Establishment of a B-lymphoblastoid cell line infected with Epstein-Barr-related virus from a cynomolgus monkey (Macaca fascicularis)

A B-lymphoblastoid cell line (Ts-B) was established from lymph nodes of an apparently healthy cynomolgus monkey. The cells were demonstrated to contain Epstein-Barr virus-related antigens. Moreover, herpesvirus particles were found in the cells by electron microscopy. The cell-free culture medium transformed lymphocytes of cynomolgus rhesus monkeys, and of Japanese monkeys.     

Establishment of a continuous embryonic cell line from Japanese flounder Paralichthys olivaceus for virus isolation

A continuous cell line, the flounder embryonic cell line (FEC), was established from gastrula-stage embryos of a marine cultured fish, the Japanese flounder Paralichthys olivaceus and cultured for more than 200 d with more than 60 passages. FEC cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with antibiotics, fetal bovine serum (FBS), sea perch serum (SPS), and basic fibroblast growth factor (bFGF). The cells were small and round, and grew actively and stably in culture. The effect of temperature, FBS concentration and bFGF on FEC cell growth was examined. Cells grew well between 24 and 30 degrees C, but had a reduced growth rate below 18 degrees C. The growth rate of FEC cells in medium containing 15% FBS was higher than that in medium containing 7.5% FBS. Addition of bFGF to the medium also significantly increased the growth rate. Chromosome analysis revealed that FEC cells have a normal diploid karyotype with 2n = 48. High survival rate was obtained after cryopreservation of cell cultures. The susceptibility of the cell line to piscine viruses was examined. Two viruses tested were shown to induce CPE (cytopathic effect) on FEC cells. FEC cell culture infected with fish iridovirus was further elucidated by electron microscopy. Many virus particles were found in the cytoplasm of the virus-infected FEC cells. These results indicated that the FEC cell line could be potentially used to isolate and study fish viruses.     

Establishment and properties of a human choriocarcinoma cell line of ovarian origin

Summary A human nongestational choriocarcinoma cell line of ovarian origin (IMa) was established in vitro. This cell line had been subcultured serially more than 22 times over 18 months. Small polygonal cells with a prominent nucleus were dominant and a sparsity of cytoplasmic organelles was an ultrastructural characteristic of the IMa cells. The production and secretion of human chorionic gonadotropin and its subunits were identified by radioimmunoassay. The IMa cells were transplantable in the hamster cheek pouch and the histological diagnosis was choriocarcinoma. A newly established ovarian choriocarcinoma cell line can be considered useful for clarifying the biological differences between nongestational and gestational choriocarcinoma cells. This research was supported in part by a Grant-in-Aid for Cancer Research from both the Ministry of Health and Welfare and the Ministry of Education, Science, and Culture of Japan.     

Epstein-Barr virus DNA recombines via latent origin of replication with the human genome in the lymphoblastoid cell line RGN1.

We show here that in a lymphoblastoid cell line Epstein-Barr virus DNA recombines with the human genome. The genetic exchange involves the oriP region of the virus. A junction between viral and human DNA from this line has been cloned and sequenced. The results indicate that the integration of Epstein-Barr virus DNA involves a region of the human genome which contains internal short repetition. An 800-bp probe has been isolated from the human part of the junction. This probe has been used to show that the human region exists as a duplication in normal cells.     

Establishment and characterization of an Epstein-Barr virus nuclear antigen-negative hairy cell line

Continuous cell lines have been established from spleen cells of patients with confirmed hairy cell leukemia (HCL). One cell line, HCL-Z1, lacks Epstein-Barr virus nuclear antigen (EBNA), grows attached to the substratum and retains typical features of hairy cells as revealed by transmission and scanning electron microscopy. HCL-Z1 differs morphologically from the three other EBNA-positive lymphoblastoid cell lines obtained (HCL-Z2, HCL-Z3, HCL-Z4) as well as from normal spleen cells or lymphocytes. The three lymphoblastoid cell lines derived from HCL patients show similar surface features as a line from a myeloma patient. Therefore, not all cell lines derived from HCL patients may be considered as representative of the patients leukemia cells.     

Marmoset lymphoblastoid cells as a sensitive host for isolation of measles virus.

B95-8, an Epstein-Barr virus-transformed marmoset B-lymphoblastoid cell line, and its derivative B95a, capable of attachment to a substrate surface, were 10,000-fold more sensitive to measles virus present in clinical specimens than were Vero cells. B95-8 and B95a cells were thus thought to be useful host cells for the isolation of measles virus. Quantitation of measles virus present in clinical specimens showed that a large quantity of virus, exceeding 10(6) 50% tissue culture infective doses per ml of a nasal-swab eluate, is shed into secretions by patients with acute measles, consistent with the contagiousness of the disease. Measles viruses isolated in B95a cells differed in some biological properties from those adapted to Vero cells. First, the viruses isolated in B95a cells did replicate in Vero cells, but release into the fluid phase was less efficient than that of Vero cell-adapted viruses. Second, minor antigenic differences were found between virus strains isolated in B95a cells and those isolated in Vero cells from the same clinical specimens. Third, the viruses isolated and propagated in B95a cells caused clinical signs in experimentally infected monkeys resembling those of human measles. It was suspected that measles virus is subject to host cell-mediated selection and that the viruses grown in B95a cells are more representative of measles virus circulating among humans than are the viruses selected in Vero cells.     

Establishment and characterization of Macaca fascicularis lymphoblastoid cell lines.

A panel of cynomolgus macaque lymphoblastoid cell lines (LCL) was established by transforming peripheral blood mononuclear cells (PBMC) with Herpesvirus papio (HVP), and selected lines were examined by flow cytometry. Results indicate that HVP-transformed macaque LCL are phenotypically heterogeneous and resemble human Epstein-Barr virus (EBV)-transformed LCL in the abundant expression of major histocompatibility complex (MHC) class I and class II molecules. At least some lines are of B cell origin.     

Mutagenic radioadaptation in a human lymphoblastoid cell line

We investigated the mutagenic radioadaptive response of human lymphoblastoid TK6 cells by pretreating them with a low dose (5 cGy) of X-rays followed by a high (2 Gy) dose 6h later. Pretreatment reduced the 2-Gy-induced mutation frequency (MF) of the thymidine kinase (TK) gene (18.3 x 10(-6)) to 62% of the original level (11.4 x 10(-6)). A loss of heterozygosity (LOH) detection analysis applied to the isolated TK(-) mutants revealed the mutational events as non-LOH (resulting mostly from a point mutation in the TK gene), hemizygous LOH (resulting from a chromosomal deletion), or homozygous LOH (resulting from homologous recombination (HR) between chromosomes). For non-LOH events, pretreatment decreased the frequency to 27% of the original level (from 7.1 x 10(-6) to 1.9 x 10(-6)). cDNAs prepared from the non-LOH mutants revealed that the decrease was due mainly to the repression of base substitutions. The frequency of hemizygous LOH events, however, was not significantly altered by pretreatment. Mapping analysis of chromosome 17 demonstrated that the distribution and the extent of hemizygous LOH events were also not significantly influenced by pretreatment. For homozygous LOH events, pretreatment reduced the frequency to 61% of the original level (from 5.1 x 10(-6) to 3.1 x 10(-6)), reflecting an enhancement in HR repair of DNA double-strand breaks. Our findings suggest that the radioadaptive response in TK6 cells follows mainly from mutations at the base-sequence level, not the chromosome level.     

Characterization of a human lymphoblastoid cell line permanently modified by simian foamy virus type 10

Simian Spumavirinae serotype, SFV10, of a Papio cynocephalus baboon, was used to infect a human lymphoblastoid cell line, LV2. Permanent growth and morphological alterations of infected cells occurred, even though no viral particles were detected. Evidence for the presence of viral genomes in the modified cell line is provided indirectly from immunological studies and induction experiments followed by coculture procedures. The permanently modified cell line obtained (LV2-FB10) is an interesting model for the investigation of the possible integration of foamy viruses into the host genomes.     

Establishment of a Vero cell line persistently infected with African swine fever virus.

A Vero cell line persistently infected with African swine fever virus was established by infecting the cells in the presence of 10 mM NH4Cl (Vero-P cell line). The virus derived from the Vero-P cultures infected Vero cells, and virus titers were comparable to those obtained in Vero cells acutely infected with African swine fever virus. The structural proteins of the virus from Vero-P cells were similar to those of the virus produced in lytic infections. Virus production was low when the Vero-P cells were growing logarithmically and increased considerably in confluent cultures when lysis appeared in a fraction of the cell population.     

Establishment and characterization of an abrin-resistant cell line

An abrin-resistant cell line, CHOR 3-4, was isolated from CHOK1 cells which were resistant to a high concentration of abrin (160 ng/ml), and had a 1000-fold higher resistance to abrin that that of CHOK1 cells. CHOR 3-4 cells were about 25-fold more resistant than CHOK1 cells to the N-glycosidase activity of abrin, which was measured by hydrolyzing the N-glycosidic bond of adenine 4324 nucleotide from 3′ end of mammalian 28S rRNA. However, the isolated polysomes of CHOR 3-4 cells had the same sensitivity to abrin as those of CHOK1 cells. On measuring the binding of 125I-abrin to CHOR 3-4 cells, it was decreased to about 20% that of CHOK1 cells. This indicates that the mechanism of the resistance of CHOR 3-4 to abrin is due to the alteration of glycoproteins or glycolipids of cell membrane.     

Xenobiotic metabolism and mutation in a human lymphoblastoid cell line

Aryl hydrocarbon hydroxylase-1 (AHH-1) cells are a human lymphoblastoid cell line competent in some aspects of xenobiotic metabolism. This cell line contains stable mixed function oxidase activity which is inducible by polycyclic aromatic hydrocarbons (PAHs) but not by phenobarbital or Arochlor 1254. Two substrates for the cellular mixed function oxidase activity, benzo[a]pyrene (B[a]P) and 7-ethoxyresorufin, have been examined. The basal and induced activities have different kinetic parameters toward these two substrates. In contrast, basal and induced activities had similar sensitivities to two cytochrome P-450 suicide substrates. B[a]P metabolism and mutagenicity were studied in this cell line. AHH-1 cells were found to produce predominantly B[a]P phenols and quinones. The major phenol metabolite cochromatographed with authentic 9-hydroxy B[a]P. AHH-1 cells were capable of forming glucuronic acid conjugates of B[a]P phenols; the major product after hydrolysis cochromatographed with 3-hydroxy B[a]P standard. AHH-1 cells did not contain detectable epoxide hydrolase activity using B[a]P-4,5-oxide as substrate. This observation is consistent with the absence of trans-dihydrodiol B[a]P metabolites in the metabolic profile. B[a]P-induced mutagenicity at the hypoxanthine guanine phosphoribosyl transferase (hgprt) locus in AHH-1 cells was found to be linearly related to phenol production during treatment and inhibited by alpha-naphthoflavone (ANF).     

Herpesvirus saimiri-induced lymphoblastoid rabbit cell line: growth characteristics, virus persistence, and oncogenic properties.

A nonproducer lymphoblastoid cell line (7710) containing the herpesvirus saimiri (HVS) genome was established from the HVS-positive spleen of a male, inbred New Zealand White rabbit (III/J strain) which had developed a well-differentiated lymphoma after inoculation of HVS and 12-O-tetradecanoylphorbol-13-acetate (TPA). Antibodies to HVS early and late antigens were detected in the serum of rabbit 7710 by indirect immunofluorescence and immunoprecipitation. The cell line was of T-cell origin, did not produce HVS, and could not be superinfected with HVS. However, HVS early antigens could be induced in the cells with n-butyric acid and TPA or TPA alone. On the other hand, late antigens were never observed, and infectious virus could not be rescued by cocultivation of 7710 cell with OMK cells. The 7710 cells were T-cell growth factor dependent, even after many in vitro passages. The 7710 cell line contained multiple copies of a nonintegrated, covalently closed circular HVS genome. As is characteristic of some other HVS-transformed nonproducer lymphoid cell lines, a large segment of unique light (L) DNA was missing in the persistent circular viral DNA present in 7710 cells. This deletion spanned at least 42.5 kilobases, corresponding to the segment between 12.3 and 50.7 map units of full-length, infectious virion L-DNA. The 7710 cells failed to induce tumors in athymic nude mice, but inbred rabbits inoculated with as few as 100 of these cells developed fatal lymphomas. Chromosomal analysis showed that tumors were due to the growth of donor cells. Cells recovered from one of the rabbits inoculated with 7710 cells also contained HVS DNA and, after in vitro culture, induced the same type of lymphoma when inoculated into two other III/J-strain rabbits. None of the previously described HVS-transformed cell lines have been able to induce tumors in either their host species or nude mice. Thus, our demonstration that the 7710 cell line is readily transplantable in syngeneic rabbits represents the first available model which allows analysis of many biological and molecular aspects of the in vivo oncogenicity of HVS.     

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.     

Establishment and characterization of an immortalized epicardial cell line

Recently, the increasing significance of the epicardium in cardiac development and regeneration is beginning to be recognized. However, because of the small proportion of primary epicardial cells and the limited cell culture time, further research on the mechanism of epicardial cells is hindered. Here, we transfected simian virus 40 Large T (SV40-LT) into primary epicardial cells to establish an immortalized cell line, named EpiSV40. We further demonstrated that EpiSV40 can be easy to culture and has the proliferation, migration and differentiation capacities comparable to primary epicardial cells. EpiSV40 can serve as an ideal in vitro model for epicardial cell research, which will booster the study of the epicardium in cardiac development and heart regeneration.