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PubMed1980

Inhibitors of DNA synthesis produced by herpesvirus-transformed cell lines.

Inhibitory factors were detected in the supernatant fluids of two herpesvirus-transformed lymphoid cell lines, one a B cell line established from a cotton-topped marmoset with EBV-induced lymphoma (1605L) and the other a HVS-transformed marmoset T cell line (C-1 Ax). The factors produced by both cell lines inhibited the uptake of 125I-iododeoxyuridine (125I-IUDR) by lymphocytes stimulated by both phytohemagglutinin and pokeweed mitogen. Both factors also suppressed the uptake of 125I-IUDR by cells of lymphoid cell lines of both B and T cell origin, indicating that neither factor was specific for lymphoid subpopulations. Furthermore, the factor produced by the 1605L cell line did not exhibit tissue specificity in the target cells that it affected, since it also suppressed 125I-IUDR uptake by the HEp-2 cell line, which is epithelial. Antibody-dependent cellular cytotoxicity, an assay that has been reported to require effector cells capable of protein synthesis but not DNA synthesis, was not suppressed by either factor, indicating that these inhibitors acted at the level of DNA synthesis. Experiments investigating the kinetics of the inhibition of PHA blastogenesis by the 1605L factor suggested that this inhibitor blocked an early stage in the DNA synthetic cycle, since maximum inhibition was observed only when the factor was present early in the culture period.

Animals↗
PubMed1980

Epstein-Barr virus RNA. V. Viral RNA in a restringently infected, growth-transformed cell line.

A continuous lymphoblastoid cell line, IB-4, was established by infection and growth transformation of normal neonatal B lymphocytes with the B95-8 isolate of Epstein-Barr virus (EBV). The IB-4 cells contained the intranuclear antigen, EBNA, but not early antigen, EA. The fragments produced by the digestion of intracellular episomal viral DNA (density, 1.700 to 1.720 g/cm3) with EcoRI restriction endonuclease were identical in size to the A, B, C, E, F, G, and H fragments of virion DNA. As expected from the previous observation that episomal intracellular DNA is circular, the fragment containing the rightward terminal sequences of EBV DNA in IB-4 cells was larger than the corresponding fragment of linear viral DNA, probably as a consequence of covalent linkage to the leftward terminal fragment. Also, two fragments, EcoRI-I and -J, which were adjacent to each other in the virion DNA, were absent from the intracellular DNA. The labeled EcoRI-J of viral DNA hybridized instead to a new fragment equal in size to EcoRI-I and -J combined. Analysis of viral RNA in IB-4 cells showed that RNAs encoded by more than 30% of the viral DNA comprised approximately 0.06% of the nuclear RNA, whereas RNAs encoded by 20% and 10% of the viral DNA comprised approximately 0.06% and 0.003% of the polyadenylated and polyribosomal RNAs, respectively. Viral mRNA (polyribosomal RNA) was encoded by DNA which mapped at 0.05 x 10(8) to 0.36 x 10(8) daltons and to a lesser extent by DNAs which mapped at 0.62 x 10(8) to 0.67 x 10(8), 0.70 x 10(8) to 0.73 x 10(8), and 1.13 x 10(8) to 1.15 x 10(8) daltons in the B95-8 genome. The most agundant nuclear viral RNAs were encoded primarily by DNA which mapped at the same loci; but RNAs encoded by many other fragments of viral DNA could also be detected among nuclear RNAs. Viral mRNA(s) (polyribosomal) was encoded by about 40% of the internal reiteration and by 25% of the BamHI-H fragments which mapped from 0.32 x 10(8) to 0.36 x 10(8) daltons, nuclear RNAs were encoded by at least 57% of the internal reiteration and 40% of BamHI-H. These data indicate that there is selective accumulation of some viral RNAs within the nucleus of IB-4 cells and that there is selective post-transcriptional processing of these RNAs. Finer mapping of the DNA which encodes mRNA (polyribosomal) in IB-4 cells indicated that some of this DNA is deleted in the DNA of the P3 HR-1 virus, the only isolate of EBV which cannot initiate growth transformation. These data, therefore, support the hypothesis that expression of this region of EBV genome is important for growth transformation or for the maintenance of restrigent infection.

B-Lymphocytes↗
PubMed1991

Early induction of ribonucleotide reductase gene expression by transforming growth factor beta 1 in malignant H-ras transformed cell lines.

Previous investigations have indicated that the suppression of proliferation by transforming growth factor (TGF) beta 1 is often lost upon cellular transformation, and that proliferation of some tumors is stimulated by TGF-beta. The present study provides the first observation of a link between TGF-beta 1 regulation of this process and alterations in the expression of ribonucleotide reductase, a highly controlled rate-limiting step in DNA synthesis. A series of radiation and T24-H-ras-transformed mouse 10T1/2 cell lines exhibiting increasing malignant potential was evaluated for TGF-beta 1 induced alterations in ribonucleotide reductase M1 and M2 gene expression. Early increases in M1 and/or M2 message and protein levels were observed only in malignant cell lines. The TGF-beta 1 induced changes in M1 and/or M2 gene expression occurred prior to any detectable changes in the rates of DNA synthesis, supporting the novel concept that ribonucleotide reductase gene expression can be elevated by TGF-beta 1 without altering the proportion of cells in S phase. T24-H-ras-transformed 10T1/2 cells were transfected with a plasmid containing the coding region of TGF-beta 1 under the control of a zinc-sensitive metallothionein promoter. When these cells were cultured in the presence of zinc, a large induction of TGF-beta 1 message was observed within 1 h. Both M1 and M2 genes were also induced, with increased mRNA levels appearing 2 h after zinc treatment, or 1 h after TGF-beta 1 message levels were clearly elevated. In total, the data suggests a mechanism of autocrine stimulation of malignant cells by TGF-beta 1, in which early alterations in the regulation of ribonucleotide reductase may play an important role.

Animals↗
PubMedDate not supplied

[Recombinant murine cell lines, transformed by various vectors based on bovine type 1 papillomavirus and expressing human tissue plasminogen activator. II. Analysis of cell lines, producing recombinant tissue plasminogen activator].

We have characterized a number of recombinant cell lines established with BPV1 and Lx1 (containing duplication of LCR-E6-E7 sequence) vectors on the basis of C127 cells. It had been shown that Lx1 based vectors possess the higher number of intracellular copies than analogous vectors on the basis of wtBPV, and most part of them is integrated into the host genome. Using various concentrations of heavy metal salts we have developed the optimized procedure for induction of recombinant tPA synthesis which is controlled by the mouse MT1 promoter. A 8-fold increase of rtPA concentration was reached in the course of induction. It had been shown that native and non-glucosylated forms of recombinant and human tPA are identical in their properties.

Animals↗
PubMedDate not supplied

Effects of benzamidine derivatives on Ha-ras-1 mRNA accumulation in a Chinese hamster cell line transformed with the activated human T24 Ha-ras-1 oncogene.

Tetra benzamidine derivatives were found to inhibit proteinase activity, cell proliferation and accumulation of the Ha-ras-1 mRNA in the FHO6T1-1 Chinese Hamster cell line, transformed with the activated human T24-Ha-ras-1 oncogene. Di- and Tri-benzamidine derivatives were also found to be potent inhibitors of proliferation of FHO6T1-1 cells. These latter compounds could be proposed as useful substrates in the synthesis of drug-conjugated monoclonal antibodies or growth factors.

Amidines↗
PubMedDate not supplied

Expression of the glucocorticoid receptor and K-ras genes in urethan-induced mouse lung tumors and transformed cell lines.

Glucocorticoids influence cell proliferation and differentiation in the lung. We examined the expression of the glucocorticoid receptor (GR) gene in urethan-induced mouse lung tumors and transformed lung cell lines to determine whether any altered responsiveness to these steroids is involved in the neoplastic development of some lung tumors. We find that a GR mRNA of similar size and amount is expressed in both normal lung and urethan-induced lung tumors. The K-ras gene is activated in urethan-induced lung adenomas and transformed lung cell lines. Both alveolar and papillary lung adenomas express slightly elevated levels of K-ras mRNA and similar levels of H-ras mRNA, but variable levels of c-myc mRNA, GR and K-ras mRNAs are concurrently expressed in a cyclic manner during the proliferation of nontransformed C10 and transformed A5 lung cell lines. Treatment of the C10 cells with dexamethasone (Dex) results in the inhibition of cell proliferation and the down-regulation of both the GR and K-ras mRNA. Dex treatment also down-regulated GR mRNA levels in A5 and LM2 cells, but no inhibitory effect was observed on K-ras mRNA levels or cell proliferation. These results suggest that glucocorticoids can inhibit K-ras expression in nontransformed lung cells. Although transformed lung cells respond to the steroid by down-regulation of the GR, the presence of an activated K-ras allele may override the inhibitory effects of these hormones on cell proliferation.

Animals↗
PubMed1985

HSV- and chemical carcinogen-induced amplification of SV40 DNA sequences in transformed cells is cell-line-dependent.

Eleven simian virus 40-transformed cell lines from 5 different species were tested for their ability to amplify integrated simian virus 40 DNA upon infection with herpes simplex virus type I or treatment with various chemical carcinogens. Four cell lines were positive only for virus-induced gene amplification and two lines were positive for both carcinogen- and virus-induced gene amplification. Individual cell lines were assayed for the presence of an intact SV40 origin of replication, the expression of a functional SV40 T-antigen, and permissivity to herpes simplex virus replication. These parameters were found to be positive in all 6 amplification-competent cell lines. The ability of herpes simplex virus to amplify SV40 DNA sequences in transformed cells is greater than that of chemical carcinogens and can be suppressed by specific inhibitors of the herpes virus-encoded DNA polymerase.

Animals↗
PubMed1999

Rat proximal tubule cell line transformed with origin-defective SV40 DNA: autocrine ANG II feedback.

The renal proximal tubule (PT) is a major site for a complete tissue renin-angiotensin system (RAS) and produces endogenous angiotensin II (ANG II). The present studies demonstrate autocrine RAS feedback in a line of origin-defective SV40 plasmid transformed immortalized rat PT cells (IRPTC) designated as line 93-p-2-1, which are highly differentiated and express all RAS components. Receptor competition assays and Southern blot following RT-PCR demonstrated that these IRPTC express AT1 and AT2 angiotensin receptor subtypes. Autocrine RAS feedback was examined following exposure to ANG II (10(-8) M), and it was noted that angiotensinogen mRNA increases significantly by 1 h and remains elevated through 24 h. The AT1 blocker losartan prevents this increase. Moreover, ANG II upregulates expression of ANG II receptor mRNA (both AT1 and AT2). Thus the present studies demonstrate positive ANG II feedback with angiotensinogen and ANG II receptors in PTC, suggesting that the main site of such intrarenal feedback in vivo is within PT. ANG II secreted by line 93-p-2-1 is increased by isoproterenol, suggesting beta-adrenergic regulation in IRPTC.

Angiotensin II↗
PubMed1986

Primary rat embryo cells transformed by one or two oncogenes show different metastatic potentials.

Second-passage rat embryo cells were transfected with a neomycin resistance gene and the activated form of the c-Ha-ras I gene, or with these two genes plus the adenovirus type 2 E1a gene. Foci of morphologically transformed cells were observed in both cases; however, the frequency of transformation was at least ten times higher with two oncogenes than with the ras gene alone. All the transformed cell lines gave rise to rapidly growing tumors when injected subcutaneously into nude mice. All but one of the cell lines transformed by the ras oncogene alone formed metastatic nodules in the lungs of animals that had been injected subcutaneously with transformed cells. When transformed cells were injected intravenously, all the ras single-gene transformants gave rise to many metastatic lung nodules. In contrast, cell lines transformed with ras and E1a did not generate metastases after subcutaneous injection and gave rise to very few metastatic lung nodules after intravenous injection. These data demonstrate that a fully malignant cell with metastatic potential, as measured in an immunodeficient animal, can be obtained from early passage embryo cells by the transfection of the ras oncogene alone.

Animals↗
PubMed2004

Inhibition of the functional expression of N-methyl-D-aspartate receptors in a stably transformed cell line by cyclosporin A.

The L(tk-) cell line L12-G10 stably transformed with the human N-methyl-D-aspartate (NMDA) receptor subunits NR1-1a/NR2A showed a Ca(2+)-dependent increase in cell death, loss of mitochondrial membrane potential, and ATP depletion after agonist stimulation. Treatment of the cells with cyclosporine A (CsA) for 4h reduced glutamate-induced cell death by 60% (IC(50) of 7.1microM). The immunophilin binding drug FK506 was not effective. Short preincubation with CsA for 10 min already decreased the glutamate-induced loss of mitochondrial membrane potential while the NMDA receptor function is not affected. However, pretreatment of the cells with CsA (30 microM) for 6h reduced membrane associated NR1-1a protein amount by approximately 85%, whereas mRNA expression remained unaffected. These results suggest, that the cytoprotective effect of CsA in L12-G10 cells is due to the inhibition of the permeability transition pore on the one hand and to the inhibition of the expression of functional NMDA receptors by an additional posttranscriptional mechanism on the other hand.

Cell Line↗
PubMed1994

Induction of apoptosis by transiently transfected metabolically stable wt p53 in transformed cell lines.

Biochemical and functional properties of wild-type (wt) and mutant p53 were studied under the same cellular environment by transient transfection. Exogenous wt p53 expressed in transformed cell lines was found to be as metabolically stable as mutant p53. Yet only mutant p53 bound to hsp70 whereas wt p53 did not, suggesting that the metabolic stability of p53 does not depend on its ability to form complexes with hsp70. The wt protein was expressed essentially in the nucleus, while mutant p53 showed both nuclear and cytoplasmic expression, as determined by immunofluorescence staining with PAb122. In addition, staining with PAb1801 revealed a number of strongly fluorescent cell fragments in cultures transfected by wt p53. Morphological features of apoptosis were observed in these cultures. Quantitative analysis by flow cytometry confirmed that only the cell population expressing wt p53 had a significant amount of cell debris. Thus, transient expression of a metabolically stable wt, but not mutant, p53 induces cell death by apoptosis. The present study demonstrates a model system to investigate the functional domains of p53 in the induction of apoptosis.

Journal Article↗
PubMed1994

Alterations in the cyclic AMP signal transduction pathway regulating ribonucleotide reductase gene expression in malignant H-ras transformed cell lines.

Ribonucleotide reductase is a highly regulated activity responsible for reducing ribonucleotides to deoxyribonucleotides, which are required for DNA synthesis and DNA repair. We have tested the hypothesis that malignant cell populations contain alterations in signal pathways important in controlling the expression of the two genes that code for ribonucleotide reductase, R1 and R2. A series of radiation and H-ras transformed mouse 10T1/2 cell lines with increasing malignant potential were exposed to stimulators of cAMP synthesis (forskolin and cholera toxin), an inhibitor of cAMP degradation (3-isobutyl-1-methylxanthine) and a biologically stable analogue of cAMP (8-bromo-cAMP). Dramatic elevations in the expression of the R1 and R2 genes at the message and protein levels were observed in malignant metastatic populations, which were not detected in the normal parental cell line or in cells capable of benign tumor formation. These changes in ribonucleotide reductase gene expression occurred without any detectable modifications in the rates of DNA synthesis, showing that they were regulated by a novel mechanism independent of the S phase of the cell cycle. Furthermore, studies with forskolin (a stimulator of the protein kinase A signal pathway) and the tumor promoter 12-0-tetradecanoylphorbol-13-acetate (a stimulator of the protein kinase C signal pathway), alone or in combination, indicated that their effects on R1 and R2 gene expression in a highly malignant cell line were greater than when they were tested individually, suggesting that the two pathways modulating R1 and R2 gene expression can cooperate to regulate ribonucleotide reduction, and interestingly this can occur in a synergistic fashion. Also, a direct relationship between H-ras expression and ribonucleotide reductase gene expression was observed; analysis of forskolin mediated elevations in R1 and R2 message levels closely correlated with the levels of H-ras expression in the various cell lines. In total, these studies demonstrate that ribonucleotide reductase expression is controlled by a complex process, and malignant ras transformed cells contain alterations in the regulation of signal transduction pathways that lead to novel modifications in ribonucleotide reductase gene expression. This signal mechanism, which is aberrantly regulated in malignant cells, may be related to regulatory pathways involved in determining ribonucleotide reductase expression in a S phase independent manner during periods of DNA repair.

1-Methyl-3-isobutylxanthine↗
PubMed2004

WRN gene and other genetic factors affecting immortalization of human B-lymphoblastoid cell lines transformed by Epstein-Barr virus.

The immortalization of human B-lymphoblastoid cell lines (LCL) transformed by Epstein-Barr virus (EBV) is accompanied by two major events: increase in telomerase activity and change in karyotype from normal diploid to aneuploidy. We investigated the effect of genetic factors on the incidence of immortalization by putting old and new data together to collect enough samples for statistical analysis. Among 50 LCL from normal individuals, 5 LCL (10.0%) were immortalized and the remaining 45 LCL were mortal. None of the 44 LCL (0%; P < 0.031 against normal individuals by chi square test) from patients having Werner syndrome (WS), a recessive genetic disorder showing premature aging, were immortalized. Among 11 LCL from a family with a tendency to have hereditary type 2 diabetes mellitus, 5 LCL (45.5%; P < 0.0040 against normal individuals, P < 0.00001 against WS patients) were immortalized. Duplicated measurements of the lifespan of 33 LCL showed a good coincidence (r=0.785) between the first and second estimations, indicating that each mortal LCL has a predetermined lifespan. These results strongly suggest that the normal WRN gene, the causative gene of WS, is essential for LCL to immortalize, and genetic factor(s) of a family having diabetes mellitus increases immortalization, implicating that host genetic factors affect immortalization of EBV and probably carcinogenesis by EBV.

Adolescent↗
PubMed1998

Relationship between ATP synthesis and 201Tl uptake in transformed and non-transformed cell lines.

201Tl tumour imaging is an established procedure, but little is known about its biological significance in transformed and non-transformed cells. In investigating the relationship between 201Tl uptake and intracellular ATP, we wished to determine whether the observed difference in delayed uptake is attributable to re-uptake via Na-K ATPase by using transformed (HeLa) and non-transformed (human fibroblast: hFB) cell lines. In each cell line, ATP was measured using the Luciferin-Luciferase method (LLM). The change in 201Tl uptake was assessed under conditions of mitochondrial suppression. Additionally, we assessed whether glycolysis is involved in 201Tl uptake under conditions of mitochondrial suppression and anaerobic incubation. Re-uptake via Na-K ATPase (HeLa vs hFB: 37.3 vs 24.2%) showed a clear difference in delayed uptake between HeLa and hFB. With HeLa, 201Tl uptake decreased biphasically with a reduction in ATP levels, whereas with hFB a linear correlation was evident. Despite the suppression of mitochondrial potential, a 5% glucose loading accelerated glycolysis with HeLa, and increased ATP (10.0 +/- 4.0%) and 201Tl uptake (16.2 +/- 3.0%). Conversely, neither ATP nor 201Tl uptake increased with hFB. Our results provide evidence that 201Tl uptake in transformed cells is related to enhanced glycolysis as well as mitochondrial ATP synthesis.

2,4-Dinitrophenol↗
PubMed1990

Metabolism of 7 beta-hydroxycholesterol in astrocyte primary cultures and derived spontaneously transformed cell lines: correlation between the esterification on C-3 -OH by naturally occurring fatty acids and cytotoxicity.

The lethal effect of 7 beta-hydrocholesterol (7 beta-OHC) on spontaneously transformed cell lines, derived from neonatal rat astrocyte primary cultures and the extent of 7 beta-OHC esterification by naturally occurring fatty acids on C-3 -OH (metabolite) was investigated. The extent of cellular death and metabolite biosynthesis matched with the 7 beta-OHC concentrations. Incubation of the cells with 10 microM 7 beta-OHC in the presence of either lipoproteins depleted fetal calf serum or with increasing serum concentrations revealed proportionality between the degree of cellular cytotoxicity and metabolite levels. The use of tetracaine or progesterone as acyl-CoA: cholesterol acyltransferase (ACAT) inhibitors indicated that ACAT was involved in metabolite production; the inhibition of metabolite biosynthesis slowed down 7 beta-OHC lethal effect. Incubation of the cells with 1 mM db-cAMP, prior 7 beta-OHC treatment, enhanced both metabolite production and cellular death. These findings support the view that the metabolite is directly implicated in the cytotoxic action.

Animals↗
PubMed1985

Evidence from two transformed cell lines that the phosphorylations of peptide tyrosine and phosphatidylinositol are catalyzed by different proteins.

Two transformed rodent cell lines (RS-1 and LSTRA) were studied in vitro to determine if their major protein tyrosine kinases catalyzed the phosphorylation of phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns4P), or diacylglycerol. RS-1 cells, transformed by Rous sarcoma virus, contain high levels of pp60src; LSTRA cells, transformed by Moloney murine leukemia virus, contain a tyrosine kinase (pp56) that is the product of an unknown cellular gene. Rates of phosphorylation of peptide tyrosine were elevated more than 20-fold in RS-1 and LSTRA particulate fractions compared to fractions from suitable control cells (N2 and YAC-1), but there was not a proportional increase in rates of phosphorylation of PtdIns, PtdIns4P, or diacylglycerol. Heat (34 degrees C) completely inactivated the LSTRA tyrosine kinase, while it enhanced the phosphorylation of PtdIns and PtdIns4P and had no effect on the phosphorylation of diacylglycerol. PtdIns4P inhibited the phosphorylation of PtdIns but had no effect on tyrosine kinase activity. An antibody, raised against a peptide with a sequence homologous to the autophosphorylation site of pp60src, immunoprecipitated tyrosine kinase activity from RS-1 and LSTRA extracts but had no effect on PtdIns kinase or PtdIns4P kinase activity. These results provide evidence that the phosphorylations of tyrosine and PtdIns are catalyzed by different proteins. An additional observation was that a monoclonal antibody that binds to pp60src and pp56 removed PtdIns kinase as well as tyrosine kinase activity from RS-1 and LSTRA particulate extracts. This antibody also removed PtdIns kinase from N2 and YAC-1 extracts, in which tyrosine kinase activity was low or undetectable. Thus, the anti-pp60src monoclonal antibody may recognize the PtdIns kinase in addition to pp60src and pp56.

1-Phosphatidylinositol 4-Kinase↗
PubMed1980

Variations in properties of virus released from morphologically different cell lines transformed in vitro by Friend leukemia virus.

The properties of the virus synthesized by each of three morphologically different cell lines originating from DBA/2J fetal liver cells transformed by the anemic strain of Friend leukemia virus in vitro were analyzed. The cells of line G-1 are malignant in syngeneic DBA/2 mice, grow in suspension, and are erythroid in origin. Cells of lines G-2 and G-3 are adherent, are epithelial in appearance, and produce no tumors in DBA/2J mice. Higher reverse transcriptase activity was detected in the culture fluid of lines G-2 and G-3, although virus from G-1 cells was more leukemogenic. Differences were also found in the virion density and size of the viral genome. RNA from the virions produced by G-2 and G-3 cells sedimented at 75 S in a sucrose gradient; virion RNA from G-1 cells sedmiented at 60 S. However, when subjected to polyacrylamide gel electrophoresis, all three virus strains showed identical RNA subunits with an estimated molecular weight of 2.6 X 10(6). Analysis of virion proteins by slab gel electrophoresis showed differences in envelope protein (gp71) components but not in the major core protein (p30). The properties of these viruses are stable and remain unchanged after passage in 3T3 cells.

Animals↗