Expression profiles of p53-, p16(INK4a)-, and telomere-regulating genes in replicative senescent primary human, mouse, and chicken fibroblast cells.

Replicative senescence is known to be an intrinsic mechanism in determining the finite life span of in vitro cultured cells. Since this process is recognized as an evolutionarily conserved mechanism from yeast to mammalian cells, we compared the senescence-associated genetic alterations in the p53, p16(INK4a), and telomere regulatory pathways using replicative senescent human, mouse, and chicken fibroblast cells. Normal human diploid fibroblast (HDF; WI38) and chicken embryonic fibroblast (CEF) cells were shown to have a more extended in vitro proliferative potential than their mouse embryonic fibroblast (MEF) counterpart. In contrast to the HDF and CEF cells, MEF cells were shown to express telomerase mRNA and maintain telomerase activity throughout their in vitro life span. Functional p53 activity was shown to increase in the replicative senescent HDF and CEF cells, but not in replicative senescent MEF cells. On the other hand, there was a gradual elevation of p16(INK4a) expression with increased cell passages which reached a maximum in replicative senescent MEF cells. Taken together, the present study demonstrates that the p53, p16(INK4a), and telomere regulatory functions may be differentially regulated during replicative senescence in human, mouse, and chicken fibroblast cells.     

构建基于杆状病毒的BV-T7杂合表达体系及利用其在哺乳动物和禽类细胞中表达eGFP基因

【目的】研究重组杆状病毒BV-T7杂合表达体系能否有效转导禽类细胞并在禽类细胞中表达外源基因(eGFP),从而构建能在禽类细胞中高效稳定表达外源基因的重组杆状病毒表达系统。【方法】本研究利用Bac-to-Bac杆状病毒表达系统,结合T7表达系统,通过对eGFP表达水平的调控来把握噬菌体T7 RNA聚合酶(T7 RNAP)的功能。利用两支重组杆状病毒,pFastBac-CMV-T7 RNAP重组杆状病毒为哺乳动物细胞启动子CMV调控的噬菌体T7 RNA聚合酶的cDNA;pFB-T7pro-IRES-GFP-T7ter重组杆状病毒为T7启动子控制的eGFP报告基因。将两支重组杆状病毒共同侵染哺乳动物OL(oligodendrocyte)细胞、鸡胚成纤维细胞和鸡胚骨骼肌细胞。【结果】两支重组杆状病毒利用T7启动子和T7 RNAP,在OL细胞、鸡胚成纤维细胞和鸡胚骨骼肌细胞中成功表达eGFP报告基因,而且未引起细胞病变,但在鸡胚原代细胞中eGFP的表达相对弱于在OL细胞中的表达。在OL细胞中重组杆状病毒对细胞的转导效率为59.5%,在鸡胚成纤维细胞和鸡胚骨骼肌细胞中转导效率分别为23.2%和33.1%。【结论】本研究构建的基于杆状病毒、T7RNA聚合酶、T7启动子(BV-T7)杂合表达体系能够在哺乳类细胞及禽类细胞中表达T7 RNAP,并利用T7RNAP继续高效而稳定地表达外源基因。这为难于体外操作的RNA病毒提供了有效的研究方法,并对新型基因工程疫苗的研制提供了一个高效而稳定的表达载体系统。     

Immunofluorescence on avian sarcoma virus-transformed cells: localization of the src gene product.

The localization of the avian sarcoma virus src gene product (termed p60src) was examined by indirect immunofluorescence in cells transformed by the Schmidt-Ruppin strain of Rous sarcoma virus, subgroup D (SR-RSV-D). Antiserum to p60src was obtained from rabbits bearing SR-RSV-D-induced tumors, and immunofluorescence was performed on chicken embryo fibroblasts (CEF) transformed with SR-RSV-D, as well as normal rat kidney (NRK) cells transformed by the same virus (termed SR-RK cells). Both acetone and formaldehyde fixation were used for the immunofluorescence tests. The specificity of the anti-tumor serum was first demonstrated in both cell systems by gel electrophoresis of immunoprecipitates prepared from 35S--methionine-labeled cells. Anti-tumor serum precipitated p60src from SR-RSV-D-transformed CEF but not from CEF infected with a transformation-defective mutant of SR-RSV-D. All viral structural proteins and precursors contained in these immunoprecipitates could be eliminated by competition with unlabeled virus. Similar experiments on SR-RK cells indicated that no viral proteins other than p60src were expressed in these cells, and this observation was supported by immunofluorescence tests using antiserum to whole virus. For immunofluorescence localization of p60src, reactions with viral structural proteins were blocked with unlabeled virus. This presaturation step, obligatory for p60src detection in the SR-RSV-D-transformed CEF, was unnecessary when antitumor serum was tested on SR-RK cells, since p60src was the only viral protein detectable in these cells. With acetone-fixed cells, p60src-specific immunofluorescence revealed a characteristic fluorescence pattern which was similar in both cell systems. The principal pattern was diffuse and situated in the cytoplasm. A clear nuclear fluorescence was never observed. Immunofluorescence on formaldehyde-fixed cells also indicated the cytoplasmic location of p60src and revealed a specific subcytoplasmic concentration of the fluorescence. With both fixation methods, an additional fluorescence pattern was seen between cells in contact, and was also found in both SR-RK cells and SR-RSV-D-transformed CEF. Immunofluorescence on viable cells suggested that p60src was not on the surface of these transformed cells. The fluorescence patterns were specific for avian sarcoma virus-transformed cells and were not found in uninfected cells, cells infected with a transformation-defective mutant of SR-RSV-D or cells transformed by an antigenically unrelated murine sarcoma virus. Furthermore, anti-tumor serum did not contain antibodies to proteins of the microtubules or intermediate filaments.     

Different localization of the product of the v-rel oncogene in chicken fibroblasts and spleen cells correlates with transformation by REV-T

Reticuloendotheliosis virus strain T (REV-T) is a highly oncogenic avian retrovirus that transforms early lymphoid cells in vivo and in vitro, but REV-T does not transform chicken embryo fibroblasts (CEF). Using antisera to p59v-rel, the v-rel oncogene product of REV-T, we show that p59v-rel is expressed at equal levels and is a phosphoprotein in REV-T infected spleen cells and CEF. Biochemical fractionation and immunofluorescence of REV-T infected nontransformed CEF show that p59v-rel is loosely associated with the nucleus. However, in REV-T transformed spleen cells p59v-rel is primarily a cytoplasmic protein. MSB-1 cells, a Marek's disease virus transformed T cell leukemic line, and E26 virus transformed myeloid cells show nuclear staining of p59v-rel when they are infected by REV-T. Our results indicate that there is a correlation between a cytoplasmic localization of p59v-rel and transformation by REV-T, and they suggest that p59v-rel cannot transform cells in which it assumes solely a nuclear location.     

Thymic dependence of cell-mediated immunity to avian sarcomas in chickens. Immunological characterization of a nonvirion antigen in virus-infected cells.

Chickens bearing tumors which have been induced by avian retroviruses express cellmediated immune responsiveness against antigens which are associated with these neoplasms. We have employed a peripheral lymphocyte stimulation test to characterize antigens which are found in the supernatant fluids of avian retrovirus-infected chicken embryo fibroblast (CEF) cells and in the plasma of birds which have been inoculated with avian myeloblastosis virus (AMV). The results indicated that the antigenic activity being measured was virus group specific, cell transformation independent, and nonvirion in nature. Paradoxically, expression of such antigen(s) was restricted to cells which were actively synthesizing progeny avian retrovirus particles, and was absent in mammalian nonproducer cells which had been transformed by avian sarcoma viruses. Ability to respond immunologically to such antigen(s) was present in animals which had been inoculated with either leukosis or sarcoma viruses. Thymectomy, but not bursectomy, was stimulatory to tumor growth and abolished sensitized lymphocyte immune responsiveness in this system.     

Contributions of differential p53 expression in the spontaneous immortalization of a chicken embryo fibroblast cell line

Background

The present study was carried out to determine whether the p53 pathway played a role in the spontaneous immortalization of the SC-2 chicken embryo fibroblast (CEF) cell line that has been in continuous culture for over three years.

Results

The SC-2 cell line emerged from an extended crisis period with a considerably slower growth rate than primary CEF cells. The phenotype of the SC-2 cells changed dramatically at about passage 80, appearing smaller than at earlier passages (e.g., passage 43) and possessing a small, compact morphology. This morphological change coincided with an increase in growth rate. Passage 43 SC-2 cells expressed undetectable levels of p53 mRNA, but by passage 95, the levels were elevated compared to primary passage 6 CEF cells and similar to levels in senescent CEF cells. However, the high level of p53 mRNA detected in passage 95 SC-2 cells did not correlate to functional protein activity. The expression levels of the p53-regulated p21^WAF1 gene were significantly decreased in all SC-2 passages that were analyzed. Examination of the Rb pathway revealed that E2F-1 and p15^INK4b expression fluctuated with increasing passages, with levels higher in passage 95 SC-2 cells compared to primary passage 6 CEF cells.

Conclusion

The present study suggests that altered expression of genes involved in the p53 and Rb pathways, specifically, p53 and p21^WAF1, may have contributed to the immortalization of the SC-2 CEF cell line.     

Characterization of the putative avian CD2 homologue.

We describe two mouse mAb recognizing the putative chicken homologue of mammalian CD2 Ag and provide evidence for both structural and functional conservation between the avian and mammalian CD2 molecules. The antibodies were T cell-specific and immunoprecipitated a single diffuse band of Mr 40,000 from lysates of surface-labeled chicken thymocytes and peripheral T cells. Removal of N-linked carbohydrate with endo-beta-N-acetylglucosaminidase F revealed the core protein size of Mr 25,000. A rabbit antiserum raised against a synthetic peptide (CD2-300), composed of 18 amino acid residues of the conserved cytoplasmic domain of human, mouse, and rat CD2, precipitated an Ag similar to chicken CD2. Sequential precipitation with CD2-300 antiserum indicated the conservation of an avian and mammalian CD2 epitope. CD2 expression on thymocytes starts at day 11 of embryonic development, and, during subsequent development, thymic gamma delta cells are all CD2+, whereas most peripheral gamma delta-T cells lack CD2. Functional conservation between the chicken and mammalian CD2 molecules was demonstrated by the induction of DNA synthesis in chicken thymocytes and peripheral T cells with the combination of anti-CD2 mAb and PMA.     

表达鸡白细胞介素2重组鸡痘病毒的构建及其体外表达产物生物活性的检测

从ConA刺激的鸡脾细胞中扩增出鸡白细胞介素2（ChIL-2）基因编码区。将该编码区Cdna序列和调控其转录的鸡痘病毒早晚期启动子（PE/L）的基因片段定向克隆到鸡痘病毒转移载体p1175中,获得重组转移载体P1175il2,然后转染已感染鸡痘病毒282E4疫苗株（wt-FPV）的鸡胚成纤维细胞（CEF）,质粒P1175il2与wt-FPV基因组DNA发生同源重组,产生了表达ChIL-2的重组鸡痘病毒Rfpv-IL2。通过在含X-gal的营养琼脂上连续挑选蓝色病毒蚀斑,获得并纯化重组鸡痘病毒Rfpv-IL2。应用XTT/PMS方法检测的Rfpv-IL2 （M.O.I 2.0）感染CEF 72小时后细胞上清中表达的重组ChIL-2生物活性,效价为3.6×105u/Ml,表明Rfpv-IL2能有效地表达ChIL-2。下一步将利用Rfpv-IL2在体内表达ChIL-2,研究ChIL-2的免疫增强作用及其作用机理。     

Expression of the Cytoskeletal-Associated Protein Filamin in Adult Rat Organs

Filamin is a well-characterized actin-associated protein first isolated from chicken smooth muscle. Subsequently, this polypeptide and its nonmuscle homolog actin-binding protein have been shown to be expressed in avian muscle tissue, mammalian smooth muscle, mammalian macrophages and other blood cell types, as well as several cultured cell lines. In this report, the occurrence of this polypeptide in adult mammalian organs has been investigated. Immunoblot analysis using three anti-filamin monoclonal antibodies showed that this protein was largely detected in adult rat organs that possess a substantial smooth muscle component. Furthermore, the limited expression of filamin in smooth muscle tissue was corroborated by immunohistochemical analysis. In contrast to avian systems, filamin was never found in detectable quantities in either mammalian cardiac or skeletal muscle. Quantitative immunoblot analysis demonstrated that filamin amounts roughly correlated with the abundance of the smooth muscle component of a given organ, comprising as much as 16.5% of the total SDS-extractable protein in bovine aorta. Work in avian systems and cells in culture has suggested that filamin is a rather ubiquitous cytoskeletal element. By contrast, this work demonstrates that filamin is highly restricted in its expression in mammalian organ systems, in situ.     

Pleiotropic action of AP-1 in v-Src-transformed cells

The activation of AP-1 is a hallmark of cell transformation by tyrosine kinases. In this study, we characterize the role of AP-1 proteins in the transformation of chicken embryo fibroblasts (CEF) by v-Src. In normal CEF, the expression of a dominant negative mutant of c-Jun (TAM67) induced senescence. In contrast, three distinct phenotypes were observed when TAM67 was expressed in v-Src-transformed CEF. While senescent cells were also present, the inhibition of AP-1 caused apoptosis in a fraction of the v-Src-transformed cells. In addition, cells containing lipid-rich vesicles accumulated, suggesting that a subpopulation of the v-Src-transformed cells underwent differentiation in response to the inhibition of AP-1. JunD and Fra-2 were the main components of this factor, while c-Jun accounted for a minor fraction of AP-1 in v-Src-transformed CEF. The downregulation of c-Jun expression by short hairpin RNA (shRNA) induced senescence in normal and v-Src-transformed cells. In contrast, a high incidence of apoptosis was caused by the downregulation of JunD, suggesting that it is required for the survival of v-Src-transformed CEF. Levels of the p53 tumor suppressor were elevated under conditions of JunD inhibition. Repression of p53 by shRNA enhanced the survival and anchorage-independent proliferation of v-Src-transformed CEF with JunD/AP-1 inhibition. The inhibition of Fra-2 had no visible phenotype in normal CEF but caused the appearance of lipid-rich vesicles in v-Src-transformed CEF. Therefore, AP-1 facilitated transformation by acting as a survival factor, by inhibiting premature entry into senescence, and by blocking the differentiation of v-Src-transformed CEF.     

Transformation-associated cytokine 9E3/CEF4 is chemotactic for chicken peripheral blood mononuclear cells.

9E3/CEF4, which is released from transformed chicken embryo fibroblasts (CEF), is a member of the platelet factor 4 family of inflammatory proteins and may be the avian homolog of interleukin-8. Since the function of 9E3/CEF4 is unknown, we examined the effect of the protein on mitogenicity and chemotaxis, as well as its expression, in fibroblasts and peripheral blood cells. 9E3/CEF4 mRNA was expressed in chicken peripheral blood monocytes, and its expression was stimulated by incubation of the monocytes with lipopolysaccharide or phorbol myristic acetate. Boyden double-membrane analysis of chemotaxis showed that 9E3/CEF4 was chemotactic for chicken peripheral blood mononuclear cells, as well as for heterophils. Untransformed CEF and CEF transformed with Rous sarcoma virus also migrated to 9E3/CEF4 protein, as measured by Boyden single-membrane analysis. 9E3/CEF4 was slightly mitogenic for CEF, causing a doubling of [3H]thymidine uptake when added to serum-starved CEF.9E3/CEF4 was found associated not only with the cell and in the culture medium of Rous sarcoma virus-transformed CEF but also with the extracellular matrix. The in vivo role of 9E3/CEF4 may be involved with chemotaxis and metastasis, rather than with direct stimulation of mitogenicity.     

78-kilodalton glucose-regulated protein is induced in Rous sarcoma virus-transformed cells independently of glucose deprivation.

To identify mRNAs with altered expression in Rous sarcoma virus (RSV)-transformed cells, we screened a chicken embryo fibroblast (CEF) cDNA library by differential hybridization. One clone, designated R1H, showed markedly elevated mRNA expression in RSV-transformed cells. Nucleotide sequence analysis indicated that R1H mRNA encodes 78-kilodalton glucose-regulated protein (GRP78). Chicken GRP78 was found to be very highly conserved in comparison with rat GRP78 (96% identity between chicken and rat amino acid sequences). In contrast to previous observations, we found that GRP78 was induced in RSV-transformed cells in the absence of glucose deprivation. When cells were grown in glucose-supplemented medium, the level of GRP78 mRNA was approximately fivefold higher in RSV-transformed CEF than in transformation-defective virus-infected or uninfected CEF. Similar changes in GRP78 protein content were also found. Using a temperature-sensitive mutant of RSV and supplemental glucose, we found a gradual increase in the level of GRP78 mRNA beginning at 4 h after shiftdown to permissive temperature. Uridine supplementation did not block the induction seen in CEF infected with a temperature-sensitive mutant. These results indicate that GRP78 is induced by p60v-src in the absence of glucose deprivation.