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At least 343 records · Page 19Linked to original sources
PubMed2004

Steps involved in immortalization and tumorigenesis in human B-lymphoblastoid cell lines transformed by Epstein-Barr virus.

Epstein-Barr virus (EBV) is closely associated with the generation of various tumors, including Burkitt's lymphoma. Human resting B cells from peripheral blood are easily transformed by EBV to actively proliferating B-lymphoblastoid cell lines (LCLs). These LCLs with normal diploid karyotypes have been believed to be "immortal", without becoming tumorigenic. A series of recent studies, however, indicate that this initial, simple concept needs extensive reconsideration. Most LCLs from normal individuals are mortal because their telomeres shorten. Some LCLs are truly immortalized by developing strong telomerase activity and aneuploidy, accompanied by various other changes: down-regulation of p16/Rb; mutation of the p53 gene; modulation of apoptosis; and sensitivity to various chemical agents. Some post-immortal LCLs additionally develop the ability to form colonies in agarose and even become tumorigenic by developing the ability to grow in nude mice. The genetic background of LCLs markedly affects the frequency of immortalization. In summary, changes of B cells after infection by EBV are roughly divided into two steps: (a) transformation of B cells into LCLs caused by EBV proteins; and (b) immortalization and tumorigenesis of LCLs mainly regulated by the factors of host cells in cooperation with EBV proteins. The new concept as reviewed here is essential for the future study of tumorigenesis by EBV.

Animals↗
PubMed1991

Molecular and biological properties of hamster tumor cell lines transformed with B77 virus: expression of v-src does not correlate with metastatic potential.

Three hamster tumor cell lines (B77Hep, B77H1De and ML cl 3.1) were investigated with the aim to determine some biological and molecular properties of cells which are connected with invasive and metastatic ability. Except B77H1De all cell lines exhibited high metastatic capacity in syngeneic adult animals. Analysis of cell lines revealed a relationship between metastatic ability and growth properties (growth rate, saturation density and colony formation in soft agar). Southern blot analysis of genomic DNA samples from high metastatic cell line (B77Hep) and low metastatic (B77H1De) showed that the metastatic potential of these cell lines did not depend on the number of integrated proviral copies. Northern blot analysis was used to determine the level of mRNA encoded by v-src gene in B77Hep and B77H1De cell lines. We found a good correlation between the number of integrated proviral copies and the level of v-src gene expression in investigated cell lines, but not with their metastatic potential. No proviral sequences were found in genomic DNA isolated from ML cl 3.1 cell line. In cell lines used in this study we found differences in expression of endogenous proto-oncogenes c-myc and c-fos.

Animals↗
PubMed1979

Nucleosomal structure of Epstein-Barr virus DNA in transformed cell lines.

Micrococcal nuclease digestion was used to analyze Epstein-Barr virus (EBV) DNA structure in nuclei of transformed cells. Digests of virus-producing (P3HR-1), non-virus-producing (Raji), and superinfected Rajii cell nuclei were fractionated by electrophoresis on agarose gels, transferred to nitrocellulose, and hybridized to 32P-labeled EBV DNA. The viral DNA of Raji nuclei produced a series of bands on electrophoresis whose lengths were integral multiples of a unit size, which was the same as the repeat length of host DNA. Viral DNA in nuclei of P3HR-1 and superinfected Raji cells produced faintly visible bands superimposed on a smear of viral DNA which dominated the hybridization pattern. No differences were detected in the patterns when total DNA digests from Raji, P3HR-1, and an EBV DNA-negative cell line (U-698M) were analyzed by ethidium bromide staining or by hybridization with the use of 32P-labeled lymphoblastoid cell DNA as probe. We conclude that the EBV episomal DNA of Raji cells is folded into nucleosomes, whereas most of the viral DNA of P3HR-1 and superinfected Raji cells is not. This pattern of DNA organization differs signficantly from that in papova group viruses.

Burkitt Lymphoma↗
PubMed1986

Monoclonal antibody 45-2D9 recognizes a cell surface glycoprotein on a human c-Ha-ras transformed cell line (45-342) and a shared epitope on human tumors.

A monoclonal antibody (45-2D9) produced after immunization of BALB/c mice with the c-Ha-ras NIH 3T3 tertiary transfectant (45-342) recognized a determinant expressed by the primary, three of three secondary, and one of three tertiary transfectants, but not by NIH 3T3 cells. The determinant was present on the cell surface and was distinct from murine leukemia virus gp70 by absorption studies. Biosynthetic labeling and immunoprecipitation studies with [35S]methionine and [3H]glucosamine demonstrated that 45-2D9 recognizes a 74,000 Mr glycoprotein with minor bands of 90,000 and 180,000 Mr on SDS-PAGE. Pulse chase studies demonstrated a 68,000 Mr precursor molecule that incorporated only [35S]methionine. The distribution of the epitope recognized by 45-2D9 was assessed by immunoperoxidase staining. The antigen was not detected on 10 primary and metastatic murine tumors or 11 transformed murine cell lines. However, a variety of surgically excised human tumors demonstrated intense staining, whereas staining of normal tissues was minimal or not detectable. Thus a human oncogene-transfected cell can express a new cell surface determinant apparently unrelated to the oncogene product, which is also selectively expressed by human tumors.

Animals↗
PubMed1988

[Transformation of rat esophageal precancerous epithelial cells and its biologic characteristics--establishment of RE25-3 esophageal carcinoma cell line].

A neoplastic transformation cell line (RE25-3) of the rat esophageal precancerous epithelium has been established by in vivo-in vitro method in our laboratory. Ten criteria were used to study the biological characteristics of the neoplastic transformation during various stages: 1. Dysplastic lesion of the esophageal epithelium was induced in wistar rat by oral administration of NSEE. The dysplastic epithelium was then removed and cultured. After several generations of subculture without carcinogen, the dysplastic epithelium showed neoplastic transformation in vitro. 2. The maximum mitotic index of the transformed cell reached 2.0% and two peaks were observed at hour 41 and 96, respectively, while the mitotic index of the normal esophageal cell was 0.6%. 3. DNA measurement was performed by MSP. The results showed disappearance of the diploid cell from the fifth subculture. Tetraploid, heteroploid and aneuploid cells were observed from the third subculture. The aneuploid cells amounted to 86.24%. 4. The number of chromosome increased as the time of culture as well as the number of subculture generation increased. Chromosome aberration was observed. 5. As the number of subculture generation increased, the damage and break of the chromosome increased. Micronuclei rate increased. 6. By incorporation of H3-TdR, DNA synthesis rate increased as the malignancy increased. 7. Growth of the RE25-3 cell was dependent on EGF in the early stage. But after 15 subculture, as neoplastic transformation developed, this dependency disappeared. 8. After 14 generations of subculture, the cell can be cultured in soft ager. 9. Keratin was proved positive within the cell by ABC immuno-histochemistry assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1975

Cytotoxic effector cells specific for B Cell lines transformed by Epstein-Barr virus are present in patients with infectious mononucleosis.

Peripheral lymphoid cells, from 12 cases of acute infectious mononucleosis (IM), were tested in a micro chromium-51 release assay for cytotoxic activity against a variety of cell lines that did or did not carry the Epstein-Barr virus (EBV) genome. Unfractionated lymphocytes from these patients were cytotoxic to both types of cell lines, as were lymphocytes from healthy individuals. If, however, lymphocytes bearing complement receptors were removed, the residual IM lymphocyte fraction was specifically cytotoxic for EBV-genome-carrying cell lines. The residual lymphocyte fraction in normal donors had no such effect. Heterophile-positive IM is caused by EBV, and these results indicate that, during the acute phase of this disease, patients harbor killer cells, probably T cells, which specifically kill EBV-genome-carrying B cells in vitro. No such specificity for EBV-genome-psitive target cells was found in normal lymphocytes stimulated in vitro with autologous EBV-genome-positive lymphoblastoid cells. Such stimulated cells were highly cytotoxic to both genome-positive and negative lines after removal of complement receptor-positive lymphocytes.

Cell Line↗
PubMedDate not supplied

[Recombinant murine cell lines transformed by various vectors based on bovine papillomavirus type 1 and expressing human tissue plasminogen activator].

We have constructed a number of vectors which include transcriptional unit of human tPA cDNA and 100% BPV-1 DNA or 100% Lx DNA (mutant BPV variant with tandem duplication of LCR-E6-E7 region). Additional HSV-1 Tk-promoter was inserted in the flanks of viral DNAa in a set of constructions. A number of recombinant cell lines have been established by means of transformation using the constructed vectors. The increased focus formation activity and the improved vector properties were demonstrated for vector construction which included Lx DNA with additional Tk promoter for activation of early viral transcription. The possibilities of BPV-based vectors design are discussed.

Animals↗
PubMed1987

Effects of growth factors on an intestinal epithelial cell line: transforming growth factor beta inhibits proliferation and stimulates differentiation.

The effects of epidermal growth factor transforming growth factor beta (TGF beta) and other growth factors on the proliferation and differentiation of a cell line derived from rat intestinal crypt epithelium (IEC-6) were defined. Incorporation of [3H]-thymidine was stimulated 1.4-2.4 fold by insulin, insulin like growth factor (IGF), platelet derived growth factor (PDGF), epidermal growth factor (EGF) and 2% fetal calf serum (FCS) respectively. Additive stimulation was observed when FCS was supplemented by insulin,IGF-I or PDGF but not EGF. Incorporation of [3H]-thymidine by IEC-6 was strongly inhibited by TGF beta with greater than 80% inhibition of incorporation at concentration approximately equal to 2.0 pM. IEC-6 cells bound 4.1 +/- 0.15 X 10(4) molecules TGF beta/cell and appeared to have only a single class of high affinity receptors (Kd approximately equal to 0.5 pM). TGF beta inhibition was unaffected by the presence of insulin or IGF-I suggesting it inhibits proliferation at a step subsequent to that at which these growth factors stimulate [3H]-thymidine incorporation. TGF beta also reduced the stimulation induced by FCS by 65%. In contrast EGF reduced TGF beta inhibition by 60%. IEC-6 cells demonstrated the appearance of sucrase activity after greater than 18 hours treatment with TGF beta. These findings suggest that TGF beta may inhibit proliferative activity and promote the development of differentiated function in intestinal epithelial cells.

Animals↗
PubMedDate not supplied

Frequent methylation of p16INK4A/p14ARF promoters in tumorigenesis of Epstein-Barr virus transformed lymphoblastoid cell lines.

BACKGROUND: The data that the p16INK4A gene is frequently inactivated in Burkitt lymphoma (BL) and that this event often accompanies the inactivation of p14ARF in several tumours prompted us to examine the genetic and methylation status of both genes in BL and B-lymphoblastoid cell lines (LCLs). MATERIALS AND METHODS: The existence of gene deletion, mutation and promoter methylation was investigated by single-strand conformational polymorphism, direct sequencing and methylation-specific PCR (MSP) analysis, respectively. RESULTS: Sequencing of each exon of both tumour suppressor genes revealed p16INK4A mutation only in 3 out of 11 BL, but 1 of them also affected the p14ARF gene. MSP analysis of promoters showed p16INK4A to be methylated and p14ARF not to be methylated in each Epstein-Barr virus-positive BL cell line. Primary B-cells and de novo established LCLs had no genetic changes or methylated promoter of either gene. LCLs achieving the stage of immortalization usually showed methylation of both p16INK4A and p14ARF promoters. CONCLUSION: Our results suggest that, in contrast to p161NK4A, inactivation of p14ARF by either genetic change or promoter methylation has no importance in the development of BL cell lines, while its methylation has a central role in the immortalization of LCLs.

B-Lymphocytes↗
PubMed1988

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.

Animals↗
PubMed1982

Human hybridomas constructed with antigen-specific Epstein-Barr virus-transformed cell lines.

A 6-thioguanine-resistant, human lymphoblastoid B-cell line (GM1500 6TG A-11; IgG secreting) was mutagen-treated with low-level gamma-irradiation and selected for ouabain resistance. One line showing 10,000-fold higher drug resistance, designated KR-4, was fused with an Epstein-Barr virus-transformed, cloned, B-lymphocyte cell line (B6) producing antitetanus toxoid (TT) antibody (IgM), and the hybrids were selected in hypoxanthine/aminopterin/thymidine medium containing 10 microM ouabain. Surviving cells, which arose at an optimal frequency of 10(-5), were subcloned by limiting dilution and screened for anti-TT production. Out of 395 final subclones, 372 were found positive for anti-TT, and seven that were selected for further study secreted specific antibody (IgM, kappa chain) at a maximum concentration of 3-6 micrograms/ml. The differential rate of anti-TT production during the logarithmic phase of cell growth was 15-fold higher in the hybridomas than in the original B6 line. The hybrid nature of the clones was confirmed by karyotype analysis, histocompatibility antigen typing, and expression of secreted and membrane-bound Ig classes. Biosynthetic labeling of the cells revealed that all hybrids secreted both IgM and IgG but that only the IgM class had specificity for TT. Because Epstein-Barr virus is a polyclonal B-lymphocyte activator, the technique we applied here may be useful for increasing the recovery of rare antigen-specific B cells in the peripheral blood and for improving the frequency and stability of hybridomas secreting a given antibody.

Antibody Formation↗
PubMed1969

Response of simian virus 40-transformed cell lines and cell hybrids to superinfection with simian virus 40 and its deoxyribonucleic acid.

Whereas normal human and monkey cells were susceptible both to intact simian virus 40 (SV40) and to SV40 deoxyribonucleic acid (DNA), human and monkey cells transformed by SV40 were incapable of producing infectious virus after exposure to SV40, but displayed susceptibility to SV40 DNA. On the other hand, mouse and hamster cells, either normal or SV40-transformed, were resistant both to the virus and to SV40 DNA. Hybrids between permissive and nonpermissive parental cells revealed a complex response: whereas most hybrids tested were resistant, three of them produced a small amount of infectious virus upon challenge with SV40 DNA. All were resistant to whole virus challenge. The persistence of infectious SV40 DNA in permissive and nonpermissive cells up to 96 hr after infection was ascertained by cell fusion. The decay kinetics proved to be quite different in permissive and nonpermissive cells. Adsorption of SV40 varied widely among the different cell lines. Very low adsorption of SV40 was detected in nonsusceptible cells with the exception of the mKS-BU100 cell line. A strong increase in SV40 adsorption was produced by pretreating cells with polyoma virus. In spite of this increased adsorption, the resistance displayed by SV40-transformed cells to superinfection with the virus was maintained.

Adsorption↗
PubMed1973

Demonstration of a cell-surface antigen associated with murine sarcoma virus by immunoelectron microscopy.

Cells transformed by murine sarcoma virus have been examined for the presence of a new virus-associated cell-surface antigen by immunoelectron microscopy. A common antigen has been detected on the surface of nonproductively transformed cells that were induced by two different strains of murine sarcoma virus, Kirsten and Moloney. This antigen shows crossreaction with cell lines transformed by murine sarcoma virus that were produced in two different mammalian species, rats and mice. Further, this antigen is distinct from previously described antigens on the surfaces of cells infected by murine leukemia virus, on the viral envelope, and on the surfaces of spontaneously transformed cell lines or cell lines transformed by x-irradiation.

Animals↗
PubMed1996

Regulation of ornithine decarboxylase in a transformed cell line that overexpresses translation initiation factor eIF-4E.

pMV7-4E cells (4E-P2), derived from NIH-3T3 cells, overexpress eIF-4E and exhibit characteristics of transformation, possibly due to translational relief of mRNAs encoding proteins that regulate cell growth. Ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, is induced in 4E-P2 cells, and this induction appears to be related to the transformed phenotype of these cells. ODC mRNA contains extensive secondary structure in its 5' untranslated region (5'UTR) and may be regulated by eIF-4E, which melts mRNA secondary structure. To better understand this regulation, cDNA constructs containing the wild-type 5'UTR of ODC or deletion mutants inserted ahead of the luciferase gene were transfected into 4E-P2 and 3T3 cells. Expression of luciferase was higher in 4E-P2 cells in all cases, suggesting that the secondary structure of the ODC 5'UTR inhibits expression in 3T3 cells, and this inhibition is overcome by the high eIF-4E levels in 4E-P2 cells. When a small open reading frame present in the 5'UTR of ODC was destroyed by a point mutation, this luciferase construct was expressed about 6-fold over that containing the wild-type 5'UTR in both cell lines, although both of these 5'UTRs contain the same predicted secondary structure. Thus, factors in addition to eIF-4E may be involved in the regulation of ODC. To examine the differences in ODC regulation by polyamines in normal and transformed cells, the effect of N1,N12-bis(ethyl)spermine (BE-3-4-3) on the synthesis and degradation of ODC was examined. ODC activity in 4E-P2 cells was 10 times less sensitive to reduction by BE-3-4-3 compared to 3T3 cells, suggesting that high ODC levels in eIF-4E-overexpressing cells are the result of decreased regulation by polyamines as well as relief of translational regulation by eIF-4E.

3T3 Cells↗
PubMed1992

Support of human cord blood progenitor cells on human stromal cell lines transformed by SV40 large T antigen under the influence of an inducible (metallothionein) promoter.

We describe the development of a human bone marrow (BM) culture system which allows study of the interaction of stromal cell lines (SCL) and highly purified hematopoietic progenitor cells. Normal BM stromal cells were electroporated with a plasmid containing the simian virus 40 (SV40) large T antigen (SV40 T Ag) under the control of a synthetic metallothionein promoter (MT4); this construct is designated MT4 SV40 T Ag. SCL in which the rate of proliferation could be controlled by altering the zinc (Zn) concentration were characterized, demonstrating that the SCL were heterogeneous with respect to G-CSF and GM-CSF production. Suppression of SCL proliferation on removal of Zn made it possible to use these lines in coculture with purified CD34+ progenitor cells from umbilical cord blood. The ability to control proliferation of SCL has allowed us to maintain the survival and expansion of colony-forming cells in culture for up to 2 months. These lines have enabled us to test for stromal cell characteristics at a clonal level and provided us with a tool to analyze the events leading to lineage commitment and hematopoietic differentiation, as demonstrated by suppression of hematopoiesis by an antibody directed against the c-kit molecule.

Antigens, CD↗