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

Summary 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/µl) were infected in vitro with Epstein-Barr virus (EBV), and CD 20⁺ 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, outgrowing 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 PCA 1 (reactive with plasma cells) which was negative on freshly explanted leukemic B cells but positive on EBV-converted clones.List of abbreviations E sheep erythrocytes - EBV Epstein-Barr virus - FCC-NHL follicular centrocytic non-Hodgkin's Lymphoma - FCS fetal calf serum - LCL lymphoblastoid cell line(s) - mab monoclonal antibody - MNC mononuclear cells - NHL non-Hodgkin's lymphoma This work was supported by the Deutsche Forschungsgemeinschaft (SFB 322/B 2)This work forms part of the Ph. D. thesis of O. Janssen     

Establishment and characterization of an amylase-producing human myeloma cell line

Two stable lines of IgA lambda-producing plasma cells (KHM-1A and KHM- 1B) that were free of the Epstein-Barr virus were established from a patient with multiple myeloma complicated by hyperamylasemia. Surface marker studies of the two cell lines showed that the cells had no surface immunoglobulins but were positive for cytoplasmic immunoglobulins (IgA lambda) and for HLA-DR and PCA-1. Secretion of IgA monoclonal immunoglobulin by the two lines was detected by a plaque- forming cell assay and by an enzyme-linked immunosorbent assay of culture media. KHM-1B cells also secreted alpha-amylase, but no such activity was detected in the culture-conditioned supernatant fluid of KHM-1A.     

Establishment and characterization of rat pancreatic beta cell lines transformed by simian virus 40

Rat pancreatic beta cells were transformed by simian virus 40 (SV40). The established beta cell lines expressed tumor antigen specific to SV40 and retained the ability to produce immunoreactive insulin (IRI) even at a high passage level. One of 12 transformed beta cell lines, SV-PB1205, was further investigated in vitro and in vivo. Firstly, effects of drugs on replication of the pancreatic beta cells were examined in in vitro experiments. Various drugs increased the uptake of 3H-thymidine into the cells. Those included D-glucose, tetragastrin and secretin. However, such effect was not observed for glucagon and growth hormone. Secondly, the SV40-transformed pancreatic beta cells were transplanted into diabetic rats. This produced such improvement of plasma glucose level at least for 2 weeks. The Significance of those experiments was discussed.     

Establishment and characterization of rat pancreatic beta cell lines transformed by simian virus 40

Rat pancreatic beta cells were transformed by simian virus 40 (SV40). The established beta cell lines expressed tumor antigen specific to SV40 and retained the ability to produce immunoreactive insulin (IRI) even at a high passage level. One of 12 transformed beta cell lines, SV-PB1205, was further investigatedin vitro andin vivo. Firstly, effects of drugs on replication of the pancreatic beta cells were examined inin vitro experiments. Various drugs increased the uptake of³H-thymidine into the cells. Those included D-glucose, tetragastrin and secretin. However, such effect was not observed for glucagon and growth hormone. Secondly, the SV40-transformed pancreatic beta cells were transplanted into diabetic rats. This produced such improvement of plasma glucose level at least for 2 weeks. The Significance of those experiments was discussed.     

Epstein-Barr virus RNA VII: size and direction of transcription of virus-specified cytoplasmic RNAs in a transformed cell line

At least three separate regions of the Epstein-Barr virus (EBV) genome encode RNA in a cell line that is growth transformed and nonpermissively infected with EBV. Six polyadenylylated cytoplasmic RNAs have been identified from these three regions. An abundant RNA 3.0-3.1 kilobases (kb) long is encoded by DNA of the internal reiteration, IR, and DNA that maps at 25.7-30 megadaltons. A second, abundant, 2.9-kb RNA is primarily encoded by DNA at 110-03 megadaltons but probably has a 3' end to the left of 110 megadaltons. A third, abundant, 3.7-kb RNA is largely encoded by DNA at 63-66 megadaltons and has a 5' end to the left of 63 megadaltons. A less-abundant 1.5-kb RNA is also encoded by IR. The least-abundant polyadenylylated RNAs identified are 2.3 and 2.0 kb. These RNAs have 3' ends mapping of 5-7 megadaltons and 5' ends mapping to the right of 7 megadaltons. The data suggest that there may be two additional polyadenylylated cytoplasmic RNAs, a 3-kb RNA mapping at 26.2-30 megadaltons and a minor RNA mapping at 102-110 megadaltons. An abundant 0.16-kb nonpolyadenylylated RNA is also present in the cytoplasm of IB-4 cells. This RNA precipitates from the cytoplasm in the presence of high concentrations of magnesium, indicating that it is complexed with protein or polyribosomes.     

Spontaneous Epstein-Barr virus transformed B-cell line sharing the identical immunoglobulin gene rearrangement with acute myeloid leukemia

Mononuclear cells from a 44-year-old patient with acute myeloid leukemia (AML) gave rise to a spontaneous permanent cell line cultured in suspension. The cell line was shown to be positive for Epstein-Barr virus nuclear antigen (EBNA). As expected, its composite phenotype was of B-cell type with B-cell antigens (CD 20, CD 21) and with monoclonal surface IgM of kappa type, but without detectable IgM secretion. Surprisingly, identical monoclonal rearrangements of the immunoglobulin heavy chain (JH) sequences could be demonstrated in the uncultured bone marrow AML cells and in the cell line that also had kappa light chain gene rearrangement. This is the first case to our knowledge of an EBNA positive B-cell line with identical monoclonal Ig heavy chain rearrangement as detected in myeloblastic leukemia cells.     

Establishment and characterization of a mantle cell lymphoma cell line

A mantle cell lymphoma (MCL) cell line (JeKo-1) was established from peripheral blood mononuclear cells of a patient with a large cell variant of MCL showing leukaemic conversion. JeKo-1 cells were Epstein-Barr virus negative and showed a B-cell phenotype with IgM⁺, IgD⁺, CD3^?, CD5⁺, CD10^?, CD19⁺, CD20⁺ and CD23^?; they overexpressed cyclin Dl, Bcl-2, c-Myc and Rb proteins. Bcl-1/J_H gene rearrangement was confirmed by polymerase chain reaction, although karyotypic analysis showed 40/41 chromosomes devoid of apparent t(11;14)(q13;q32) translocation. JeKo-1 cells were highly tumourigenic in SCID mice.     

Establishment and characterization of an opisthorchiasis-associated cholangiocarcinoma cell line （KKU-100）

AM: To establish and characterize a new cholangiocarcinoma cell line from a patient living in the Opisthorchis viverrini (O. viverrini) endemic area of Northeast Thailand. METHODS: Fresh liver biopsy and bile specimens were obtained from a 65-year-old Thai woman with cholangiocarcinoma of the ports hepatis. After digestion, the cells were cultured in Ham's F12 media. The established cell line was then characterized for growth kinetics, cell morphology, imm-unocytochemistry and cytogenetics. Tumorigenicity of the cell line was determined by heterotransplanting in nude mice. RESULTS: The primary tumor was a poorly differentiated tubular adenocarcinoma. Examination of the bile revealed malignant cells with O. viverrin eggs. The cholangiocarcinoma cell line KKU-100 was established 4 mo after the primary culture-population doubling time was 72 h. KKU-100 possesses compact and polygonal-shaped epithelial cells. Immunocytochemically, this cell line exhibited cytokeratin, EMA, CEA, and CA125, but not a-fetoprotein (AFP), CA19-9, desmin, c-met, or p53. Such protein expressions parallel those of the primary tumor. Cytogenetic analysis identified aneuploidy karyotypes with a modal chromosome number of 78 and marked chromosomal structural changes. Inoculation of KKU-100 cells into nude mice produced a transplantable, poorly differentiated aden-ocarcinoma, similar to the original tumor. CONCLUSION: KKU-100 is the first egg-proven, Opisthorchis- associated cholangiocarcinoma cell line, which should prove useful for further investigations of the tumor biology of this cancer.     

Establishment and characterization of an arsenic-sensitive monoblastic leukaemia cell line (SigM5)

Few human monoblastic cell lines have been characterized to date. We have established the SigM5 cell line from a patient with acute monoblastic leukaemia (FAB M5a). Original leukaemic cells had a karyotype of 47,XY,+8, whereas the cell line showed a stemline clone of 81,XX,Y,Y,1,4,6,7,+8,+8,9,10,10,11,13,16,19[cp], with a minor sideline also present. Cytochemical staining was strongly positive with alpha-naphthylbutyrate acetate esterase, particulate positive with Sudan black and weakly positive for myeloperoxidase. Cells were positive for CD13, CD15, CD18, CD23, CD33, CD38, CD45, CD68 and myeloperoxidase. CD14 expression was 3-15%. SigM5 constitutively secreted interleukin (IL)-2, IL-8, IL-10, tumour necrosis factor (TNF)-alpha, ferritin, lysozyme, N-elastase and neopterin upon stimulation with interferon (IFN)-gamma. Cells expressed the proinflammatory mediator macrophage migration inhibitory factor (MIF). All NADPH oxidase subunits were constitutively present, but nitroblue tetrazolium reduction was only detectable upon activation with IFN-gamma. SigM5 monoblasts were sensitive to arsenic trioxide (As2O3) previously not described to induce apoptosis in monoblastic cells. Differing considerably in morphology, immunophenotype and sensitivity to arsenics from the widely used cell lines U937, HL-60 and THP-1, SigM5 is a new monoblastic cell line useful for studying leukaemogenesis, monocyte differentiation and tumour cell susceptibility to arsenic compounds.     

Establishment and characterization of a diethylnitrosamine-initiated woodchuck hepatocyte cell line

Woodchucks free from woodchuck hepatitis virus were treated with diethylnitrosamine in vivo for 2 months, and then hepatocytes obtained by enzymatic perfusion were cultured with the hepatopromoter phenobarbital. This in vivo-in vitro procedure gave rise to proliferating epithelial cell foci, from one of which the presently described hepatocyte cell line (WLC-3) was established and characterized. WLC-3 cells possess morphological and biochemical features of differentiated hepatocytes, including glucose-6-phosphatase activity and albumin production. Histopathological analysis of the tumor which developed transitorily in nude mouse subcutis after inoculation of the cell line revealed glandular structures comprising cells of hepatocellular-like morphology. This is the first established woodchuck hepatocyte cell line free from woodchuck hepatitis virus and is therefore expected to be useful for studying the mode of gene expression and viral proliferation of woodchuck hepatitis virus and the mechanisms underlying woodchuck hepatitis virus-related hepatocarcinogenesis.     

Establishment and characterization of a Kaposi's sarcoma-associated herpesvirus- and Epstein-Barr virus-negative malignant lymphoma cell line (OHK) with primary effusion lymphoma immunophenotype

A novel cell line, designated OHK, was established from ascites of a 59-year-old Japanese woman with diffuse large B-cell lymphoma showing a peculiar serosal tropism, as seen in primary effusion lymphomas (PEL). OHK exhibited a large pleomorphic morphology with irregular nuclei and distinct nucleoli, and included immunoblastic and Reed-Sternberg-like giant cells. On ultrastructural examination, rich intermediate filaments, and well-developed Golgi apparati and rough endoplasmic reticulum, were seen. Immunophenotypically, OHK lacked T and B cell-associated antigens, and had CD10, CD30, CD33 and CD138 antigens. Although OHK cells did not express immunoglobulin (Ig) protein, Southern blot analysis demonstrated clonal rearrangements of Ig heavy and light chain genes. These observations suggest that OHK cells are derived from preterminally differentiated B cells, and that they have features of PEL. Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus were not detected. OHK displayed hyperploid karyotypes with multiple structural abnormalities, and produced some cytokines such as macrophage-colony-stimulating factor (M-CSF), granulocyte-CSF, interleukin 6 and transforming growth factor beta 1. In particular, vascular endothelial growth factor (VEGF), whose stimulation of vascular permeability is thought to be critical to the pathogenesis of PEL, was also produced in large quantities. These results indicate that OHK may be a useful tool for the investigation of PEL.     

HIV-1慢性感染Jurkat细胞系的建立及其特性研究

目的建立艾滋病病毒Ⅰ型（HIV-1）慢性感染Jurkat细胞系,研究其生物学特性。方法参照文献方法改良,建立HIV-1ⅢB慢性感染Jurkat细胞系。其细胞系特性采用以下方法检测：光学显微镜观察细胞形态;流式细胞仪检测HIV-1ⅢB感染细胞的阳性率;噻唑蓝（MTT）法检测抗病毒药物对慢性感染细胞的毒性作用;酶联免疫吸附试验（ELISA）检测药物对慢性感染细胞中病毒复制的影响;合胞体形成方法检测药物对HIV-1ⅢB慢性感染细胞与正常细胞融合的阻断作用。结果成功建立了HIV-1慢性感染Jurkat细胞系（Jurkat/HIV-1ⅢB）,感染细胞阳性率〉90%。检测影响病毒复制各周期的抗病毒药物对Jurkat/HIV-1ⅢB细胞的作用,发现Jurkat/HIV-1ⅢB细胞具有HIV-1慢性感染细胞的生物学特征。结论成功建立Jurkat/HIV-1ⅢB细胞系,为抗HIV-1药物的研发和病毒感染机制研究提供了新的工具。     

Establishment and characterization of human hepatocellular carcinoma cell line FHCC-98

AIM: To establish a novel human hepatocellular carcinoma (HCC) cell line FHCC-98 from HCC tissue and to provide a suitable model for studying HCC occurrence, progress and metastasis.METHODS: Serially passaged cells were cultured and their morphologies were observed under light and electron microscope. Cytogenetic study was conducted by using flow cytometry and chromosome analysis. Expressions of tumor markers such as α-fetoprotein (AFP), cytokeratin (CK) and hepatoma metastasis-associated factor HAb18G/CD147 on the FHCC-98 cells were detected by immunocytochemistry or Western blotting. Lactic dehydrogenase (LDH) isoenzymes were detected by polyacrylamide gel electrophoresis (PAGE).Xenograft was performed by inoculating FHCC-98 cells into the flanks of nude mice.RESULTS: Morphology of FHCC-98 cells was the same as that of other malignant cells. The expressions of the cells were positive for HAb18G/CD147 and CK, and negative for AFP. Its population doubling time was 21.4 h. The cell DNA was tetraploid and the major chromosomes were triploid by cytogenetics analysis. The tumorigenicity in nude mice was 100%. PAGE showed four bands representing LDH2, LDH3,LDH4 and LDH5.CONCLUSION: FHCC-98 is a novel HCC cell line and an ideal cell model for further exploring the mechanism of hepatocellular carcinoma invasion and metastasis.     

Establishment and characterization of a new human eosinophilic leukemia cell line

A human eosinophilic leukemia cell line, designated as EoL, was established from the peripheral blood of a patient with Philadelphia chromosome-negative eosinophilic leukemia (EL). The EoL cell line grows in single cell suspension with a doubling time of 48 hours for about one year. The reactivity of these cells was tested with a panel of monoclonal antibodies; they were found to express surface IA antigen, myeloid antigen (IF10, MY9) and membrane receptors for interleukin 2 (IL-2, Tac antigen). Under standard culture conditions, a small percentage of cells having more typical eosinophilic characteristics was present. These cells had cytoplasmic granules and were positive for Luxol-fast-blue and eosinophil peroxidase. Under culture conditions to induce the maturation of myeloid cells, such as alkaline medium or addition of dimethyl sulfoxide (DMSO), the frequency of cells with typical eosinophilic features increased to about 40%. In addition, cytogenetic studies showed that cultured cells and original leukemic blasts presented similar chromosome abnormalities. EoL seems to be a unique leukemic line committed to the eosinophilic lineage and can provide a useful in vitro model for the study of malignant eosinophilic properties.     

Functional analysis of an Epstein-Barr virus-nontransformed human B cell line

A human B cell line, IW.2, was established from the culture of peripheral blood lymphocytes of a patient with chronic lymphocytic leukemia. IW.2 was shown to express IgM, B1 (CD20), and 12 (a major histocompatibility complex class II antigen) molecules on the cell membrane by analysis of flow microfluorometry (FMF) as well as a receptor for the third component of complement (C3R), but not to express an Epstein-Barr virus nuclear antigen. The cell growth of IW.2 was markedly inhibited when treated with either B cell stimulatory factor from phytohemagglutinin-conditioned medium or 12-O-tetradecanoylphorbol-13-acetate. Interestingly, IW.2 cells were capable of generating a significant amount of both IgM and IgG at the same time when stimulated with these reagents, followed by a decrease in the expression of IgM, B1, and I2 molecules on the cell membrane as well as C3R. The results suggest that IW.2 cells may be a model for the study of the maturation of human B cells.     

Establishment of anti-thyroglobulin antibody-producing cell line by EB virus transformation

We established an anti-thyroglobulin antibody-producing cell line using the peripheral lymphocytes from a patient with chronic thyroiditis. The method was based on preselection by "panning", transformation by EB virus and twice clonings by "limiting dilution". The cells of the cloned cell line (Yo3CTX10) had neither E-receptor nor IgG X Fc-receptor but had C3-receptor as shown by rosetting. We could not detect surface immunoglobulin, but we could detect cytoplasmic immunoglobulin (IgG lambda) with FITC-stainings. This cell line has continuously secreted anti-thyroglobulin antibody for 8 months, which was IgG lambda as shown by immunoelectrophoresis and solid phase radioimmunoassay. The pattern obtained using the purified antibody showed two sharp bands (H, L-chains) in SDS polyacrylamide gel electrophoresis, but it showed six sharp bands in isoelectric focusing electrophoresis. Thus we could establish an oligoclonal cell line producing IgG lambda anti-thyroglobulin antibody.     

Establishment and characterization of human hilar bile duct carcinoma cell line and cell strain

We established and characterized a human hilar bile duct carcinoma (HBDC) cell line from cells isolated from the ascites of a 75-year-old Japanese woman. Histopathological findings were confirmed to be poorly differentiated adenocarcinoma (pat BsrlCm according to the Japanese Society of Biliary Surgery General rules for surgical and pathological studies on cancer of the biliary tract, 4th edn.). Using a semi-agarose method, a daughter-cell strain was also established. Both the cell line and the strain were transplanted into scid or nude mice with a 100% inoculation rate. The population doubling times of the cell line and the strain were 32.25 and 35.78 h, respectively. The cell line and strain strongly expressed human epithelial antigen (HEA)-125 and cytokeratin (CK)-19 but did not express desmin and partly expressed vimentin. High values tumor markers (carbohydrate antigen [CA19-9], Span-1, KMO-1) were detected in culture supernatants from both the cell line and the cell strain and the concentrations paralleled the patient's serum data. DNA analysis revealed that the cell strain was diploid, whereas the cell line was aneuploid, with a DNA index (DI) of 0.85. Chromosomal analysis of the cell line and the strain revealed a range of numerical abnormalities (76–93 and 74–88, respectively) as well as structural abnormalities. The establishment of this HBDC cell line and strain may provide some benefit for fundamental biological research.     

Establishment of two Epstein-Barr virus negative Burkitt cell lines from a patient with AIDS and B-cell lymphoma

To analyze the pathogenesis of B-cell lymphomas in patients with acquired immunodeficiency syndrome (AIDS), we studied two cell lines, Es I and Es III, established from one such lymphoma for the presence of sequences of the Epstein-Barr virus (EBV) and the human immunodeficiency virus [HIV; lymphadenopathy-associated virus (LAV/HTLV- III)] as well as for the presence of cytogenetic abnormalities and monoclonal rearrangements of immunoglobulin and T-cell receptor genes. Both cell lines expressed the same IgM, kappa phenotype as the original lymphoma. The karyotype of Es I was 46, XY, t(8;14), 2 p+, inv (6p), 17p-, and the cells of Es III had an additional i(7q). Immunoglobulin gene studies demonstrated the identical monoclonal rearrangements in both cell lines. Neither EBV nor HIV sequences were detectable in the malignant B cells at the genomic level, leading to the conclusion that mechanisms other than transformation by EBV or HIV may have contributed to the B-cell lymphoma in this patient and possibly also to the generally increased frequency in patients with AIDS.