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1.
Revertants of v-fos-transformed fibroblasts have mutations in cellular genes essential for transformation by other oncogenes 总被引:18,自引:0,他引:18
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. 相似文献
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Behavior of myc and ras oncogenes in transformation of rat embryo fibroblasts. 总被引:47,自引:19,他引:28
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H Land A C Chen J P Morgenstern L F Parada R A Weinberg 《Molecular and cellular biology》1986,6(6):1917-1925
The requirements for transformation of rat embryo fibroblasts (REFs) by transfected ras and myc oncogenes were explored. Under conditions of dense monolayer culture, neither oncogene was able to transform REFs on its own. However, the introduction of a ras oncogene together with a selectable neomycin resistance marker into REFs allowed killing of the normal nontransfected cells and the outgrowth of colonies of ras transformants, 10% of which survived crisis and became tumorigenic. These cells expressed greater than 10-fold-higher levels of ras p21 than tumorigenic cells cotransfected with ras and myc oncogenes. The myc oncogene similarly was unable to induce tumorigenic conversion of REFs unless especially refractile colonies of oncogene-bearing cells, produced by use of a cotransfected selectable marker, were picked and subcultured. Tumorigenic conversion of REFs by single transfected oncogenes appears to require special culture conditions and high levels of gene expression. 相似文献
4.
P G Milner 《Biochemical and biophysical research communications》1991,180(1):423-430
Transformation of normal rat kidney fibroblasts (NRK) by the simian sarcoma virus (SSV) occurs as a result of expression of p28v-sis, a homologue of platelet-derived growth factor-B chain. Chromatographic separation revealed that the bulk (85%) of the mitogenic activity in SSV-transformed NRK cells was not due to p28v-sis but rather two distinct endothelial cell growth factors that eluted off heparin-Sepharose between 1 and 2 M NaCl. Protein purification and Northern blot analysis revealed that one of these growth factors was the 18 kd form of bFGF, the expression of which was found to increase 15-fold with SSV-transformation of NRK cells. The pure 18 Kd bFGF had no effect on NRK cell growth but was a potent neurotrophic agent for fetal rat cortical neurones and a potent growth factor for fetal bovine heart endothelial cells, suggesting a paracrine but not autocrine role for this protein. The second endothelial cell growth factor activity in SSV-transformed NRK cells was due to an 18 Kd protein which could be distinguished immunologically, biochemically, and mitogenically from bFGF. 相似文献
5.
Synthesis and secretion of avidin was studied in cultured chicken embryo fibroblasts infected with transforming retroviruses (Rous sarcoma virus, its mutants temperature-sensitive for transformation, OK-10 virus) or a nontransforming retrovirus (RAV-1). Avidin was detectable in both transformed and untransformed cultures, and was identical to chicken egg white avidin by several criteria: biotin-binding, heat-induced biotin exchange, subunit size (mol. wt. 15 600), immunoprecipitation of metabolically labeled proteins and immunoblotting. Transformation increased the production of avidin up to 50-fold, but several experiments suggested that the induction was not a direct consequence of virus-induced cell transformation. The production of avidin seemed to relate to cellular damage both in cultures of virus-transformed and of normal fibroblasts. It may represent a response to cellular damage and viral transformation may activate the process. 相似文献
6.
Complementation by BCL2 and C-HA-RAS oncogenes in malignant transformation of rat embryo fibroblasts. 总被引:8,自引:3,他引:5
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The BCL2 (B cell lymphoma/leukemia-2) and C-HA-RAS oncogenes encode membrane-associated proteins of 26 and 21 kilodaltons, respectively. Although RAS proteins have long been known for their ability to bind and hydrolyze GTP, recent investigations suggest that BCL2 encodes a novel GTP-binding protein (S. Haldar, C. Beatty, Y. Tsujimoto, and C. M. Croce, Nature [London] 342:195-198, 1989). Cotransfection of BCL2 and HA-RAS oncogenes resulted in morphological transformation of early-passage rodent fibroblasts, rendering these cells tumorigenic in animals and enabling them to grow in semisolid medium. In contrast, cotransfection of BCL2 with oncogenes that encode nuclear proteins (E1A and C-MYC) did not produce malignant transformation, whereas HA-RAS did complement with these genes. These findings suggest that proteins encoded by oncogenes such as BCL2 and HA-RAS, although having similar subcellular locations and perhaps similar biochemical properties, can regulate distinct complementary pathways involved in cellular transformation. 相似文献
7.
L W Jiang V M Maher J J McCormick M Schindler 《The Journal of biological chemistry》1990,265(9):4775-4777
The pH of the intralysosomal compartment of fibroblasts in culture was monitored by measuring the fluorescence emission intensity at 530 nm of fluid phase pinocytosed fluorescein-conjugated dextrans (FITC-dextrans) excited at 488 and 457 nm. Following the procedure of Ohkuma and Poole (Ohkuma, S., and Poole, B. (1978) Proc. Natl. Acad. Sci. U. S. A. (1978) 75, 3327-3331), a relationship was established between the fluorescence emission intensity of the FITC-dextrans and pH. This correlation was used to determine the intralysosomal apparent pH (pHapp) of a series of fibroblast cultures. The mean intralysosomal pHapp values of nontransformed mouse 3T3 fibroblasts and an infinite life-span human fibroblast cell strain, designated MSU-1.1, was 5.0. In distinction that of 3T3 fibroblasts transformed to the malignant state by Kirsten murine sarcoma virus and MSU-1.1 cells transformed by transfection of the v-Ki-ras or T24 H-ras oncogene was 6.1. These measurements suggest that ras transformation results in a significant perturbation of lysosomal pH. 相似文献
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Extracellular matrix overexpression is a common final pathway that leads to ventricular remodeling. Fibronectin plays a pivotal
role in this progress. In the work presented here, we explored the possibility of direct inhibition of fibronectin synthesis
in rat cardiac fibroblasts by small interference RNA (siRNA). We found that siRNA could successfully suppress the fibronectin
overexpression stimulated by angiotensin II. To overcome the limitations of plasmid-based siRNA, we subcloned the H1 promoter
into pLXIN, a commercially available retroviral vector. We found that H1 promoter worked very well to form the small hairpin
RNA (shRNA) on the retroviral vector, and the fibronectin expression was dramatically down regulated by shRNA. We think the
retroviral shRNA delivery system that we have constructed may have potential roles in treating ventricular remodeling. 相似文献
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Effective suppression of fibronectin synthesis by retrovirus delivered shRNA in rat cardiac fibroblasts 总被引:2,自引:0,他引:2
WU Di HONG Quan CHEN Xiangmei FU Bo LI Aipingi LIU Hang BAI Xueyuan & WANG Jianzhong . Department of Nephrology General Hospital of PLA Beijing China . Department of Cardiology General Hospital of PLA Beijing China 《中国科学:生命科学英文版》2004,47(4):368-375
Ventricular remodeling plays a central role in chronic heart failure. Fibrosis is an important manifest of ventricular remodeling. Fibrosis is a disproportion-ate accumulation of extracellular matrix (ECM). Fi-brosis stiffens the ventricles and impedes both con-traction and relaxation. Furthermore, fibrosis results in reduced capillary density and an increased oxygen diffusion distance that can lead to hypoxia of myo-cytes. Thus, inhibiting the fibrosis profoundly affects myocyte metabolism … 相似文献
13.
v-src transformation of rat embryo fibroblasts. Inefficient conversion to anchorage-independent growth involves heterogeneity of primary cultures 总被引:3,自引:0,他引:3
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《The Journal of cell biology》1994,126(2):475-483
To clarify whether a single oncogene can transform primary cells in culture, we compared the transforming effect of a recombinant retrovirus (ZSV) containing the v-src gene in rat embryo fibroblasts (REFs) to that in the rat cell line 3Y1. In the focus assay, REFs exhibited resistance to transformation as only six foci were observed in the primary cultures as opposed to 98 in 3Y1 cells. After G418 selection, efficiency of transformation was again somewhat lower with REFs compared to that with 3Y1 cells, but the number of G418-resistant REF colonies was much greater than the number of foci in REF cultures. Furthermore, while 98% of G418-resistant colonies of ZSV-infected REFs were morphologically transformed, only 25% were converted to anchorage- independent growth, as opposed to 100% conversion seen in ZSV-infected 3Y1 cells. The poor susceptibility of REFs to anchorage-independent transformation did not involve differences in expression and subcellular distribution of p60v-src, or its kinase activity in vitro and in vivo. It rather reflected a property of the primary cultures, as cloning of REFs before ZSV infection demonstrated that only 2 out of 6 REF clones tested were permissive for anchorage-independent growth. The nonpermissive phenotype was dominant over the permissive one in somatic hybrid cells, and associated with organized actin filament bundles and a lower growth rate, both before and after ZSV infection. These results indicate that the poor susceptibility of REFs to anchorage-independent transformation by p60v-src reflects the heterogeneity of the primary cultures. REFs can be morphologically transformed by p60v-src with high efficiency but only a small fraction is convertible to anchorage- independent growth. REF resistance seems to involve the presence of a suppressor factor which may emerge from REF differentiation during embryonic development. 相似文献
14.
Complete transformation of embryonal rat fibroblasts by polyomavirus occurs during passage in vitro 总被引:1,自引:0,他引:1
The tumorigenicity of secondary rat embryo fibroblasts transfected with a plasmid harboring a replication origin-defective polyomavirus was found to increase during in vitro propagation. Thus, polyomavirus-transfected cells were found to be more than 10,000-fold more tumorigenic when injected into syngenic rats at 3 months after transfection compared to those injected at an earlier time point. Furthermore, most clones of polyomavirus-transfected cells did not grow in semisolid medium at 52 days after transfection but did grow at 95 days. Addition of glucocorticoid hormones, but not of 25% fetal calf serum, to the growth medium of the early passage cells resulted in limited anchorage-independent growth. An altered level of expression of a number of proteins was found in cells analyzed at different times after transfection. Notably, the expression of a component of the actin filament system, tropomyosin 2, was shown to decrease during growth in vitro. The development of a more fully transformed phenotype at late passages correlated with loss of the requirement for large T-antigen for growth. Thus, cells transfected with a polyomavirus mutant encoding a thermolabile large T-antigen did not grow at the restrictive temperature at 6 weeks after transfection, but grew well at 5 months after transfection. We suggest that these phenomena may be explained by assuming that establishment of rodent fibroblasts, and thereby sensitivity to transformation by middle T-antigen, is not an immediate consequence of expression of large T-antigen but occurs after a period of growth in vitro. 相似文献
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Activated Ha-ras can cooperate with defective simian virus 40 in the transformation of nonestablished rat embryo fibroblasts. 总被引:17,自引:8,他引:9
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We studied the ability of activated Ha-ras to cooperate with simian virus 40 (SV40) in the transformation of nonestablished rat embryo fibroblasts. Cotransfection with Ha-ras greatly accelerated the rate of focus induction by wild-type SV40. Moreover, a series of transformation-defective SV40 mutants could be partially complemented by Ha-ras. This was true not only for mutants retaining an intact N-terminal immortalization-competent domain, but also for a nonkaryophilic SV40 mutant. In the latter case, all detectable T antigen was cytoplasmic, indicating that efficient transformation can be achieved through the interaction of two nonnuclear proteins. By employing cell lines derived with various SV40 mutants, it was determined that the ability to complex with p53 depends on the integrity of a relatively large region in the C-terminal half of large T. Finally, we report that nonkaryophilic SV40 large T forms a complex with the major heat shock protein HSP70, and we discuss its possible implications. 相似文献
17.
Efficient immortalization and morphological transformation of human fibroblasts by transfection with SV40 DNA linked to a dominant marker 总被引:10,自引:0,他引:10
Lynne V. Mayne Anne Priestley Michael R. James Julian F. Burke 《Experimental cell research》1986,162(2):530-538
Immortal cell lines are essential for genetic and biochemical studies. Unlike rodent cells, which will form continuously growing cultures either spontaneously or after infection with an oncogenic virus (e.g., Simian Virus 40 (SV40)), human cells fail to form continuous cell lines spontaneously and in only rare cases from cell lines after oncogenic virus infection. We have used a plasmid (pSV3gpt) containing both the SV40 early region encoding T antigen and the bacterial gene xanthine-guanine phosphoribosyl transferase (gpt) to achieve high efficiency morphological transformation and immortalization of primary human skin fibroblasts. Transfection of this plasmid into primary human skin fibroblasts derived from a normal individual, two Cockayne's syndrome patients, and an immuno-deficient patient and selection for the gpt gene resulted in an altered cell morphology and growth properties characteristic of previously described SV40-transformed cells. Transfected cultures subsequently senesced, entered crisis and in each case formed a rapidly growing culture. The high efficiency of immunortalization described here (four out of four cell strains) is in contrast to previously described procedures utilizing focal overgrowth. We suggest that the use of a dominant selectable marker linked to the SV40 early region increases the probability of establishing an immortal human cell line. 相似文献
18.
Expression in rat fibroblasts of a human transforming growth factor-alpha cDNA results in transformation 总被引:49,自引:0,他引:49
Acquisition of the ability to produce and respond to a growth factor may result in increased cellular proliferation and could lead to malignant transformation. The fact that a large variety of tumor cells secrete transforming growth factor-alpha (TGF-alpha) suggests involvement of TGF-alpha in cellular transformation and provides supporting evidence for the autocrine stimulation model. In order to determine directly the role of TGF-alpha in tumorigenicity, we introduced a human TGF-alpha cDNA expression vector into established nontransformed Fischer rat fibroblast (Rat-1) cells. Synthesis and secretion of human TGF-alpha by these cells results in the loss of anchorage-dependent growth and induces tumor formation in nude mice. Anti-human TGF-alpha monoclonal antibodies prevent TGF-alpha expressing Rat-1 cells from forming colonies in soft agar. 相似文献
19.
Fukui Y Tanaka T Tachikawa H Ihara S 《Biochemical and biophysical research communications》2007,356(2):512-516
SWAP-70 is a phosphatidylinositol trisphosphate (PtdIns(3,4,5)P(3)) binding protein, which acts in F-actin rearrangement. The role of SWAP-70 in oncogenic transformation of mouse embryo fibroblasts (MEFs) by v-Src was examined by use of MEFs defective in SWAP-70. v-Src morphologically transformed MEFs lacking SWAP-70, but growth of the transformed cells in culture was slower than that of cells supplemented with exogenous SWAP-70. The v-Src-transformed MEFs deficient in SWAP-70 were unable to grow in soft agar while those expressing SWAP70 readily formed colonies, suggesting that SWAP-70 is required for anchorage independent growth of v-Src transformed MEFs. When transplanted in nude mice, tumors formed by the v-Src transformed SWAP-70(-/-) MEFs were smaller than those formed by cells expressing exogenous SWAP-70. These results suggest that SWAP-70 may be required for oncogenic transformation and contributes to cell growth in MEFs transformed by v-Src. 相似文献
20.
Stepwise transformation of rat embryo fibroblasts: c-Jun, JunB, or JunD can cooperate with Ras for focus formation, but a c-Jun-containing heterodimer is required for immortalization. 总被引:3,自引:1,他引:2
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L Vandel N Montreau E Vial C M Pfarr B Binetruy M Castellazzi 《Molecular and cellular biology》1996,16(5):1881-1888