after 14 days when compared with 65% for control cells. In addition to enhanced culture viability, Mcl-1-expressing cell lines were isolated that consistently showed increases in antibody production of 20,35% when compared with control cultures. The quality of the antibody product was not affected in the Mcl-1-expressing cell lines, and Mcl-1-expressing cells exhibited 3-fold lower caspase-3 activation when compared with the control cell lines. Altogether, the expression of Mcl-1 represents a promising alternative cell engineering strategy to delay apoptosis and increase recombinant protein production in CHO cells. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Mutagenicity of non-homologous end joining DNA repair in a resistant subset of human chronic lymphocytic leukaemia B cellsBRITISH JOURNAL OF HAEMATOLOGY, Issue 5 2006Ludovic Deriano Summary Non-homologous end joining (NHEJ) is an important determinant of genomic stability in mammalian cells. This DNA repair pathway is upregulated in a subset of B-cell chronic lymphocytic leukaemia (B-CLL) cells resistant to DNA damage-induced apoptosis. Using an in vitro assay for double-strand breaks (DSB) end ligation, we studied the fidelity of DSB repair in B-CLL cells which were resistant or sensitive to in vitro DSB-induced apoptosis with concomitant patients' resistance or sensitivity to chemotherapy, respectively. The fidelity of DNA repair was determined by DNA sequencing of polymerase chain reaction products cloned in pGEM-T vector. Sequence analysis of DNA end junctions showed that the frequency of accurate ligation was higher in sensitive B-CLL cells and control cell lines, than in resistant cells where end joining was associated with extended deletions. Upregulated and error-prone NHEJ in resistant cells could be a quite possible mechanism underlying both genomic instability and poor clinical outcome. [source] | ||||||||||||||||||||||