Analysis of a transformed cell line using antisense c-fos RNA

Simian sarcoma virus (SSV)-infected NIH-3T3 cells (SSV-NIH-3T3), express a homologue of platelet-derived growth factor, (PDGF) a powerful inducer of the c-fos gene. We have used these cells to test the hypothesis that autocrine stimulation by PDGF-like molecules leads to c-fos expression which is functional in the transformed phenotype. We have transfected SSV-NIH-3T3 cells with a c-fos antisense-RNA expression vector, pSVsof, or control plasmids. pSVsof-transfected cells exhibit markedly decreased c-fos mRNA and protein levels, restored density-dependent growth arrest and reduced (three of five clones) tumorigenicity compared to control lines. The results confirm that c-fos cooperates in the transformed phenotype of SSV-NIH-3T3 cells.     

Deficient polymerization in vitro of a point-mutated beta-actin expressed in a transformed human fibroblast cell line

HUT-14 cells, tumorigenic human fibroblasts, express a mutant beta-actin which has a single amino acid substitution at position 244 (glycine to aspartic acid), in addition to normal beta- and gamma-actin. In order to characterize the biochemical function of the mutant beta-actin, actins were extracted and purified from HUT-14 cells. The partially purified actin fraction contained beta-, gamma-, and mutant beta-actins in the ratio of 1:1:1, the same ratio as in the cells. When the actin of this fraction was purified through a polymerization step, mutant beta-actin was always less incorporated into actin filaments than beta- and gamma-actin. When the polymerization ability of purified HUT-14 actins was examined by sedimentation technique, it was lower than those of muscle and of cytoplasmic actins from another human cell line (HUT-11) which expresses only normal beta- and gamma-actin, in the ratio of 2:1. The deficient polymerization of mutant beta-actin was also observed by examining the ratio of beta-, gamma-, and mutant beta-actins incorporated into actin filaments. The ratio of mutant beta-actin in polymerized actins under all conditions examined was always less than that before polymerization. These results indicate that the single amino acid substitution at position 244 caused the reduction of incorporation of the mutant beta-actin into actin filaments in vitro.     

A transformed murine Leydig cell line expresses the ETA receptor subtype

We recently demonstrated that transformed murine Leydig cells (MA-10) responded to endothelin-1 (ET-1) via increased steroidogenesis. This study addresses the endothelin receptor subtype present on this cell line and whether or not the cells produce ET-1. The expression of the preproendothelin-1 (PPET-1) gene was investigated by Northern blot analysis, and PPET-1 mRNA was found to be <0.2% of that present in pulmonary endothelial cells. The medium from MA-10 cells, maintained under serum-free conditions, was analyzed by radio-immunoassay to determine immunoreactive-ET-1 production and ET-1 levels were found to be below the sensitivity of the assay (<10 pg/ml). The data from competitive binding experiments with [¹²⁵I]ET-1 and unlabeled ET-1, ET-3 and receptor subtype selective ligands yielded a single class of high affinity binding sites with ET_A receptor subtype characteristics. The results of this study demonstrate that MA-10 cells possess the ET_A receptor subtype but do not produce significant quantities of ET-1 under basal conditions.     

Natural killer cell regulation of murine embryonic pulmonary fibroblast survival in vivo

We examined the role of the natural killer (NK) cell in controlling the survival of embryonic pulmonary fibroblasts in vivo. In vitro, both primary embryonic fibroblasts and an embryonic fibroblast line (10T1/2) were lysed by syngeneic C3H/HeN splenocytes threefold more efficiently than primary adult fibroblasts. The membrane phenotype of the effector cells was typical of NK cells. It was asialo GM1+, Lyt2.1-, Lyt 1.1-, Thy 1.2-. The cytotoxicity of the effector cell could be enhanced by IFN-alpha/beta but was deficient in the C3H/HeJ bg/bg mutant. Iododeoxyuridine (131I-dUrd)-labeled embryonic fibroblasts were injected intravenously into syngeneic mice with either enhanced or deficient NK function and their survival in the lung was quantitated. Enhanced fibroblast survival was detected in the NK deficient C3H/HeJ beige (bg/bg) mutant strain compared to its normal littermate C3H/HeJ (bg/+). A second method of NK depletion by pretreatment with rabbit anti-asialo GM1 antiserum also produced a striking increase in fibroblast survival. Poly(I:C) significantly enhanced the elimination of pulmonary fibroblasts from the lung between 4 and 24 hr after injection. Poly(I:C) did not enhance clearance of pulmonary fibroblasts in the C3H/HeJ (bg/bg) mutant, but did so in the normal littermate C3H/HeJ (bg/+). In conclusion, we have shown that the survival of embryonic pulmonary fibroblasts was inversely correlated with in vivo NK activity suggesting a possible role for this cytotoxic cell in the control of fibroblast growth in vivo.     

转化的大鼠胚胎成纤维细胞系差异表达基因的筛选研究

来源于转化的大鼠胚胎成纤维细胞系的两株细胞,A1－5细胞与B4细胞相比表现出非常强的抗辐射性并伴随不同寻常强的G2延迟效应;用PCR选择性抑制消减杂交方法对这两株细胞进行差减,希望找到对A1－5细胞表现出的不同寻常的表型起关键作用的某一个或某一些基因。结果得到了160个差减转化子,逐个进行序列测定,并进行Dot blot杂交,共得到35个差异表达基因片段（EST）。通过对美国国家生物技术信息中心（NCBI）的非冗余序列库（NT）、鼠EST库及人EST库的BLAST进行同源检索,发现其中21个代表了尚未登录的新基因,另外14个分别与已知基因高度同源。     

Loss of insulin binding and insulin receptor mRNA in a transformed human fetal fibroblast cell line

Insulin binding and insulin receptor gene expression have been assessed in cultured fetal (WI38) and SV40 transformed fetal (WI38/VA13) human fibroblasts to determine whether transformation influences the expression of insulin receptors. The transformed cell line had virtually no insulin binding and extremely low levels of insulin receptor mRNA. No apparent gene deletion or rearrangement was detected and therefore the marked decrease in insulin receptor gene expression seen in WI38/VA13 cells is an important example of negative regulation of insulin receptor gene expression. This cell line could serve as a model for studies of the mechanism for negative regulation of insulin receptor gene expression. Overexpression of the insulin receptor gene in these cells may reveal insights into the role of the insulin receptor in tumor biology.     

"Chain" karyotypic evolution of embryonic stem cell line R1 in vitro

Cytogenetic anomaly frequencies were analysed in three sublines of ES R1 line in its five clonal sublines, obtained from two cell colonies after transformation of ES R1 cells by plasmid with gene lif. Cell transformation did not increase cytogenic anomalies, however, the initial sublines of ES R1 line, as well as its transformed clonal descendants bore a redundant quantity of the chromosome 8 material within the structure of various Robertsonian translocations even in cells with diploid chromosome quantity (2n = 40). In the initial sublines ES R1 and its clonal descendants a common Rb (8; 15) was revealed. It was supposed that selection for the increase in ES cell sensitivity to cytokines (in particular, LIF) under cultural conditions was accompanied by an increase in chromosomal copies, carrying genes of mapk andjak/stat, through which downstream effectors of cytokine signals for preservation of cell pluripotention and propagation are realized. Genes of chromatid separation and chromosome segregation control (for example, separase gene Esp1 in chromosome 15) may be passively involved in this process, thus promoting acceleration of karyotype evolution in ES cells.     

转化的大鼠胚胎成纤维细胞系与p53val135功能关系的研究

A1-5是一株典型的温度敏感的p53^val135细胞系,许多实验室用它来研究p53的功能,我们首次报道了A1-5细胞表现出非常强的抗辐射性并伴随不同寻常强的G2延迟效应,B4是另一株温度敏感的p53^val135细胞系,A1-5与B4细胞都 是来源于同样的原代转化的大鼠胚胎成纤维细胞系,只是来自不同的转染实验,本阐明了A1-5细胞非常强的抗辐射性及不同寻常强的G2停滞效应与p53val135的功能无关,A1-5细胞系具有的特殊表型是研究放射敏感性新性质的独特的模型。     

Effect of c-fos overexpression on the murine macrophage cell line P388DI

In order to study the role of Fos on the regulation of proliferation in the monocyte-macrophage lineage we realized a stable transfection of the murine P388D1 cell line by the murine c-fos gene under the control of the human metallothionein IIA promoter. Several clones have been selected by geneticin: they show a variable number of integrated transgene (two to ten copies). Their expression has been analyzed in the presence or absence of cadmium chloride as inducer (5 × 10⁻⁶ M). In one clone especially, the c-fos mRNA and Fos protein levels were respectively 6and 10-fold increased. The study of cell growth by tritiated thymidine incorporation indicates a negative effect of the overexpressed Fos protein in the absence of any other stimulus.     

Two-dimensional gel electrophoresis database of murine R1 embryonic stem cells

Embryonic stem (ES) cell lines represent a population of undifferentiated pluripotent cells capable of multilineage differentiation in vitro. Although very useful for studying developmental processes, human ES cell lines have also been suggested as a potential and unlimited source for cellular transplantation and the treatment of human disease. The proteomic basis of embryonic stemness (pluripotentiality and multilineage differentiation) and the transitions that lead to specific cell lineages however, remain to be defined. As an important first step in defining these processes, we have performed a proteomic analysis of undifferentiated mouse R1 ES cell lines using pH 3-10, 4-7 and 6-11 two-dimensional electrophoresis gels, matrix-assisted laser desorption/ionization and tandem mass spectrometry. Of the 700 gel spots analyzed, 241 distinct protein species were identified corresponding to 218 unique proteins, with a significant proportion functionally related to protein expression.     

Generation and characterization of a conditionally immortalized lung clara cell line from the H-2Kb-tsA58 transgenic mouse

The Clara cell is believed to be the progenitor of the peripheral airway epithelium, and it produces the surfactant proteins SP-A and SP-B, in addition to the 10-kDa Clara cell secretory protein (CCSP or CC10). To date, attempts to develop Clara cell lines have been unsuccessful. Most such attempts have involved the in vitro insertion of a transforming viral oncogene. We have reported previously the characterization of a differentiated conditionally immortalized murine lung Type II epithelial cell line, T7, from the H-2Kb-tsA58 transgenic mouse. We have also used this mouse model to derive Clara cell lines. In this model, the need for in vitro gene insertion is circumvented by the creation of a transgene, in which the large tumor antigen of a temperature-sensitive strain (tsA58) of the simian virus 40 (SV40) is fused with the major histocompatibility complex promoter H-2Kb. The promoter is active in a wide range of tissues and is induced by interferons (IFN). From the lungs of animals harboring the hybrid construct, we isolated and characterized Clara cells. The cells contain dense secretory granules and mitochondria typical of Clara cells, and express SP-A, SP-B, SP-D, and the Clara cell secretory protein, CC10. Withdrawal of the IFN and elevation of the incubation temperature permit normal cell differentiation similar to that of Clara cells in vivo. This cell line should be very useful for the investigation of normal Clara cell function and gene expression.     

MiR-223 suppresses cell proliferation by targeting IGF-1R

To study the roles of microRNA-223 (miR-223) in regulation of cell growth, we established a miR-223 over-expression model in HeLa cells infected with miR-223 by Lentivirus pLL3.7 system. We observed in this model that miR-223 significantly suppressed the proliferation, growth rate, colony formation of HeLa cells in vitro, and in vivo tumorigenicity or tumor formation in nude mice. To investigate the mechanisms involved, we scanned and examined the potential and putative target molecules of miR-223 by informatics, quantitative PCR and Western blot, and found that insulin-like growth factor-1 receptor (IGF-1R) was the functional target of miR-223 inhibition of cell proliferation. Targeting IGF-1R by miR-223 was not only seen in HeLa cells, but also in leukemia and hepatoma cells. The downstream pathway, Akt/mTOR/p70S6K, to which the signal was mediated by IGF-1R, was inhibited as well. The relative luciferase activity of the reporter containing wild-type 3'UTR(3'untranslated region) of IGF-1R was significantly suppressed, but the mutant not. Silence of IGF-1R expression by vector-based short hairpin RNA resulted in the similar inhibition with miR-223. Contrarily, rescued IGF-1R expression in the cells that over-expressed miR-223, reversed the inhibition caused by miR-223 via introducing IGF-1R cDNA that didn't contain the 3'UTR. Meanwhile, we also noted that miR-223 targeted Rasa1, but the downstream molecules mediated by Rasa1 was neither targeted nor regulated. Therefore we believed that IGF-1R was the functional target for miR-223 suppression of cell proliferation and its downstream PI3K/Akt/mTOR/p70S6K pathway suppressed by miR-223 was by targeting IGF-1R.     

Expression and possible functions of the cholinergic system in a murine embryonic stem cell line

The expression of a cholinergic system during embryonic development is a widespread phenomenon. However, no precise function could be assigned to it during early pre-neural stages and there are only few studies that document when it precisely starts to be expressed. Here, we examined the expression of cholinergic components in a murine embryonic stem cell line by RT-PCR, histochemistry, and enzyme activity measurements; the acetylcholine (ACh) content was measured by HPLC. We have demonstrated that embryonic stem cells express ACh, acetylcholine receptors, choline acetyltransferase (ChAT), acetyl- and butyryl-cholinesterase (AChE and BChE). Butyryl-cholinesterase (BChE) expression was higher than AChE. The cholinesterase activity was down-regulated by adding specific inhibitors to culture medium. Inhibition of BChE led to a reduction of proliferation. This is the first demonstration that mouse embryonic stem cells express the full molecular equipment of a cholinergic system. Locally produced ACh might function as an intercellular signal, modulating the proliferation of stem cells.     

Hexose sugar transport activity in a mouse fibroblast cell line temperature sensitive for expression of the transformed phenotype

A temperature-sensitive mouse fibroblast cell line was used to examine the relationship between hexose sugar uptake rates and the control of cell growth. The cell line used (ts-H6-15) is a derivative of SV-3T3 cells, exhibiting a transformed phenotype at 32°C and a normal phenotype at 39°C. For cells actively growing at either temperature, a marked decrease in the rate of 3-0-methyl-D-glucose (3-0-MeG) transport is observed as cell population density increases. At all cell population densities tested, 3-0-MeG transport rates (at a common assay temperature) were greater in H6-15 cells grown at 32°C than at 39°C, with the enhancement being maximal at the lowest cell densities. The effect of low serum-arrest on H6-15 cells revealed that cells growing at 39°C arrest in G1, while cells at 32°C stop more randomly throughout their cycle. Under conditions of low serum-arrest the rate of 3-0-MeG transport remained as high as in actively growing cells at both 32°C and 39°C. However, 2-deoxyglucose uptake rates were growth state-dependent at 39°C, indicating perhaps metabolic as well as membrane-level control of sugar accumulation. These results further demonstrate that rates of hexose sugar transport by themselves are not always absolutely correlated with rates of cell proliferation and, thus, may not be reliable predictors of cell growth potential.     

Structure of bovine papillomavirus type 1 DNA in a transformed mouse cell line

Linearized bovine papillomavirus type 1 (BPV-1) DNA was introduced into mouse C127 cells, where it recircularized and replicated as an intact monomeric, extrachromosomal circular form in the resulting transformants. These cells contained a mixture of complex high molecular weight forms that were converted to a linear form of approximately BPV-1 size upon digestion with an enzyme that cuts once within the BPV-1 genome. Further analysis of one of these cell lines revealed that these high molecular weight forms consisted of two components. One was detected on agarose gels as a diffuse smear of slow-migrating material representing linear forms that were tightly associated with host chromosomes, probably by integration. The second component was composed of discrete-sized oligomeric open and supercoiled extrachromosomal circular forms of up to approximately 48 X 10(3) base-pairs (6 tandemly linked BPV-1 genomes) in size. No catenated (interlocked) forms could be detected.     

Fibronectin receptor overexpression and loss of transformed phenotype in a stable variant of the K562 cell line.

A variant of the K562 erythroleukemia cell line, FA-K562, was selected by cycles of adhesion to solid-phase plasma fibronectin (FN). FA-K562 expresses fourfold more cell-surface alpha 5 beta 1 fibronectin receptor (FNR) than parental K562. In addition to expected differences in adhesion to FN, other differences between FA-K562 and K562 implicate this FNR in the regulation of cell growth and morphology. FA-K562 proliferates slowly in liquid culture, its cloning efficiency in soft agar is only approximately 10% compared with approximately 85% for parental K562, and it is nontumorigenic in nude mice. The reduced soft agar growth potential of FA-K562 involves FNR function, because either glycine-arginine-glycine-aspartate-serine (GRGDS) or monoclonal anti-alpha 5 antibody in the agar medium increased cloning efficiency of FA-K562 about fivefold. Morphologically, FN-adherent FA-K562 become fibroblastoid in appearance, assemble filamentous actin, and differ from K562 in vimentin staining intensity and pattern. Soluble GRGDS peptide inhibits both FA-K562 adhesion to FN and the associated cytoskeletal changes. These findings link the alpha 5 beta 1 FNR to both the transformed phenotype and morphology of FA-K562.