V-fos基因对NIH3T3细胞转化的研究

本文报道了用含v-fos基因的pFBJ-2质粒转染NIH 3 T 3细胞,获得了转化细胞株。对细胞株的研究结果表明:(1)Southern杂交检测到细胞基因组中有v-fos基因的整合;(2)点杂交测得有v-fos mRNA的表达;(3)出现一系列转化表型,包括细胞形态的改变,异常的增长速率,在软琼脂上的贴壁不依赖性生长,对低血清浓度培养液的适应性以及细胞膜表而超微结构的变化等,提示v-fos基因能使NIH 3 T 3细胞发生转化,并在体外转化过程中起决定性作用。     

Viral and cellular fos proteins: a comparative analysis

The FBJ murine osteosarcoma virus (FBJ-MuSV) induces osteosarcomas in mice and transforms fibroblasts in vitro. It contains an oncogene termed v-fos derived from a normal cellular gene by recombination with an associated helper virus. The product of the v-fos gene is a 55,000 dalton protein, p55v-fos. This protein was found in the nuclei of cells containing amplified levels of the v-fos gene, and also in the nuclei of virus-transformed cells. The c-fos protein was localized in the nuclei of normal mouse amnion cells and in the nuclei of cells transformed by a recombinant plasmid that expresses the c-fos gene product. However, p55c-fos undergoes more extensive post-translational modification in the nucleus than p55v-fos. Immunofluorescence data indicate that the level of p55c-fos in normal mouse amnion cells is similar to that found in fibroblasts transformed by the v-fos or c-fos proteins.     

Augmented excretion of procathepsin L of a fos-transferred highly metastatic rat cell line

We previously reported that v-fos transfer to a src-transformed rat 3Y1 cell line (SR-3Y1-2) enhanced lung metastasis accompanied with an increase in invasiveness. When analyzing factors relating to the high invasiveness, with special reference to typical lysosomal proteases (cathepsins), involving degradation of stroma, we found that the excretion of procathepsin L was significantly larger in the fos-transferred highly metastatic cell line (fos-SR-3Y1-202) than that in the recipient cell lines. The cathepsin D-induced enzyme activity of the excreted procathepsin L in fos-SR-3Y1-202 was about 4 and 13 fold that of SR-3Y1-2 and 3Y1, respectively. The increase in the excretion of procathepsin L from fos-SR-3Y1-202 may play a role in the high invasiveness induced by transfer with the v-fos oncogene.     

Generation of a murine monoclonal antibody that detects the fos oncogene product

A hybridoma producing a monoclonal antibody (MoAB) recognizing both the cellular and viral forms of fos has been generated by somatic cell hybridization techniques from spleen cells of mice immunized with a synthetic peptide corresponding to amino acids 128-152, a consensus region, of both the v-fos and c-fos oncogene products. Three proteins with molecular weights of 55,000, 44,000, and 42,000 were detected by immunoblotting. While MoAB 2G9C3 failed to immunoprecipitate fos from Finkel-Biskis-Jenkins murine osteosarcoma-virus-infected fibroblasts, both the 55,000 v-fos protein and the 39,000 cellular protein were coprecipitated using polyvalent rabbit antibodies to the same peptide. Whereas no cell surface membrane expression of fos was detected, after membrane permeabilization by a brief exposure to lysolecithin it was possible to specifically detect internal fos by immunofluorescence flow cytometry. Immunohistochemical staining of FBJ virus-infected cells revealed intense, nuclear staining.     

Growth factor requirements of oncogene-transformed NIH 3T3 and BALB/c 3T3 cells cultured in defined media.

We report conditions for the efficient growth of NIH 3T3 and BALB/c 3T3 cells cultured in a defined medium supplemented with either platelet-derived growth factor (PDGF) or pituitary-derived fibroblast growth factor (FGF). The oncogenes v-mos, v-src, v-sis, and c-H-ras Val 12 can induce morphological transformation of these cells and can release them from the mitogen requirement for growth, while the oncogene v-fos cannot abrogate the PDGF-FGF requirement. The radically different behavior of normal and transformed NIH 3T3 cells in PDGF-FGF-free defined medium can form the basis of a sensitive new fibroblast transformation assay.     

A sensitive enzyme-linked immunosorbence assay for the c-fos and v-fos oncoproteins

The c-fos nuclear oncoprotein is rapidly induced when the growth of normal cells is initiated by mitogens, and it is also synthesized in several cell systems in response to stimuli that do not cause cell proliferation. When expressed inappropriately, c-fos, and its retroviral counterpart v-fos, can transform susceptible cells in vivo and in vitro. We have developed a simple and sensitive ELISA for the c-fos and v-fos proteins. Fos proteins are captured from cell lysates by an antibody specific for an amino-terminal peptide substantially conserved between v-fos and c-fos; the captured proteins are recognised by a second antibody against a different peptide sequence also conserved in the two proteins. The second antibody has been conjugated to alkaline phosphatase to provide an enzyme label; bound alkaline phosphatase is measured with a sensitive cycling enzyme system that generates a coloured end-product. We show that the fos ELISA is immunologically specific and use it to monitor increased c-fos expression in serum-stimulated HeLa cells and human fibroblasts, and in mitogen-stimulated murine thymocytes.     

Transformation of T lymphocytes by the v-fos oncogene

Activation of T lymphocytes through the T cell antigen receptor has been shown to stimulate a rapid and transient accumulation of c-fos mRNA and protein. Transfection of a normal murine T lymphocyte clone with the FBJ-v-fos oncogene resulted in generation of a cell line that was morphologically transformed, had lost the requirement for IL-2 for proliferation, and was tumorigenic in adult syngeneic mice; however, the transformed cells retained the ability to proliferate in response to IL-2. The transformed cells did not show constitutive expression of IL-2 or c-fos mRNA, although the promoter regions of both IL-2 and c-fos genes contain AP-1 sites that are expected to be targets for binding of Fos/Jun complexes. In contrast, the transformed T cells showed increased constitutive expression of IL-2R alpha and c-myc mRNA; these genes may represent cellular targets for transformation by v-fos and physiologic activation by c-fos. We discuss the possibility that these transformed cells behave as cells partially activated through the TCR, and that transformation occurs through a mechanism independent of IL-2.     

Expression of transfected recombinant oncogenes increases radiation resistance of clonal hematopoietic and fibroblast cell lines selectively at clinical low dose rate

To determine the effect of oncogene expression on gamma radiation sensitivity of hematopoietic compared to fibroblastic cells, we selected clonal sublines of an interleukin-3 (IL-3)-dependent hematopoietic progenitor cell line 32D cl 3 and NIH/3T3 embryo fibroblastic cells following transfection with each oncogene linked to the mycophenolic acid resistance gene. Each mycophenolic acid-resistant subclone demonstrated high levels of specific poly(A)+ mRNA for each oncogene. The parent line 32D cl 3 demonstrated similar radiosensitivity at 116 cGy/min (D0 126, n 1.17) compared to 5 cGy/min (D0 123, n 1.65). This pattern was not altered in subclones of 32D cl 3 cells transfected with the epidermal growth factor (EGF) receptor gene and grown in EGF (at 116 cGy/min D0 104, n 0.998, at 5 cGy/min D0 115, n 1.09), or in 32D cl 3 cells expressing the v-sis oncogene (at 116 cGy/min D0 122.4, n 1.79, at 5 cGy/min D0 135, n 1.43). In contrast, expression of the transfected oncogenes v-erb-B, v-abl, or v-src conferred significant radioresistance at 5 cGy/min dose rate (D0 194, n 1.77; D0 165.5, n 1.56; D0 171, n 1.28, respectively). With the exception of v-sis, oncogene expression resulted in nonautocrine factor independence of 32D cl 3 subclones, and production of donor origin tumors in syngeneic new-born or adult mice. Two rare spontaneous factor-independent subclones of 32D cl 3 were also tested. Nonautocrine clone 32D cl 2 demonstrated significantly increased radioresistance at low dose rate (D0 186, n 1.63), while autocrine (IL-3 producing) subclone 32D cl 4 revealed no significant increase in radioresistance at 5 cGy/min. The parent fibroblast cell line NIH/3T3 showed an intrinsic relative radioresistance at low dose rate (at 5 cGy/min D0 157.3, n 1.81, compared to 116 cGy/min D0 134.3, n 1.57). Expression in NIH/3T3 of transfected oncogenes v-abl, v-fms, v-fos, or H-ras increased radioresistance at low dose rate (D0 208.6, n 1.61; D0 206.6, n 1.51; D0 167.5, n 1.85; and D0 206.8, n 1.08, respectively). Thus expression of each of several oncogenes induces resistance to gamma irradiation at 5 cGy/min in hematopoietic and fibroblast cell lines. These data may help explain the clinical recurrence of oncogene-expressing leukemia and lymphoma cells after marrow stem cell ablative doses of low-dose-rate total-body irradiation.     

An avian transforming retrovirus isolated from a nephroblastoma that carries the fos gene as the oncogene.

A new avian transforming retrovirus, NK24, was isolated from a chicken with a nephroblastoma. This transforming virus induced fibrosarcomas with osteogenic cell proliferation and nephroblastomas in vivo and transformed fibroblast cells in vitro. From extracts of NK24-transformed cells, anti-gag serum immunoprecipitated a 100-kilodalton nonglycosylated protein with no detectable protein kinase activity. An NK24 provirus present in infected quail cells was molecularly cloned and subjected to nucleotide sequence analysis. The genome of NK24 was 5.3 kilobases long and had a 1,126-base-pair sequence of cellular origin in place of a viral sequence of avian leukosis virus containing the 3' half of the gag gene and the 5' half of the pol gene. Although the entire env gene was retained, it appeared to be inactive, possibly owing to the loss of function of its splice acceptor site as a result of a second deletion of 1,598 bases in the 3' half of the pol gene that extended to the acceptor site. Nucleotide sequence analysis revealed that the NK24 virus contained the fos gene, previously identified as the oncogene of FBJ and FBR murine osteosarcoma viruses. Unlike the v-fos gene products of FBJ and FBR, which suffer a structural alteration at their carboxyl termini, the NK24 v-fos gene product seemed to have the same carboxyl-terminal structure as the chicken c-fos gene product. A comparison of the structures of the products of the NK24 v-fos and mouse c-fos genes suggested that the fos gene product consists of highly conserved regions and relatively divergent regions.     

Revertants of v-fos-transformed fibroblasts have mutations in cellular genes essential for transformation by other oncogenes

Morphologic revertants of FBJ murine sarcoma virus (v-fos)-transformed rat-1 fibroblasts were isolated using a novel selection procedure based on prolonged retention of rhodamine 123 within mitochondria of v-fos-transformed versus normal fibroblasts. Two classes of revertants were isolated: class I revertants have sustained mutations in cellular genes, and a class II revertant has a nonfunctional v-fos provirus. Somatic-cell hybridization studies suggested that the revertant phenotype was recessive to the transformed phenotype. Class I revertants were also resistant to retransformation by v-gag-fos-fox, v-Ha-ras, v-abl, and v-mos, but could be retransformed by the trk oncogene and polyoma virus middle T antigen. These results suggest that the class I revertants sustained mutations in one or more cellular genes essential for transformation by some, but not all, oncogenes. Our data suggest the existence of common biochemical pathways for transformation.     

蛋白激酶Cα过表达导致正常人胚肺成纤维细胞生长失调的研究

采用自行构建的过表达ＰＫＣα亚类的正常人胚肺细胞模型,观察到细胞生长加速,血清依赖性明显下降,细胞形态发生变化,单层培养丧失接触抑制性,出现岛状生长,与对照组细胞相比,贴壁依赖性下降,在软琼脂中能形成小集落,细胞中微丝发生部分解聚,进一步检测观察到细胞中与转化密切相关的ｒａｓ癌基因表达明显增强,抑癌基因ｐ５３表达下降．首次表明在正常人胚肺细胞中ＰＫＣα的过表达直接导致细胞增殖加速,并可诱导出现部分转化特征．因此,ＰＫＣα的过表达与活化可能在细胞多阶段致癌过程中发挥着重要作用,而癌基因表达的增强与抑癌基因表达减弱可能是其作用分子机理之一．     

Transformation by the oncogene v-fms: The effects of castanospermine on transformation-related parameters

The effects of castanospermine on various parameters associated with transformation were examined in cells expressing the viral oncogene v-fms. Fischer rat embryo (FRE) cells transformed by the oncogene v-fms and grown in the presence of castanospermine reverted to a more normal cell morphology and accumulated fms protein within the endoplasmic reticulum. Treated cells attained contact inhibition of cell growth at a much lower cell density compared to the untreated controls. No effect of castanospermine on cell growth was observed for FRE cells transformed by a different oncogene v-fgr. Castanospermine-treated SM-FRE (v-fms transformed) cells reexpressed extracellular matrix fibronectin and exhibited an extensive actin-containing cytoskeleton similar to that of normal nontransformed FRE cells. Castanospermine treatment of SM-FRE cells resulted in a sixfold decrease in [3H]deoxyglucose uptake compared to that of the nonreverted SM-FRE cells. Again, no effect was observed in FRE cells transformed by the oncogene v-fgr (GR-FRE). These results further characterize the reversion caused by castanospermine and indicate that cell surface expression coordinately controls anchorage independent growth, cell morphology, contact inhibition of growth, and hexose uptake.     

Neoplastic transformation and characterization of human fibroblasts by treatment with60Co gamma rays and the human c-Ha-ras oncogene

Summary Human fibroblasts (KMST-6) immortalized by treatment with⁶⁰Co gamma rays were further neoplastically transformed by transfection of the c-Ha-ras oncogene from human lung cancer. The ras-transfected cells formed undifferentiated fibrosarcoma in nude mice. One of the tumors was recultured and a neoplastic human fibroblast line, KMST-6/RAS, was established. To analyze multistep carcinogenesis of human cells, the cellular characteristics of these genetically matched immortalized (KMST-6) and neoplastic (KMST-6/RAS) cell lines were studied in detail. KMST-6/RAS cells showed an increased saturation density, colony formation on confluent monolayers of normal human fibroblasts, proliferation in neomycin-containing medium, anchorage-independent growth, and enhanced expression of the transfected c-Ha-ras oncogene, whereas the immortalized cells did not demonstate these characteristics. Unexpectedly, growth of KMST-6/RAS cells was serum-dependent, although they were neoplastic. Interestingly, the neoplastic cells did not show the criss-crossing or piling up growth pattern characteristic of transformed rodent fibroblasts.     

Antisense RNA-mediated suppression of Bmi-1 gene expression inhibits the proliferation of lung cancer cell line A549

The oncogene Bmi-1 regulates cell proliferation and senescence. It is reported that it controlled the self-renewal of leukemic and breast cancer stem cell and was overexpressed in some solid tumors and hematologic malignancies. In this study, the effects of inactivation of Bmi-1 mediated by a plasmid-expressing antisense Bmi-1 RNA on the proliferation of lung cancer cell line A549 were investigated. As a result, when the plasmid was stably introduced into the cell line, the Bmi-1 protein level was specifically downregulated, and the cell proliferation was significantly inhibited as shown by the cell growth curve and colony forming assay. The cells were found mostly in the phase of G(0)/G(1) and cells in S phase were significantly decreased. Our results suggest that targeting Bmi-1 might be a therapeutic potential for the treatment of non-small-cell lung cancer.