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NIH3T3 Fibroblasts (nih3t3 + fibroblast)
Selected AbstractsThe transcriptional programme of contact-inhibitionJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2010Monika Küppers Abstract Proliferation of non-transformed cells is regulated by cell,cell contacts, which are referred to as contact-inhibition. Vice versa, transformed cells are characterised by a loss of contact-inhibition. Despite its generally accepted importance for cell-cycle control, little is known about the intracellular signalling pathways involved in contact-inhibition. Unravelling the molecular mechanisms of contact-inhibition and its loss during tumourigenesis will be an important step towards the identification of novel target genes in tumour diagnosis and treatment. To better understand the underlying molecular mechanisms we identified the transcriptional programme of contact-inhibition in NIH3T3 fibroblast using high-density microarrays. Setting the cut off: ,1.5-fold, P,,,0.05, 853 genes and 73 cDNA sequences were differentially expressed in confluent compared to exponentially growing cultures. Importing these data into GenMAPP software revealed a comprehensive list of cell-cycle regulatory genes mediating G0/G1 arrest, which was confirmed by RT-PCR and Western blot. In a narrow analysis (cut off: ,2-fold, P,,,0.002), we found 110 transcripts to be differentially expressed representing 107 genes and 3 cDNA sequences involved, for example, in proliferation, signal transduction, transcriptional regulation, cell adhesion and communication. Interestingly, the majority of genes was upregulated indicating that contact-inhibition is not a passive state, but actively induced. Furthermore, we confirmed differential expression of eight genes by semi-quantitative RT-PCR and identified the potential tumour suppressor transforming growth factor-, (TGF-,)-1-induced clone 22 (TSC-22; tgfb1i4) as a novel protein to be induced in contact-inhibited cells. J. Cell. Biochem. 110: 1234,1243, 2010. Published 2010 Wiley-Liss, Inc. [source] Use of Green Fluorescent Protein-Conjugated ,-Actin as a Novel Molecular Marker for in Vitro Tumor Cell Chemotaxis AssayBIOTECHNOLOGY PROGRESS, Issue 6 2000Louis Hodgson To study the dynamics of actin cytoskeleton rearrangement in living cells, an eukaryotic expression vector expressing a ,-actin-GFP fusion protein was generated. The expression construct when transfected into NIH3T3 fibroblast, A2058 human melanoma and 293T human embryonic kidney carcinoma cell lines expressed ,-actin-GFP fusion protein, which colocalized with endogenous cellular actin as determined by histoimmunofluorescence staining. The ,-actin-GFP was also observed to be reorganized in response to treatments with the chemoattractant type IV collagen. Cells extended pseudopodial protrusions and altered the morphology of their cortical structure in response to type IV collagen stimulation. More importantly, ,-actin-GFP accumulated in areas undergoing these dynamic cytoskeleton changes, indicating that ,-actin-GFP could participate in actin polymerization. Although ectopic expression of ,-actin-GFP lead to minor side effects on cell proliferation, these studies suggest that this strategy provides an alternative to the invasive techniques currently used to study actin dynamics and permits real-time visualization of actin rearrangements in response to environmental cues. [source] Secreted CREG inhibits cell proliferation mediated by mannose 6-phosphate/insulin-like growth factor II receptor in NIH3T3 fibroblastsGENES TO CELLS, Issue 9 2008Ya-Ling Han Cellular repressor of E1A-stimulated genes (CREG) is a recently described glycoprotein that plays a critical role in keeping cells or tissues in mature, homeostatic states. To understand the relationship between CREG and its membrane receptor, mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R), we first generated stable NIH3T3 fibroblasts by transfection of pDS_shCREGs vectors, which produced an approximately 80% decrease in CREG levels both in the lysate and in the media. We used fluorescence activated cell sorting and a bromide deoxyuridine incorporation assay to identify whether CREG knockdown promoted the cell proliferation associated with the increase of IGF-II in NIH3T3 fibroblasts. Proliferation was markedly inhibited in a concentration-dependent manner by re-addition of recombinant CREG protein into the media, and this was mediated by the membrane receptor M6P/IGF2R. We subsequently confirmed the direct interaction of CREG and M6P/IGF2R by both immunoprecipitation-Western blotting and immunofluorescence staining. We found that expression of CREG correlated with localization of the receptor in NIH3T3 fibroblasts but did not affect its expression. Our findings indicated that CREG might act as a functional regulator of M6P/IGF2R to facilitate binding and trafficking of IGF-II endocytosis, leading to growth inhibition. [source] | ||||||||||