共查询到20条相似文献,搜索用时 34 毫秒
1.
Cooperation of middle and small T antigens of polyomavirus in transformation of established fibroblast and epithelial-like cell lines. 总被引:8,自引:7,他引:1
We have reported recently that small T antigen of polyomavirus stimulates the growth of NIH 3T3 cells beyond their saturation density and induces weak anchorage-independent growth (T. Noda, M. Satake, T. Robins, and Y. Ito, J. Virol. 60:105-113, 1986). We examined whether small T antigen would cooperate with middle T antigen in the in vitro transformation of NIH 3T3 (fibroblasts) and NRK-52E (epitheliallike) cells. The small-T-antigen gene, when cotransfected with the middle-T-antigen gene, had no additional effect on the efficiency or size of dense foci formation induced by the middle-T-antigen gene on a monolayer of NIH 3T3 cells. However, the small-T-antigen gene dramatically increased the rate of growth of NIH 3T3 cells transformed by middle T antigen in semisolid medium. Introduction of the small-T-antigen gene into middle-T-antigen-transformed cells did not disturb the integrated middle-T gene, alter expression of the middle-T gene, or enhance middle-T-antigen-associated tyrosine protein kinase activity. For NRK-52E cells, the expression of middle T antigen alone resulted in small, slow-growing foci on a monolayer. These cells did not show anchorage-independent growth, despite the fact that middle-T-antigen-associated tyrosine protein kinase activity was clearly detected in these cells. NRK-52E cells expressing both middle and small T antigens formed faster growing foci on a monolayer than middle-T-antigen-expressing cells did and grew in semisolid medium, even when the amounts of middle T antigen and its associated kinase activities were lower than those of middle-T-antigen-expressing cells. We conclude that small T antigen cooperates with middle T antigen in the in vitro transformation of established cell lines of fibroblast and epitheliallike cells, that it does not share the middle-T-antigen function even though they are structurally related, and that it has a significantly more important role in the transformation of NRK-52E cells than that of NIH 3T3 cells. 相似文献
2.
Anchorage-independent colony formation of SV40 transformed BALB/c-3T3 cells in serum-free medium: role of cell- and serum-derived factors 总被引:6,自引:0,他引:6
D B McClure 《Cell》1983,32(3):999-1006
The colony-forming response of SV40 transformed BALB/c-3T3 cells in agarose suspension culture was studied in a serum-free medium (with insulin, transferrin and serum albumin as the only macromolecular supplements) that was optimized for colony formation of fibronectin-attached monolayer cultures. In this serum-free medium, the SV3T3 cells fail to form colonies in agarose suspension. However, they can be induced to anchorage-independent colony formation by the growth factors that are additionally required by their untransformed counterparts for proliferation in monolayer culture. The SV3T3 cells are also rendered anchorage-independent for colony formation in serum-free medium by conditioned medium from dense monolayer serum-free SV3T3 cultures. These experiments suggest that it is the cell-substrate interaction that is responsible for the growth factor autonomy of fibronectin-attached transformed cells. 相似文献
3.
Summary Phenotypic changes (increased longevity, decreased growth factor requirements, altered cell surface features, growth in semisolid
agarose, and SV40 T antigen expression) suggesting in vitro transformation were displayed by human normal colon mucosal epithelial
cells transfected with pSV3gpt, a pBR322 recombinant containing the SV40 “early” T antigen coding region and the dominant
selectable marker bacterial gene, xanthine-guanine phosphoribosyltransferase. In contrast, control cultures which received
neither DNA nor the recombinatn pSV2gpt (which is identical to pSV3gpt but lacks the SV40 T antigen region) were not phenotypically
altered. 相似文献
4.
Role of the Three Polyoma Virus Early Proteins in Tumorigenesis 总被引:21,自引:4,他引:17
A modified polyoma virus genome which can encode the middle T protein but not the large or small T proteins transforms rat cells in culture with an efficiency about 20% that of the wild-type genome. Although middle T-transformed cells grow as tumors when transplanted into nude mice or syngeneic rats, the middle T gene alone is totally inactive when used in a more stringent and rigorous assay for tumorigenicity such as the injection of DNA into newborn rats. Thus, functions other than those expressed by middle T antigen are required for the elaboration of all the properties associated with tumorigenesis. To assess whether a complementary function could be exerted by the large or the small T antigen, we constructed plasmids containing two modified early regions which independently encoded middle T and one of the two other proteins. Both recombinants were tumorigenic in newborn rats. Cell lines derived by transfer of these plasmids under no special selective conditions did not acquire the property of growth in low-serum medium but exhibited the same tumorigenic properties as wild-type polyoma DNA-transformed cells. Furthermore, a recombinant which encoded the middle and small T antigens, but not the large T antigen, was tumorigenic in newborn rats. Although the small T antigen provides a complementary function for tumorigenicity, it cannot complement the middle T antigen for an efficient induction of transformation of cultured cells. This suggests that the complementary function exerted by the small T antigen is different from that of the N-terminal fragment of the large T protein. 相似文献
5.
Influence of transfected SV40 early region on growth and differentiation of human endothelial cells.
Endothelial cells isolated from human umbilical veins show a limited in vitro life span of about 40 population doublings. Cell division is dependent on the presence of endothelial cell growth factor in the culture medium. We have transfected primary endothelial cells with a plasmid containing the early region of SV40 virus. Large T positive cells were obtained which grew in the absence of endothelial cell growth factor at low serum concentrations and showed a prolonged lifespan. Expression of von Willebrand factor and SV40 large T antigen was detected simultaneously in transfected cells. 相似文献
6.
7.
Cell transformation in vivo seems to be a multistep process. In in vitro studies certain combinations of two oncogenes, a cytoplasmic gene product together with a nuclear gene product, are sufficient to transform primary rodent cells. Polyoma virus large T antigen can immortalize and, in cooperation with polyoma virus middle T antigen, transform primary cells. On the other hand mutant mouse p53 can also immortalize and, in cooperation with an activated Ha-ras oncogene, transform primary cells. In the present study we analyzed whether mutant p53 can replace polyoma virus large T antigen in a cell transformation assay with polyoma virus middle T antigen. Transfection of mutant p53 alone resulted in a cell line which had retained the actin cable network, grew poorly in medium with low concentration of serum, and failed to grow in semisolid agar. Cotransfection of mutant p53 together with polyoma virus middle T led to cells which grew in medium containing low serum concentration, grew well in semisolid agar, and displayed an altered morphology with the tendency to overgrow the normal monolayer. By these criteria these cells were considered fully transformed. The rate of p53 synthesis was similar in both cell lines. However, only p53 from the transformed cell line turned out to be stable. Cells transformed by mutant p53 and polyoma virus middle T expressed nearly the same amount of the c-src-encoded pp60c-src protein as cells transformed by the same p53 and cotransfected activated Ha-ras oncogene. However, only the polyoma virus middle T/p53-transformed cells exhibited an elevated level of pp60c-src-specific tyrosine kinase activity. Thus, despite different mechanisms leading to cell transformation, mutant p53 can replace polyoma virus large T antigen and polyoma virus middle T can replace the activated Ha-ras oncogene in cell transformation. 相似文献
8.
H Spieker-Polet K Hagen M Teodorescu 《Journal of immunology (Baltimore, Md. : 1950)》1985,134(5):2827-2834
It has been proposed that anti-Ig antibody activates B cells in a way analogous to the antigens, i.e., it delivers its signal by cross-linking and clustering the Ig receptors on the surface of one cell. However, the cross-linking of different B cells which may deliver to each other a signal has not been considered. Thus, we examined the effect of preventing cell contacts on the response of rabbit B cells to anti-Ig allotype antibody by using a solid agarose medium. First, we examined the 14C-uridine incorporation in liquid medium and in liquid or solid agarose in cultures stimulated with anti-Ig antibody (Ab). The response was high in liquid agarose or liquid medium but was absent when the agarose-containing medium was solidified at the start of the cultures. If the agarose was solidified 6 hr after the start, a good response was obtained. Moreover, if the cells were sedimented at the start before solidifying the agarose-containing medium, a good response was also obtained. Similar results were obtained when T cells were activated by Con A. To examine whether B cells require contacts with other B cells or with non-B cells, we examined their response to anti-Ig Ab in the absence of macrophages or T cells, and found that purified B cells from lymphoid organs or from thoracic duct responded well in the absence of T cells and/or macrophages. The response was also absent when the cells were cultured in agar at a "local" concentration close to 10(8) cell/ml. Also, concentrated supernatants of anti-Ig-stimulated cells did not increase the response to anti-Ig in solid agarose. These two last observations suggest that the lack of response in solid agarose is not due to a lack of diffusible factors or to a lack of feeder cells. Therefore, because the only difference between the cultures that responded to anti-Ig or Con A and those that did not was the distance between the cells, we concluded that the contact between B cells or between T cells is essential to their activation by their respective mitogens. We speculate that the anti-Ig Ab or the antigen cross-links B cells, which then provide each other with the activating signal or with one of the activating signals. 相似文献
9.
Lin-Chang Chiang Janet Silnutzer James M. Pipas David W. Barnes 《In vitro cellular & developmental biology. Plant》1985,21(12):707-712
Summary NIH3T3 cells grow in a serum-free basal nutrient medium supplemented with fibronectin, transferrin, insulin, epidermal growth
factor (EGF) and high density lipoprotein (HDL). The individual omission from the serum-free medium of insulin, EGF, or HDL
results in greatly reduced cell growth. These growth-restrictive conditions can be used to select for cells transformed with
SV40, the polyomavirus middle T antigen gene, the activated humanras gene, and the mouse c-myc gene.
This work was supported by grants ES-00-210 from the National Institutes of Health (NIEHS) and MV-182 from the American Cancer
Society.
Dr. L.-C. Chiang is a Visiting Research Scientist from PPG Industries, Inc.
Editor’s statement This paper represents a new approach to the identification of oncogenes that would escape the screening
methods currently in use. Inherent in the method is the assignment of function to oncogenes. 相似文献
10.
Loss of epidermal growth factor receptors and release of transforming growth factors do not correlate with sarcoma virus-transformation in clonally-related NIH/3T3-derived cell lines. 总被引:1,自引:0,他引:1
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Transformation of NIH/3T3 cells by Kirsten murine sarcoma virus (MSV) caused a dramatic reduction in the number of cell-surface receptors for epidermal growth factor (EGF). However, the number of EGF receptors remained at a very low level in a non-tumourigenic revertant cell line isolated from the virus-transformed cells, indicating that an increase in EGF receptors is not a requirement for the phenotypic reversion of Kirsten MSV-transformed 3T3 cells. Serum-free conditioned medium from normal and virus-transformed cell lines contained similar amounts of cell growth-promoting activity as assayed by the ability to stimulate DNA synthesis in quiescent Swiss 3T3 cell cultures. However, the concentrated conditioned medium from these cell lines showed no evidence of beta-transforming growth factor (TGF) activity as assayed by promotion of anchorage-independent growth of untransformed normal rat kidney (NRK) fibroblasts in agarose. The cellular release of alpha-TGF activity was assayed by measuring the ability of concentrated conditioned medium to inhibit the binding of 125I-EGF to Swiss 3T3 cells. Conditioned medium protein from the virus-transformed cell line inhibited 125I-EGF binding but only to the same extent as conditioned medium protein prepared from the untransformed cell line. The alpha-TGF secretion by these cell lines was estimated to be 30-45-fold lower than the level of alpha-TGF released by a well-characterized alpha-TGF-producing cell line (3B11). These results suggest that the induction of TGF release is not a necessary event in the transformation of NIH/3T3 cells by Kirsten MSV. 相似文献
11.
Truncated forms of the polyomavirus middle T antigen can substitute for the small T antigen in lytic infection. 总被引:10,自引:8,他引:2
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Cloned polyomavirus genomes encoding the small T antigen or truncated forms of the middle T antigen facilitated the growth of genomes encoding only the large T antigen in mouse 3T6 cells. We conclude that an N-terminal domain of the middle T antigen, in the appropriate cellular location, can substitute for the small T antigen during lytic infection. 相似文献
12.
Murine T cell clones, which were retrieved from an A.TH anti-A.TL(lak) T cell line and had been long-term cultured in the medium supplemented with T cell growth factor (TCGF) and mitomycin C(MMC)-treated feeder cells of either Is or Ik haplotype, were found to survive in TCGF-free medium for a long time, quite in contrast to so far reported TCGF-dependent T cell clones. When T cells of these clones at the full growth in the TCGF-medium were transferred to TCGF-free medium, they survived at resting state for a long time, and half-life, i.e., the time when 50% of the transferred cells were still viable, of some clones reached 20 days. The cloned T cells at the resting state retained full responsiveness to the specific lak antigen but lost the responsiveness to TCGF as determined by [3H]thymidine uptake, whereas the same T cells harvested from TCGF-medium did not show the antigen-specific responsiveness. The cloned T cells at the resting state showed marked DNA synthesis in response to the specific antigen but never entered the phase of the cell division. Addition of TCGF to the antigen-activated cloned T cells at their peak DNA synthesis triggered the cell division without time lag. Thus, it was confirmed at a single clone level that two sequential signals, one via the antigen-receptor reacting with specific antigen and another via the TCGF-receptor accepting TCGF, are required for clonal expansion of T cells reacting with antigen. The mitogen-responsiveness among five clones was examined at their resting state; two clones responded to Con A and PHA only in the presence of accessory cells (MMC-treated, T cell-depleted syngeneic spleen cells), and one clone responded well to Con A and PHA in the absence of accessory cells. Thus, most of our clones retained physiologic characteristics of T cells directly collected from mice even after long-term culture in TCGF-medium. 相似文献
13.
Primary hamster embryo cells infected with bovine papilloma virus (BPV) or treated with BPV DNA-calcium phosphate precipitates
showed striking morphological alterations characteristic of transformed cells. Long, spindle-shaped cells grew into dense
foci, eventually overgrowing monolayers of normally shaped cells. Samples of these cells were tested for anchorage-independent
growth in dilute agarose medium. Cells were able to grow in agarose to form colonies which, when removed from agarose and
transferred to liquid medium, established clones. Mockinfected cultures inoculated with plain medium displayed normal cell
morphology and growth properties. This is the first report of BPV-transformed cells demonstrating anchorage-independent growth
in agarose and the establishment of BPV transformed clones. 相似文献
14.
Middle T antigen as primary inducer of full expression of the phenotype of transformation by polyoma virus. 总被引:32,自引:20,他引:12
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
A large number of polyoma virus-transformed cells of rat, mouse, and hamster origin were examined for presence of T-antigen species. The results showed that all lines of cells contained middle and small T antigens, but not all contained a full-sized large T antigen, in some cell lines large T antigen was absent, whereas in others it was present as truncated forms lacking various lengths of the carboxy-terminal part of the protein. Cells transformed by the new viable deletion mutants of polyoma virus, dl-8 and dl-23, formed larger and smaller colonies or foci, respectively, when they were suspended in semisolid medium or plated as monolayers together with untransformed cells on a plastic surface. The deletions in the DNA of these mutants resulted in the shortening of the large and middle T antigens simultaneously without affecting the size of the small T antigen. Variation of large T-related proteins in dl-8 and dl-23-transformed cells seemed to be the same as that observed in wild-type-transformed cells. Regardless of the amount and size of large T-related protein in mutant-transformed cells, the phenotype of the cells was entirely dependent on the mutant used. The results suggest that (i) persistence of large T antigen is not universally required for the maintenance of the transformation phenotype, (ii) small T antigen alone may not be sufficient for inducing the full expression of the transformation phenotype, and (iii) middle T antigen is implicated as being primarily responsible for the full expression of the phenotype of transformation. The results also provide the evidence that the carboxy-terminal region of middle T antigen and a part of large T antigen are encoded in the genome in the same DNA segment around map units 88 to 94 in different reading frames. 相似文献
15.
A transforming growth factor related to epidermal growth factor is expressed by fetal mouse salivary mesenchyme cells in culture 总被引:1,自引:0,他引:1
T G Ram V Venkateswaran S A Oliver H L Hosick 《Biochemical and biophysical research communications》1991,175(1):37-43
Fetal mouse salivary mesenchyme cells secrete a protein with an apparent MW of 15 Kd that is immunologically related to epidermal growth factor (EGF). Conditioned medium collected from these cells in culture stimulates the growth of primary mouse mammary epithelial cells cultured within collagen gels, competes for binding to EGF receptor sites on these mammary epithelial cells and stimulates the anchorage-independent growth of normal rat kidney fibroblast cells within soft agarose. Prior immunoprecipitation of salivary mesenchyme cell conditioned medium with anti-EGF antibodies effectively removes or attenuates all of these effects confirming that an EGF-like factor is involved in these responses. 相似文献
16.
Mutation causing premature termination of the polyoma virus medium T antigen blocks cell transformation 总被引:34,自引:16,他引:18
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
We used site-specific mutagenesis to introduce a termination codon, TGA, into the reading frame for the polyoma virus medium T antigen. We induced this mutation in a region of the polyoma genome in which the overlapping coding regions for the large and medium TE antigens are translated in different reading frames. Therefore, the mutation terminated translation of the medium T antigen, but it caused only a single amino acid substitution in the large T antigen and did not affect the small T antigen. Cells infected by the mutant virus produced normal-size small and large T antigens. The infected cells produced a 28,000-dalton fragment of the 48,000-dalton medium T antigen, whose size and tryptic peptide map were consistent with its being a truncated N-terminal fragment terminating at the new termination codon of the mutant. Immunoprecipitates of mutant-infected cell extracts did not show medium-T-antigen-associated protein kinase activity. The mutant virus replicated normally in mouse 3T6 cells and induced cellular DNA synthesis in resting mouse 3T3 cells, but it failed to transform rat or hamster cells, as judged by focus formation and growth in agar. The mutant complemented a tsA mutant which affects the large T antigen for transformation, implying that the mutant defect for transformation was in the medium T antigen. These results imply that the small T antigen and the large T antigen together are insufficient to cause transformation and support the conclusion that the medium T antigen is essential for cell transformation by polyoma virus. 相似文献
17.
Polyomavirus middle tumor antigen increases responsiveness to growth factors. 总被引:1,自引:0,他引:1
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
L Raptis 《Journal of virology》1991,65(5):2691-2694
The middle tumor antigen (mT) of polyomavirus is unable to transform a clone of NIH 3T3 cells to anchorage independence (L. Raptis and J.B. Bolen, J. Virol. 63:753-758, 1989). However, this oncogene increased the responsiveness of these cells to the growth factors (alpha-like and beta-type transforming growth factors) produced by cells possessing the whole transforming region of polyomavirus. This resulted in the growth of NIH 3T3 cells, expressing mT under control of the dexamethasone-regulatable mouse mammary tumor virus promoter, in agar medium supplemented with these growth factors upon addition of the inducer. Therefore, mT, a transforming oncogene, is able to enhance the responsiveness of established cells to growth factors, a property previously attributed primarily to myc and other establishment type oncogenes. 相似文献
18.
Common elements in growth factor stimulation and oncogenic transformation: 85 kd phosphoprotein and phosphatidylinositol kinase activity 总被引:121,自引:0,他引:121
D R Kaplan M Whitman B Schaffhausen D C Pallas M White L Cantley T M Roberts 《Cell》1987,50(7):1021-1029
The phosphorylation of proteins on tyrosine in vivo and in vitro was examined in 3T3 cells stimulated by platelet-derived growth factor (PDGF) and transformed by polyoma middle T antigen (MTAg) by using an antibody directed against phosphotyrosine (P-tyr). Two common events were observed upon PDGF stimulation or MTAg transformation of cells: the appearance in the immunoprecipitates of an 85 kd phosphoprotein, and increased phosphatidylinositol (PI) kinase activity. In PDGF-stimulated cells, the 85 kd phosphoprotein and PI kinase activity appeared rapidly, within 1 min of growth factor addition. The PI kinase activity and 85 kd phosphorylation were also increased in anti-P-tyr immunoprecipitates from cells transformed by v-fms and v-sis, but not by SV40 T antigen. The presence of the tyrosine-phosphorylated 85 kd protein correlated with PI kinase activity during several purification steps. These results suggest that the 85 kd phosphoprotein, a putative PI kinase, is a substrate for both the PDGF receptor and MTAg/pp60c-src tyrosine kinase activities. 相似文献
19.
Significance of the gastrin homology and surrounding sequences in polyomavirus middle T antigen for cell transformation.
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Deletion of residues 305 to 327 of polyomavirus middle T antigen, including the (Glu)6-Tyr-315 sequence that is a preferred site of phosphorylation in vitro by pp60c-src, markedly altered viral transformation of rat cells. The efficiency of transformation by the deletion mutant depended on how it was introduced into cells, and the resulting transformants displayed limited growth rates in monolayer and in suspension. Substitution of the polyomavirus residues 305 to 327 with a homologous region (containing [Glu]5-Ala-Tyr) from porcine gastrin did not restore wild-type transforming activity. These mutant middle T antigens interacted with pp60c-src and were phosphorylated in vitro. Thus, although a sequence of consecutive glutamic acid residues followed by a tyrosine is a dominant structural element which strongly influences the physical properties of middle T antigen, its presence did not ensure the biological activity of the protein. Other elements in this region of middle T antigen also contributed substantially to the transforming capacity of polyomavirus. 相似文献