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Increased uracil-DNA glycosylase, AP-DNA binding protein and deoxyribonuclease activities in tumor and SV40-transformed cell lines of human origin.

The activities of three human DNA metabolizing enzymes--uracil-DNA glycosylase, apurinic/apyrimidinic(AP)-DNA binding protein (an AP-DNA endonuclease) and the major cellular deoxyribonuclease (presumably DNase III and/or DNase IV)--were measured in logarithmic growing (diploid non-established) fibroblast strains, tumor-derived cell lines and SV40-transformed cell lines. The levels of activity of uracil-DNA glycosylase and DNase were increased, on average, 5- to 6-fold in tumor cell lines and 10-fold in SV40-transformed cell lines compared to those observed in normal fibroblast strains. AP-DNA binding activity was only 2- to 3-fold higher in both tumor-derived and SV40-transformed cell lines. Measurements in serum-deprived (and hence growth-retarded) SV40-transformed cells indicated that the observed increase in enzyme activity was only partially due to a higher proportion of S-phase cells in the rapidly growing transformed lines. Cell extract mixing experiments indicated that the relatively low levels of activity of the three enzymes in normal fibroblasts could not be ascribed to the presence of an inhibitory factor(s) in the crude extract.

Cell Line

Fucolipid patterns of cell lines transformed by highly and weakly tumorigenic simian virus 40 and herpes simplex virus.

The fucolipids of simian virus 40 (SV40)-transformed Balb/c mouse and herpes simplex virus (HSV)-transformed hamster cells of varying degrees of tumorigenicity were examined. In the virus-transformed cell lines and in a spontaneously transformed cell line, as compared to non-transformed cell lines, there was a decrease in the incorporation of fucose radioactivity into fucolipid IV. Fucolipid IV is the least chromatographically mobile, presumably most complex fucolipid. Only in the highly transplantable SV40- and HSV-transformed cell lines was the decrease in incorporation of radioactivity in fucolipid IV as marked as that observed in oncornavirus-transformed cell lines. Similarly, only in the highly tumorigenic DNA-virus-transformed cell lines was there a significant increase in incorporation of radioactivity from radioisotopically-labeled fucose into fucolipid III as has been observed in oncornavirus-transformed cell lines.

Animals

Effect of interferon gamma on the sensitivity of bovine-papilloma-virus(BPV1)-transformed cell lines to cell-mediated cytotoxicity.

The effect of interferon gamma (IFN gamma) on the immunogenicity and immunosensitivity of mouse cell lines transformed by bovine papillomavirus type 1 (BPV1) DNA was examined in a syngeneic mouse model. The overnight incubation of BPV1-transformed cell lines with 100 IU/ml IFN gamma did not affect their ability to induce the generation of cytotoxic effector cells but it clearly increased their sensitivity to lysis by interleukin-2-induced lymphokine-activated killer (LAK) cells and by non-specific LAK-type effector cells induced by BPV-1-transformed cell lines. The treatment of two allogeneic lymphoid tumour cell lines, P815X2 and YAC-1, with IFN gamma either decreased or had no effect on their sensitivity to LAK-cell-mediated lysis.

Animals

Susceptibility of normal and transformed cell lines to cytostatic and cytocidal effects exerted by macrophages.

Activated, nonimmune macrophages exerted profound effects on the proliferation and viability of eukaryotic target cells in vitro. Pronounced macrophage-mediated cytostasis was exerted on every rapidly proliferating cell line examined, irrespective of transformation, species derivation, cell type, or growth characteristics. However, the magnitude of cytostasis effected differed markedly among the 40 cell lines tested. There was no evident correlation between susceptibility to cytostasis and degree of transformation. Transformed cell lines with high and with low malignant attributes were affected equally. A comparable pattern was discerned for cytocidal effects of macrophages, in which the susceptibility of transformed targets was independent of the degree of malignancy.

Cell Line

Differential sensitivity of astrocyte primary cultures and derived spontaneous transformed cell lines to 7 beta-hydroxycholesterol: effect on plasma membrane lipid composition and fluidity, and on cell surface protein expression.

The cytotoxicity of 7 beta-hydroxycholesterol (7 beta-OHC) was investigated on rat astrocyte primary cultures and spontaneously transformed cell lines derived from them. Confluent astrocyte primary cultures (normal cells) were unaffected by 20 microM 7 beta-OHC over a period of 72 h whereas 30 microM markedly affected the viability of the transformed cells within the first 72 h. Both cell types incorporated 18% of the total amount of 7 beta-OHC added to the cultures at concentrations of 20 microM or 30 microM. Cellular fractionation after incubation with 20 microM or 30 microM 7 beta-OHC indicated that the plasma membrane incorporated 2 or 6 fold more 7 beta-OHC than the intracellular one's respectively. Plasma membrane cholesterol (CH) and phospholipid (PL) analysis showed that 20 microM 7 beta-OHC did not affect CH/PL in normal cells; in contrast, plasma membranes of transformed cells displayed a significant CH/PL decrease, which was more pronounced with 30 microM 7 beta-OHC treatment. Fluorescence anisotropy measurements indicated that 20 microM 7 beta-OHC slightly fluidified the plasma membrane of normal cells whereas it has not effect on that of the transformed cells one; however, an increase in plasma membrane fluidity was observed when the transformed cells were treated with 30 microM 7 beta-OHC. Lactoperoxidase catalyzed radioiodination of cell surface proteins and subsequent autoradioelectrophoretic analysis demonstrated that the labelled protein pattern was unchanged when both cell types were incubated with 30 microM 7 beta-OHC. These findings demonstrate that 7 beta-OHC is lethal to highly proliferating cultured glial cells. The high accumulation of 7 beta-OHC in the plasma membrane and its decrease in fluidity, by themselves, do not seem to be involved in the processes leading to cellular death. However, increase of plasma membrane fragility associated with the decrease of CH/PL, which occurs exclusively in plasma membranes isolated from 7 beta-OHC treated transformed cells together with high 7 beta-OHC uptake, are probably implicated in 7 beta-OHC cytotoxicity. The possibility of an additional action mechanism is discussed.

Animals

Molecular association between transplantation antigens and cell surface antigen in adenovirus-transformed cell line.

A rat cell line (A2T2C4) transformed with adenovirus type 2 elicited cytotoxic T lymphocytes in syngeneic rats. Cytotoxicity was abolished by a rabbit antiserum directed against the major histocompatibility (AgB) antigens and by a syngeneic rat antiserum raised against the virus-transformed cell line. The syngeneic antiserum immunoprecipitated surface proteins with apparent molecular weights of 45,000, 19,000, 17,000, and 12,000 from the A2T2C4 cells but it displayed no reactivity against primary rat fibroblasts and spleen cells. The rabbit antiserum against AgB antigens precipitated a 19,000-dalton component from the A2T2C4 cells which was not observed in primary rat fibroblasts. Sequential immunoprecipitation revealed identity between the major polypeptides recognized by the two antisera. Because the rabbit anti-AgB antigen serum was specific for the transplantation antigen subunits and because the syngeneic rat antiserum against the A2T2C4 cells failed to react with the AgB antigens in normal cells, it is concluded that the 19,000-dalton component is coprecipitated with the AgB antigens. Antisera directed specifically against beta2-microglobulin and the alloantigenic AgB antigen subunit also coprecipitated the 19,000-dalton component. These results indicate that the AgB antigen subunits form a ternary complex with a virus-coded protein on the surface of the virus-transformed A2T2C4 cells. This molecular complex may be recognized by the cytoloytic T lymphocytes

Adenoviruses, Human

Selective killing of human T cell lymphotropic virus type I-transformed cell lines by a damavaricin Fc derivative.

n-Pentyl ether of damavaricin Fc (n-pentyl DvFc) preferentially killed human T-cell lymphotropic virus type I (HTLV-I)-transformed cell lines. The mechanism of action of the drug was investigated using MT-4 cells. Cytotoxic action was diminished by the removal of n-pentyl DvFc from the culture or by the addition of sulfhydryl compounds such as 2-mercaptoethanol and dithiothreitol. The killing activity of n-pentyl DvFc was also diminished by membrane-acting agents including quinidine and diphenylhydantoin. Influx and subsequent efflux of Ca2+ were observed when either HTLV-I infected (MT-4 cells) or uninfected cells were treated with n-pentyl DvFc. An efflux of K+ was observed in HTLV-I infected MT-4 cells immediately after the exposure of the cells to n-pentyl DvFc. The K+ efflux, however, was not observed in the uninfected T cells. n-Pentyl DvFc seems to act primarily on the cell surface of MT-4 cells, leading to the perturbation of membrane function. The restoration of cell growth, however, is critically dependent on the presence of dithiothreitol and 2-mercaptoethanol, implying a role for a free sulfhydryl group in the killing activity.

Anti-Bacterial Agents

Assignment of the integration site for simian virus 40 to chromosome 17 in GM54VA, a human cell line transformed by simian virus 40.

GM54VA human cells transformed by simian virus 40 (SV40) were fused with peritoneal macrophages obtained from three different mouse strains. All 27 hybrid clones studied were positive for SV40 tumor antigen in 100% of their cells and contained human chromosome 17. Human chromosome 17 was the only human chromosome present in five of the hybrid clones. Fusion of GM54VA cells and either thymidine kinase (EC 2.7.1.75)-deficient mouse or Chinese hamster fibroblasts resulted in the growth in hypoxanthine-aminopterin-thymidine medium of hybrid clones positive and negative for SV40 tumor antigen. Counterselection of the hybrid clones positive for tumor antigen in medium containing 5-bromodeoxyuridine resulted in the growth of hybrid cells that were negative for tumor antigen. These experiments indicate that negative for tumor antigen. These experiments indicate that SV40 is integrated in only one of the two parental human chromosomes 17. Because the genome of SV40 has been assigned to human chromosome 7 in two other SV40-transformed human cell lines, at least two different integration sites for SV40 would seem to be present in human cells: one located in human chromosome 7 and the other located in human chromosome 17.

Alleles

Tumorigenicity and karyotype of rat embryo cell lines transformed by BK virus.

A rat embryo cell line transformed by BK virus was used to induce tumors in rats. Cell lines were established from these tumors. Other sublines were obtained by in vitro cloning of the parental line. Growth characteristics and karyotypes were compared to the tumorigenicity of these cell lines. The in vitro cloned sublines had a low tumorigenicity. Tumorigenicity of the tumor cell lines varied from high to undetectable. The tumor cell line with the highest tumorigenicity also had the highest saturation density in vitro, but otherwise there was little correlation between tumorigenicity and the in vitro characteristics of the cells. Karyotype analysis was done for two cell lines with high or low tumorigenicity which both had a near-diploid complement of chromosomes. The findings were in agreement with the expression-supression model of Rabinowitz and Sachs (1970). The suppression chromosomes seemed to be confined in group A, the expression chromosomes in group B.

Animals

Oncogenicity of a nude mouse cell line transformed by a human papovavirus.

Primary cultures of NIH nude mouse (nu/nu) kidney cells were transformed with a human papovavirus (MMV). The transformed cell line expressed T-antigen, and MMV DNA was found to be associated with the cell DNA. When NIH nu/nu mice were inoculated with the transformed cells, they developed tumors at the injection site but failed to generate detectable levels of T-antibody. A control group of nu/+ littermates rejected the tumor inoculum but mounted an antibody response to T-antigen. It was proposed that nude mouse cells may be a suitable system to test oncogenicity of in vitro transformed cells.

Animals

Cell-mediated immunity to Theileria-transformed cell lines.

In East and Central Africa the protozoan parasite Theileria parva causes a disease of cattle called East Coast fever (ECF). In Kenya alone between 60,000 and 85,000 cattle die from ECF every year. Infected animals can recover from ECF either naturally or after treatment with tetracyclines or menoctone and are subsequently able to resist challenge with the homologous strain of parasite. That this acquired resistance is due to cell-mediated rather than humoral immunity has been suspected but never decisively shown. A major difficulty in studying immunity to ECF has been the lack of inbred animals for studying Theileria-specific immunity in the absence of allogeneic histocompatibility barriers. We have avoided this problem by measuring cell-mediated immune responses in a syngeneic system in vitro. Unidirectional mixed lymphocyte cultures (MLC) were set up using bovine peripheral blood lymphocytes (PBL) as responder cells and autologous cell lines transformed in vitro by T. parva as stimulator cells. In these cultures, DNA synthesis was induced in PBL from both normal and Theileria-immune animals. However, cytotoxic lymphocytes were induced only in cultures containing responder lymphocytes from Theileria-immune cattle. The results show that Theileria-transformed cells express antigens which are recognized by effector cells and provide evidence that cell-mediated cytotoxic mechanisms function in immunity to ECF.

Animals

The induction of IL-6 and gelatinase B by IL-1 in mouse cell lines transformed with bovine papillomavirus: decreased production in tumorigenic cells.

Six cell lines, that were cloned from murine C127 cells infected by bovine papillomavirus type 1 (BPV1), were found to differ in the degree of transformation in vitro and of tumorigenicity in vivo. In these cell lines the degree of tumorigenicity was inversely correlated with IL-6 induction by IL-1 beta. Whereas the parental C127 cell line produced 15-30 U/ml of IL-6 spontaneously, none of the transformed cell lines produced significant levels of IL-6 constitutively. On induction by human IL-1 beta the parental C127 cell line produced up to 300 U/ml of IL-6, whereas the fully transformed ID14 cell line failed to produce any. The less transformed cell lines produced lower yields of IL-1 beta-induced IL-6, dependent on their degrees of transformation and tumorigenicity. Gelatinase B (96 kDa), a matrix metalloproteinase inducible by IL-1 beta, was dose-dependently regulated in the parental C127 cell line and in the weakly transformed cell line Tlc. These data suggest that transformation processes by BPV1 generally impair IL-1-regulated gene transcription. This impairment seems not to be located at the IL-1 beta receptor level, since in all the cell lines studied the numbers and affinities of the IL-1 beta binding sites were found to be comparable. This impairment seems not to be mediated by transformation-induced inactivation of the protein kinase C pathway since phorbol 12-myristate 13-acetate (PMA) induced IL-6 production equally well in all C127 cell-derived clones. It is suggested that BPV1 transformation can change the expression of host genes that might play a functional role in tumor immune surveillance and tumorigenicity in vivo.

Animals

The preferential cytotoxicity of reovirus for certain transformed cell lines.

The susceptibility of a variety of cell lines of different mammalian origin to cytotoxic (CT) induction by either ultraviolet light-irradiated reovirus type 2 (UVR2) or viable reovirus type 2 plus the protein synthesis inhibitor, cycloheximide, was examined. The following groups of cells were found to be susceptible to CT-induction: certain tumor cells and spontaneously transformed cell lines of human origin and certain virally and spontaneously transformed cell lines of murine origin. The following groups of cells were found to be resistant: normal human diploid cell lines, primary and continuous cell cultures of subhuman primates, primary mouse cells, normal rat kidney cells and baby hamster kidney cells. Susceptibility to CT-induction could not be related to the adsorption of virus to cells, nor to the capacity of the cell to support virus replication.

Animals

In vitro traits of adenovirus-transformed cell lines and their relevance to tumorigenicity in nude mice.

Six independently isolated adenovirus 2-transformed rat cell lines and one adenovirus 5-transformed human cell line have been examined in vitro for serum growth requirements, saturation density, anchorage-independent growth, proteolytic enzyme activity and the presence of LETS glycoprotein and T antigen. This series of adenovirus-transformed cell lines exhibits an oncogeni spectrum ranging from being tumorigenic in immunocompetent rats through to nontumorigenic in adult nude mice. The relevance of the in vitro findings to growth potential in vivo is discussed.

Adenoviruses, Human

Establishment and characterization of a new Epstein-Barr virus transformed cell line from a human B cell lymphoma.

We have established a new cell line from a patient with centrocytic B cell lymphoma. Highly purified peripheral blood B cells from patient DUL (WBC counts 158,000/microliters) were infected in vitro with Epstein-Barr virus (EBV), and CD20+ B cells were cloned into 96 well culture plates with the aid of a cell sorter autoclone device. As shown by GTG-banding and Southern blot analysis, out-growing EBV-positive clones had the same chromosomal abnormalities and identical monoclonal IgH gene rearrangement as the original EBV-genome-negative leukemic B cell clone. Surface marker analysis with a panel of monoclonal antibodies revealed identical patterns on EBV-negative and -positive clones, with the exception of PCA1 (reactive with plasma cells) which was negative on freshly explanted leukemic B cells but positive on EBV-converted clones.

Antigens, Surface

The presence of non-isotype-specific antibodies in polyclonal anti-IgE reagents: demonstration of their binding to specifically selected Epstein-Barr virus-transformed cell lines.

Polyclonal anti-human IgE reagents were earlier shown to contain variable amounts of nonisotype-specific antibodies depending on the strategy used for their preparation. The presence of these antibodies in two commercial anti-IgE reagents was demonstrated in this work by (a) their binding to human Ig-surface-positive lymphoblastoid cells specifically selected by one of the polyclonal anti-human IgE reagents and (b) their binding to the non-IgE immunoglobulins secreted by those lymphoblastoid cells. Peripheral blood B lymphocytes from two normal and two atopic patients were immortalized with Epstein-Barr virus (EBV) and then selected for cells that rosette with anti-IgE-coated erythrocytes. Selection was repeated four times and cells were then cloned. The cloned cells formed rosettes and their supernatants agglutinated erythrocytes coated with rabbit anti-IgE. The immunoglobulins of these clones were positive in an ELISA for IgE, using two different polyclonal anti-human IgE reagents. They were shown, however, to be 19 S IgMs. This discrepancy was due apparently to substantial contamination of anti-non-IgE-isotype-specific antibodies in the polyclonal anti-IgE reagents used both in the selection of cells and in the ELISA. The human monoclonal B-cell lines which were applied here as targets amplified the non-IgE-isotype specific antibody contamination present in the polyclonal anti-human IgE reagents. Because of the normally very low frequency of IgE-positive cells, the use of polyclonal anti-IgE reagents to detect these cells has to be carefully evaluated.

Animals

Concatameric replication of Epstein-Barr virus: structure of the termini in virus-producer and newly transformed cell lines.

The linear form of Epstein-Barr virus (EBV) DNA has homologous direct tandem repeats of approximately 500 bp at each terminus (TR). After infection, EBV DNA circularizes via the TR to form the intracellular episomal DNA. To analyze the mechanism of the synthesis of linear DNA through possible replicative intermediates, the terminal fragments were identified in the total intracellular DNA and the covalently closed circular DNA from a productively infected cell line after induction of replication or after treatment with an inhibitor of viral DNA synthesis. These studies indicate that some of the fused terminal fragments detected in the total intracellular DNA are replication-dependent forms which are selectively excluded from the covalently closed circular fraction and are eliminated after treatment with acyclovir. The EBV terminal restriction enzyme fragments were identified in three producer cell lines, each with a characteristic number of TR in the intracellular episomal DNA. Identification of the termini in cell lines established with the three virus strains revealed that the newly transformed cell lines had a greater number of TR than did the template DNA in the producer cell line. The increase in the number of TR in progeny episomes indicates that linear DNA is produced from concatameric replicative intermediates rather than from amplified catenated circular intermediates.

Cell Line

Anti-tumor surveillance of non-contact-inhibited transformed cell lines.

In order to determine if the correlated expression of transformation and tumorigenicity is affected by the agents used to induce transformants or by the immune status of the host used to test the tumorigenicity of transformants, we derived a series of cloned cell lines from foci of transformed cells induced by treatment of the contact-inhibited mouse cell line B/C-N7.ICI with the DNA demethylating agent 5-azacytidine (5-AZC) or the DNA demethylating and mutating agent benzo(a)pyrene dihydrodiol epoxide (BPDE). The transformed cell lines were injected into syngeneic nude and normal mice to determine their tumorigenicity. The results of this analysis showed that 93% of the transformants induced by 5-AZC treatment grew as tumors when injected into nude mice. Of those lines capable of growing as tumors in nude mice, 86% were also tumorigenic when injected into normal mice. In contrast, only 64% of BPDE-induced transformants grew as tumors in nude mice, and of those, only 44% were also tumorigenic in normal mice. The existence of non-contact-inhibited transformants that are tumorigenic only in nude mice indicates that host anti-tumor immune surveillance mechanisms are operative in normal mice. Further, the difference in both the percentage of transformed cell lines that are tumorigenic and the percentage of tumorigenic transformants that are susceptible to immune surveillance when transformants are induced by BPDE as compared to 5-AZC indicates that the transforming agent can affect both the correlation between the expression of transformation and tumorigenicity, and the interaction between the immune system and tumorigenic transformants.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide