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At least 217 records · Page 12Linked to original sources
PubMed1985

Characterization of stable Epstein-Barr (EB) virus transformed cell lines and mouse-human hybridomas producing a large quantity of anti-tetanus toxoid (TT) monoclonal antibody.

Antigen-specific human monoclonal antibodies (hMoAb) were investigated with particular emphasis on the stability and scale-up of production. Peripheral blood lymphocytes from a healthy donor previously vaccinated with tetanus toxoid (TT) were resensitized in vitro with the antigen in the presence of pokeweed mitogen (PWM). Three days after the stimulation, lymphocytes were infected with Epstein-Barr (EB) virus. Among EB virus transformants, several lymphoblastoid cell clones producing anti-TT hMoAb were established. One of these clones, 3G6, produced about 10 micrograms/ml of IgM (K) in the culture supernatant. Furthermore, 3G6 cells were fused with murine myeloma cells (X63-Ag8 X 653) and three heterogeneous hybridoma clones (HE719, HE810 and HE811) were obtained after careful hybrid selection in hypoxanthine-aminopterin-thymidine-ouabain containing medium and cloning by limiting dilution technique. These hybrid clones contained human HLA and mouse H-2d antigens together with mouse-human mixed karyotype. When these mouse-human hybridomas were injected i.p. into pristane-treated nude mice of BALB/c origin, ascites were found and a large amount of anti-TT hMoAb up to 0.5 mg/ml were produced there. These hybridomas have retained their anti-TT antibody-producing capabilities over 10 months. The approach described here has a potential application for the production of other antigen-specific hMoAb.

Animals↗
PubMed2003

Alpha-lipoic acid induces p27Kip-dependent cell cycle arrest in non-transformed cell lines and apoptosis in tumor cell lines.

alpha-Lipoic acid is a naturally-occurring co-factor found in a number of multi-enzyme complexes regulating metabolism. We report here that alpha-lipoic acid induces hyperacetylation of histones in vivo and has differential effects on the growth and viability of normal versus transformed cell lines. The human tumor cell lines FaDu and Jurkat, as well as a Ki-v-Ras-transformed Balb/c-3T3 murine mesenchymal cell line, all initiated apoptosis following exposure to alpha-lipoic acid. In contrast, treatment of non-transformed cell lines with alpha-lipoic acid resulted only in reversible cell cycle arrest in G0/G1. Treatment with butyrate, another short-chain fatty acid, induced a G0/G1 arrest in both transformed and non-transformed cell lines. alpha-Lipoic acid caused a post-translational elevation in the levels of the cyclin-dependent kinase inhibitor p27Kip1. Studies using p27Kip1-deficient MEF cells demonstrated that p27Kip1 was required for the alpha-lipoic acid-mediated cell cycle arrest. The mechanism of apoptosis was independent of Fas-mediated signaling, as alpha-lipoic acid-treated Jurkat cell mutants deficient in Fas or FADD retained sensitivity to apoptosis. The differential selectivity of the pro-apoptotic effects of alpha-lipoic acid for transformed cells supports its potential use in the treatment of neoplastic disorders.

3T3 Cells↗
PubMed1992

The expression of the mouse VpreB/lambda 5 locus in transformed cell lines and tumors of the B lineage differentiation pathway.

The expression of RNA transcripts from two pre B lymphocyte related genes, VpreB and lambda 5, has been studied in a series of transformed cell lines which appear frozen at different states of B lineage differentiation, from early progenitors to surface Ig positive B cells. In the HAFTL-1 cell line, which arose from fetal liver by transformation with a retrovirus containing the Hras oncogene, Northern analysis of poly A+ mRNA as well as in situ hybridization of RNA in single cells revealed that lambda 5 and VpreB are already expressed at the progenitor stage and increase in expression as the progenitors differentiate to precursor (preB) cells, or are turned off as the progenitors differentiate to myeloid cells. Continued rearrangements of Ig genes in pre B cell lines leading to Ig expression on the surface of NFS-5 pre B cells do not influence the continued expression of VpreB and lambda 5. Surface Ig-positive B lineage cell lines also express the pre B-related genes. Both Ly1+ as well as Ly1- pre B cells are VpreB- and lambda 5-positive. Lipopolysaccharide (LPS) stimulation of 70Z/3 pre B cells does not turn off lambda 5 expression. It therefore appears that, at least in transformed cell lines, the expression of VpreB and lambda 5 is not directly regulated by the expression of microH, kappa L, or lambda L chains, LPS reactivity, or the Ly1 surface antigen. Fusion of plasmacytoma cells with normal pre B cells to generate pre B hybridomas leads to down-regulation of VpreB/lambda 5 expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Serum-responsive expression from the murine thymidine kinase promoter is specifically disrupted in a transformed cell line.

Thymidine kinase (TK) gene expression is controlled in normal cells at both the transcriptional and posttranscriptional levels. Together, these regulatory systems mediate the 20-50-fold induction of TK mRNA observed as cells traverse the G1-S boundary of the cell cycle. Previously, we have reported that a "Yi" protein complex was observed to bind the mouse TK promoter in a cell cycle-dependent manner in nontransformed cells (Q-P. Dou, J. L. Fridovich-Keil, and A. B. Pardee, Proc. Natl. Acad. Sci. USA, 88: 1157-1161, 1991) and bound constitutively in transformed cells (D. W. Bradley, Q-P. Dou, J. L. Fridovich-Keil, and A. B. Pardee, Proc. Natl. Acad. Sci. USA, 87: 9310-9314, 1990). Nonetheless, TK mRNA levels in these cells continue to exhibit a marked S-specific induction (> 10 fold), raising the question: what is the status of TK promoter-mediated, as opposed to posttranscriptional, gene regulation in these transformed cells? To address this question, we have used cell synchrony experiments involving both transformed and nontransformed cells stably transfected with a TK promoter-beta-globin reporter gene construct. We have found that, in marked contrast to the tight regulation of reporter gene expression observed in nontransformed cells (J. L. Fridovich-Keil, J. M. Gudas, Q-P. Dou, I. Bouvard, and A. B. Pardee, Cell Growth & Differ., 2: 67-76, 1991), reporter gene expression in the transformed cells is constitutive and, therefore, closely parallels the presence of Yi DNA-binding activity. These data are fully consistent with other recently published observations concerning differential controls of TK transcriptional and posttranscriptional regulation (J. M. Gudas, J. L. Fridovich-Keil, and A. B. Pardee, Cell Growth & Regul., 4: 421-430, 1993) and support the hypothesis that, in transformed cells, endogenous TK is regulated predominantly at the posttranscriptional level.

3T3 Cells↗
PubMed1994

Type C Niemann-Pick disease fibroblasts and their transformed cell lines are hypersensitive to HMG-CoA reductase inhibitors.

The deficiency of exogenous cholesterol transport within fibroblasts of Niemann-Pick disease type C (NPC) has been addressed. In this report we confirmed that the endogenous synthesis of cholesterol within cells was markedly increased in NPC fibroblasts and those transformed by origin-defective simian virus 40. The transformed fibroblasts and those of the primary culture were hypersensitive to 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors as a consequence of their dependence on endogenous cholesterol biosynthesis. The transformed fibroblasts should help further biochemical and genetic research in this condition.

Cell Line, Transformed↗
PubMed1984

Idiotypic determinants on human thyroglobulin autoantibodies derived from the serum of Hashimoto patients and EB virus transformed cell lines.

Rabbit anti-idiotypic reagents were prepared against three different human serum autoantibodies to thyroglobulin (Tg). Two of the rabbit antisera recognized private idiotypes (IdI) whilst a third antiserum recognized both an IdI and a cross-reactive idiotype (IdX) which was expressed in 50% of Hashimoto patients tested. A fourth anti-idiotype was produced against an IgM anti-Tg secreted by a patient's Epstein-Barr (EB) virus transformed lymphocytes. This antiserum only reacted with the immunizing IgM anti-Tg and therefore also recognized a private idiotype. Both the private and the public idiotypes appear to be restricted to the anti-Tg set of antibodies; this would favour the view that the IdX represents a collection of idotopes with the ability to bind Tg as their common feature, rather than a common structure based upon closely similar germ line derived amino acid sequences.

Animals↗
PubMed2003

In vitro establishment of tumorigenic human B-lymphoblastoid cell lines transformed by Epstein-Barr virus.

We studied tumorigenic and phenotypic characteristics of pre- and postimmortal human B-lymphoblastoid cell lines (LCLs) transformed by Epstein-Barr virus (EBV): preimmortal LCLs showed low telomerase activity and a normal diploid karyotype while postimmortal LCLs showed much higher telomerase activity and maintained a clonal aneuploidic state. Among five postimmortal LCLs tested, LCLs N0005 and N6803 formed colonies in agar medium and showed marked aneuploidy, and N6803 was transplantable into nude mice indicating that it had a complete malignant phenotype, but all preimmortal LCLs and the remaining three postimmortal LCLs lacked these characteristics. The products of tumor suppresser genes, p16(INK4A) and pRb, were downregulated in these two LCLs, and the p53 gene was mutated in N0005 LCL. We believe these results showed for the first time that some postimmortal EBV-transformed LCLs can become tumorigenic, contrary to previous reports, and that these LCLs provide an in vitro model of tumorigenesis induced by EBV.

Aneuploidy↗
PubMed1990

Effect of IL-6 on proliferation and IG production of human EBV-transformed cell lines in serum free culture media.

To optimalize growth and Ig production of EBV transformed B cells for large scale tissue culture, we analyzed five stable monoclonal EBV-B cell lines for their responsiveness to interleukin (IL)-6 in standard medium with 5% FCS and in several serum-free media. As we previously demonstrated these cell lines were found to be heterogeneous with regard to their production of Il-1, Il-6 and lymphotoxin. We could not demonstrate an effect of Il-6 on proliferation on any of the cell lines in either standard medium with 5% FCS or in any of the serum-free culture media. In standard medium, cell lines capable of Il-6 production showed a slight elevation of Ig production in response to exogenous Il-6. Only one cell line, that did not secrete any of the lymphokines tested, responded strongly to Il-6 with a 12 times elevated IgM production. In contrast, in serum-free medium supplemented with Bovine Serum Albumin (BSA), Il-6 raised Ig secretion of all cell lines. This study shows that in some serum-free culture media EBV cell lines can be propagated as well as in standard medium with FCS. Responsiveness of these cells to cytokines is heterogeneous and is at least partially influenced by the culture medium used.

B-Lymphocytes↗
PubMed2001

Phorbol ester tumour promoter mediated altered expression and regulation of matrix metalloproteinase-2 in a H-ras transformed cell line capable of benign tumour formation.

The matrix metalloproteinases (MMPs) are thought to play key roles in tumour formation and malignant progression. The present study demonstrates alterations in the regulation of matrix metalloproteinase-2 (MMP-2) expression in response to the phorbol ester tumour promoter, PMA, in a H-ras transformed cell line, NR3, which is capable of benign tumour formation. PMA treatment of NR3 cells resulted in decreased expression of MMP-2 mRNA levels. Following a lag period, an accompanying change in gelatinolytic activity was also found. These PMA-mediated alterations in MMP-2 mRNA levels were independent of de novo protein synthesis and involved both transcriptional and post-transcriptional events. Most notably, PMA regulates MMP-2 mRNA expression through a mechanism involving message de-stabilization. Additionally, protein kinase C mediated events were found to play a role(s) in the regulation of MMP-2 message expression in NR3 cells. This study demonstrates several novel aspects regarding the regulation of MMP-2 expression in a H-ras transformed cell line and thereby provides further insight into the altered growth regulatory programs associated with H-ras mediated cellular transformation.

Animals↗
PubMed1995

Xeroderma pigmentosum variant: generation and characterization of fibroblastic cell lines transformed with SV40 large T antigen.

Xeroderma pigmentosum variant (XPV) fibroblasts from the XP4BE strain (CRL1162) were transformed with the SV40 large T antigen with the purpose of generating immortalized cell lines that are defective in post-replication repair (PRR). Two transformation and selection protocols were used and at least two independent clones were obtained, which behaved in culture as immortal cell lines. Fingerprinting analyses were used to demonstrate their origin from XP4BE cells and to compare their genetic profiles. These cell lines were shown to be hypersensitive to killing by uv when compared to SV40-transformed fibroblasts derived from foreskins of normal neonates. One of the XPV transformed cell lines (CTag) was characterized further as a potential source of cell-free extracts with capability for catalyzing the T antigen-dependent in vitro replication of plasmid DNA carrying the SV40 origin of replication. In this assay system, CTag extracts were shown to be as active as those produced from HeLa cells. In vitro replication of uv-damaged plasmid DNA by protein extracts from PRR-defective (XPV) and PRR-proficient cells might allow the identification and characterization of protein factors that contribute to normal replication of uv-damaged DNA. Ultraviolet irradiation of plasmid DNA templates caused dose-dependent inhibition of in vitro replication by both HeLa and CTag extracts.

Antigens, Polyomavirus Transforming↗
PubMed1984

Isolation and characterization of four adenovirus type 12-transformed human embryo kidney cell lines.

Four transformed cell lines were established from cultures of human embryo kidney (HEK) cells microinjected or transfected with cloned adenovirus 12 (Ad12) EcoRI-C DNA (0 through 16.5 map units of the left-hand end of the viral genome). Each cell line showed a different growth pattern. Southern blotting demonstrated that all of the cell lines contained Ad12-specific DNA sequences, but in the microinjected isolates these were at a much lower copy number than in the transfected isolate. Two cell lines (Ad12 HEK 1 and 3) appeared to contain tandemly repeated Ad12 EcoRI-C DNA fragments. Immunoprecipitation and Western blotting confirmed that Ad12 early region 1 (E1) proteins were being expressed by all four of the transformed cell lines, but indicated that E1A polypeptide expression was considerably less than E1B polypeptide expression. All of the Ad12-transformed HEK cell lines were tumorigenic when inoculated intracranially into athymic nude mice.

Adenoviruses, Human↗
PubMed1969

In vitro transformation by adenovirus-simiam virus 40 hybrid viruses. IV. Properties of clones isolated from cell lines transformed by adenovirus 2-simiam virus 40 and adenovirus 12-simiam virus 40 transcapsidant hybird viruses.

Clones were isolated from hamster cells transformed by the adenovirus 2-SV40 and adenovirus 12-SV40 transcapsidant hybrid viruses. The clones were characterized with respect to their cytomorphology, virus and antigen content, and the histomorphology of tumors induced by transplantation of the clonal sublines to hamsters. Three different cellular and colonial morphologies were observed. Clones with an SV40 morphology gave rise to tumors predominantly with an SV40 histology, whereas clones with an adenovirus morphology produced typical adenovirus tumors upon transplantation of the transformed cells. Clones which had features of both SV40 and adenovirus transformed cells gave rise to "intermediate" and adenovirus tumors. The results indicate that multiple events occur during transformation and tumorigenesis by the transcapsidant virus populations and provide an explanation for the multiplicity of findings which have been reported with these virus populations.

Adenoviridae↗
PubMed1982

Human monoclonal autoimmune antibody produced in vitro: rheumatoid factor generated by Epstein-Barr virus-transformed cell line.

The infection of selected lymphocytes from a rheumatoid arthritis patient with Epstein-Barr virus resulted in an immortalized cell line that secretes a monoclonal rheumatoid factor (RF). The cloned line has been growing for more than 24 months, and constantly produces a monoclonal IgM, lambda, 19S, RF (1-2 micrograms/ml/106 cells). The RF agglutinates human and rabbit IgG (but not IgM) and also protein A-coated erythrocytes, but fails to do so to mouse, goat and swine IgG-coated erythrocytes. When bound to immune complexes, this monoclonal RF dose not bind complement. In the cell supernatant RF is the only immunoglobulin and it comprises approximately 5% of the total proteins. The affinity of RF to aggregated human IgG, as detected in inhibition experiments, is higher than that of Fc receptors found on human non-T lymphocytes, K562 and Daudi cell lines.

Antibodies, Monoclonal↗
PubMed1994

Application of a transformed cell line constitutively expressing HSV-1 polypeptides for the detection of HSV antibodies in human sera by an enzyme immunoassay.

We have previously reported the construction of a cell line BA4, constitutively producing the glycoproteins gD, gG, and alpha 4, the major regulatory protein of HSV-1. These cells have been selected in stepwise increasing concentrations of methotrexate and shown to produce much higher amounts of gD than non-selected cells. Extracts of the selected cells were used in an enzyme linked immunosorbent assay to detect HSV antibodies in human sera obtained from Greek blood donors. We report here that (i) the assay developed is able to distinguish HSV antibody positive from negative human sera and (ii) that its application in an epidemiological survey showed that the incidence of HSV infection in the general population in Greece is 90.4%.

Animals↗