Characterization of lymphoma-derived cell lines: comparison of cell lines positive and negative for Epstein-Barr virus nuclear antigen. II. Surface markers

Fifteen of 16 lymphoma-derived cell lines and the Faji and P3HR1 cell lines were characterized with regard to certain surface markers, particularly immunoglobulins, complement receptors, Epstein-Barr virus (EBV) receptors, and Fc receptors. Ten lines positive for EBV nuclear antigen (EB-pos) were stained weakly or not at all by antihuman immunoglobulin fluorescein isothiocyanate conjugates, whereas EBV nuclear antigen negative (EB-neg) cell lines stained brightly. EB-pos lines frequently manifested Fc receptors, particularly for 7S antibody, whereas EB-neg lines did not. Receptors for the C3b component of complement and for EBV, which correlated significantly with each other, were expressed to a much lesser extent by EB-neg lines than by EB-pos lines. These findings are pertinent to an understanding of the infrequent association of this virus with American undifferentiated lymphomas of the Burkitt's and non-Burkitt's types.     

Characterization and comparison of two newly established Epstein-Barr virus-negative lymphoma B-cell lines. Surface markers, growth characteristics, cytogenetics, and transplantability

Two Epstein-Barr virus (EBV)-negative lymphoma B-cell lines, HBL-1 and HBL-2, were established from a pleural effusion and a lymph node biopsy of two patients with diffuse large cell lymphoma. HBL-1 and HBL-2 showed the characteristics of activated B-cells in B-cell lineage, as did original lymphoma cells. Chromosome analyses revealed that HBL-1 exhibiting 14q+ marker-positive lymphoid cancer showed a new subclass of 14q32 translocation resulting from a translocation between chromosomes 14 and 16, which had been masked in a complex translocation involving five chromosomes, and that HBL-2 had a 14q+ marker chromosome, the result of an 11;14 translocation [t(11;14)(q13;32)]. Successful heterotransplantation into athymic nude mice demonstrated tumorigenicity of HBL-1 and HBL-2. The transplantability and tumor growth rate of HBL-2 were higher and more rapid than those of HBL-1. HBL-1 and HBL-2 appear useful for facilitating therapeutic investigations as well as immunologic and oncogenic studies in B-cell lymphomas.     

Studies of Epstein-Barr virus (ebv)-associated nuclear antigen. I. assay in human lymphoblastoid cell lines by direct and indirect determination of 125I-IgG binding.

A quantitative assay for EBNA in human lymphoblastiod cell lines has been developed. The assay employs EBNA-positive and -negative 125-I-IgG preparations as reagents and can be used in a direct or indirect manner. EBNA specificity has been demonstrated in a number of ways. The antigenic relationship between EBNA present in Raji cells and in a number of other human lymphoblastiod cell lines of diverse origins and between the cell-associated antigen present in FT Raji cells and that present in isolated Raji nuclei has been studied. The possibility of carrying out blocking titrations for anti-EBNA determination has been demonstrated and the effect of glutaraldehyde and formaldehyde upon antigenic activity in FT Raji cells has been studied.     

Phenotypic and cytogenetic characteristics of a new Epstein-Barr virus negative cell line (SKW 4) derived from a B-cell lymphoma

A new Epstein-Barr virus nuclear antigen (EBNA) negative cell line SKW 4 has been established in vitro from a patient with diffuse histiocytic lymphoma. The SKW 4 seems to be an authentic human tumour cell line as evidenced by its EBV negativity, monoclonality and aneuploidy tested during early in vitro passage. The cell line expresses surface mu and kappa-chains, HLA-DR antigen, C3 and Fc receptors and B-cell lineage antigens. The karyotypic analyses demonstrated many numerical and structural aberrations. No Burkitt lymphoma associated translocations (t8;14, t2;8, t;22) were detected, but most of the markers found are those commonly associated with various types of human cancer. The SKW 4 thus represents the most common type of 'histiocytic lymphoma', that with a B-lymphoid cell phenotype, but is unique among HL derived lymphoma lines in its strong expression of a Helix pomatia A agglutinin binding surface glycoprotein of an apparent molecular weight of 75 000 daltons.     

Epstein-Barr virus strain-specific differences in transformed cell lines demonstrated in growth characteristics, induction of viral antigens and ADCC susceptibility

Paired lymphoid cell lines were established by transformation with both B95-8 (B) and QIMR-WIL (Q) EBV strains, of cells either from cord blood or from peripheral blood or spleens of patients with leukemias or lymphomas. The morphologies of the transformed cell clumps differed consistently between B-transformed lines (B-lines) and Q-transformed lines (Q-lines) even after 1 year in culture. When the B- and Q-lines were compared for superinfection with P3HR-1 EBV, B-lines had a higher frequency of EA induction than did the Q-lines. The shape of dose-response curves for superinfection indicated that a much higher multiplicity of infection by P3HR-1 EBV was required for EA expression in Q-lines than in B-lines. This difference in superinfection was independent of the EBV receptor concentration on the two types of lines and reflected apparent control of the level of EA induction by the resident EBV genome of the transformed line. Transforming EBV could be rescued by P3HR-1 EBV superinfection of both B- and Q-lines originating from cells of patients with Hodgkin's disease and hairy cell leukemia but not from cord blood. The cord blood lines transformed with virus produced spontaneously from these B- or Q-lines, showed that the cell lines contained antigen-determining information of the resident genome in the original B- or Q-lines, respectively. Superinfected B-lines were more susceptible to ADCC with anti-EBV-positive sera than were superinfected Q-lines. These experiments demonstrate that distinct biology. differences exist in paired cell lines transformed by different EBV strains.     

False negative and prozone reactions in tests for antibodies to Epstein-Barr virus-associated nuclear antigen

Titration of anti-complementary human sera may yield false negative or prozone reactions in anti-complement immunofluorescence (ACIF) tests for antibodies to Epstein-Barr virus-associated nuclear antigen (EBNA) when the human complement (C') is added to the serum dilutions prior to charging of the target cell smears (two-stage ACIF test). This effect of anti-complementary sera and with it a source of error is avoided by using a three-stage ACIF technique; that is, when the target cell smears are successively overlayed with serum dilutions, C' and fluorescein isothiocyanate-conjugated antibodies to human βC/βA globulins. The two- and three-stage ACIF procedures yield comparable anti-EBNA titers with non-anti-complementary sera.     

Epstein-Barr virus integration in human lymphomas and lymphoid cell lines.

BACKGROUND. Epstein-Barr virus (EBV) is maintained as an episome in most infected cells. The presence of fused terminal restriction enzyme fragments distinguishes the circular DNA form from the linear virion form. METHODS. EBV genomic structure was analyzed in 8 lymphoid cell lines and 21 human lymphoma specimens by the Southern blot technique. RESULTS. Evidence of viral integration into host chromosomal DNA was identified in four cell lines. In the Namalwa and BL30-B95.8 cell lines, integration occurred through the terminal repeat (TR) sequences. In the BL41-P3HR1 and BL41-B95.8 cell lines, there was loss of left-end viral genomic sequences, including ori-P sequences required for episome maintenance, implying that integration was required for viral genome persistence. Integration was not detected in four other cell lines (Raji, Daudi, B95.8, and BL30-P3HR1). In 21 EBV-containing human lymphomas, including 18 immunodeficiency-related lymphomas, fused TR sequences were identified without evidence of viral genomic integration. CONCLUSIONS. These findings suggest that, although viral integration is common in Burkitt lymphoma cell lines infected in vitro, integration is not common in human lymphomas that develop in vivo in normal or immunodeficient people.     

Lymphoblastoid cell lines and burkitt-lymphoma-derivee cell lines differ in the expression of a second epstein-barr virus encoded nuclear antigen

Twenty-seven Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines and 27 EBV-carrying Bur-kitt-lymphoma-derived lines were analyzed for expression of the second EBV-encoded nuclear antigen (EBNA-2) by immunoblotting and anticomple-ment immunofluorescence with EBNA-2-specific sera. While all lymphoblastoid cell lines expressed EBNA-2, only 10 of the 27 BL lines were EBNA-2-positive. Comparison of the EBNA-2 coding Bam HI W-Y- and H-fragments of EBV-DNA in the different cell lines by restriction enzyme analysis suggests that EBNA-2 negativity is due either to sequence diversity or to a deletion in the BamHI WYH region.     

Effect of nickel sulfate on cellular proliferation and Epstein-Barr virus antigen expression in lymphoblastoid cell lines

Nickel is found in high levels in the environment of the high-risk areas for nasopharyngeal carcinoma (NPC) of China. Epstein-Barr virus (EBV) is associated with NPC and the interaction of nickel and EBV may be a contributive cofactor to the development of NPC. The study of the in vitro effect of nickel sulfate on cell proliferation and EBV-antigen expression demonstrated that nickel increases cell proliferation of some EBV-positive lymphoblastoid cell lines and increases early antigen expression of Raji cells. Nickel exerted variable effects on viral capsid antigen (VCA): increasing VCA-positive cells in B95-8 cells while decreasing VCA in P3HR-1 cells. It is proposed that the uptake of nickel in NPC high-risk areas could be one of the factors responsible for cancer development in the nasopharynx in China.     

Detection of Epstein-Barr virus (EBV)-associated nuclear antigen in human lymphoblastoid cell lines by means of an 125-I-IgG-binding assay

The elution at low pH of human EBV-positive ¹²⁵I-IgG bound to preparations of EBV-carrying non-producer lymphoblastoid cell lines has been investigated. Specific binding was demonstrated. A number of approaches were used to confirm the specificity. The stoichiometry of binding and elution was studied. The sub-cellular location of the EBV-associated antigen (s) detected by this method was shown to be predominantly nuclear. This confirms other studies demonstrating the presence of a nuclear antigen in non-producer lymphoblastoid cell lines carrying the EBV genome.     

The status of the p53 gene in human papilloma virus positive or negative cervical carcinoma cell lines.

We have analyzed p53 gene alterations in five cervical cancer derived cell lines. Two of the five cervical cancer cell lines, HTB31 (C-33A) and 32 (HT-3), harbored missense mutations in codons 273 and 245 respectively, whereas the other three tumor cell lines, HTB33 (ME180), 34 (MS751) and 35 (SIHA), did not reveal any mutation in the p53 coding sequence spanning codons 126-307. Although all the tumor cell lines express comparable levels of p53 RNA, only HTB31 and HTB32 contain high or detectable levels respectively of p53 protein. The other three tumor cell lines, where neither p53 mutation nor the expression of p53 protein could be detected, were found to harbor human papilloma virus (HPV) 16 or 18. The inactivation of the wild-type p53 function resulting from a missense mutation, or the lack of detectable wild-type p53 protein due to the translational/post-translational deregulation of p53 protein levels may be the contributing factor in the tumorigenicity of these five cases of cervical cancer. The lack of detectable p53 protein in HTB33, 34 and 35 associates with the presence of either HPV16 or -18 in these cell lines.     

Inhibition of estrogen receptor positive and negative breast cancer cell lines with a growth hormone-releasing hormone antagonist

GHRH antagonists have been shown to inhibit growth of various human cancer cell lines xenografted into nude mice including estrogen receptor negative human breast cancers. Previous observations also suggest that GHRH locally produced in diverse neoplasms including breast cancer might directly affect proliferation of tumor cells. In the present study we demonstrate that a novel highly potent GHRH antagonist JMR-132 strongly inhibits the proliferation of both estrogen receptor negative SKBR 3 and estrogen receptor positive ZR 75 human breast cancer cell lines in vitro. The proliferation in vitro of ZR 75 and SKBR 3 was increased after direct stimulation with GHRH(1-29)NH2. The GHRH antagonist JMR-132 had a significant antiproliferative activity in the absence of GHRH and nullified the proliferative effect of GHRH in these cell lines. SKBR 3 and ZR 75 expressed the GHRH ligand as well as the pituitary type of GHRH-receptor, which likely appears to mediate the antiproliferative mechanisms in these cell lines. These in vitro results suggest that JMR-132 is a potent inhibitor of breast cancer growth, independent of the estrogen receptor status. Further investigations on the combination treatment with endocrine agents affecting the estrogen pathway and GRHR antagonists are needed in order to improve the treatment of breast cancer.     

Procoagulant activity of human neuroblastoma cell lines, in relation to cell growth, differentiation and cytogenetic abnormalities

Procoagulant activity (PCA) was investigated in relation to cell growth, differentiation, and cytogenetics in seven human neuroblastoma cell lines. Before 5-bromodeoxyuridine (BrdUrd) treatment, PCA was notably heterogeneous, with the highest activity in NCG (S-type in morphology) 40- to 100-fold greater than the lowest activity in SK-N-DZ (N-type). PCA was not related to 1p abnormalities. After BrdUrd treatment at 5 micrograms/ml for 6 days, PCA increased 6.8-fold in GOTO and 2.7-fold in SK-N-DZ with associated growth inhibition and morphological changes (I-type morphology converted to S-type in GOTO and N-type converted to an advanced N-type in SK-N-DZ). In contrast, only growth suppression was observed in 2 other cell lines, and no changes in PCA, growth or morphology were induced in the remaining 3 cell lines.     

Cellular localization of an Epstein-Barr virus (EBV)-associated complement-fixing antigen in producer and non-producer lymphoblastoid cell lines

Anti-complement immunofluorescence (ACIF) was used to study the complementfixing antigens of human lymphoblastoid cell lines. These cell lines carry the Epstein-Barr virus (EBV) genome although only producer cultures synthetize EBV-specific antigens (virus capsid antigen, VCA and early antigen, EA) detectable by direct and indirect immunofluorescence, usually in less than 5% of the cells. The ACIF test revealed an antigen localized in the nucleus of the lymphoblastoid cells. In contrast to EA and VCA, this antigen was present in over 90% of the cells of both producer and non-producer cultures. The antigen was shown to be specific for EBV by comparing the reactions of 52 sera in the ACIF test. Sera giving the nuclear reaction contained antibodies to VCA, EA or antigens detectable by complement fixation tests on cell extracts, but sera without EBV antibodies failed to give the reaction. Weak, equivocal or discordant reactions occurred with six sera with low titres in VCA, EA or complement fixation tests. Cell lines derived by transformation of human and primate lymphocytes by EBV gave the nuclear reaction. Control cells with no known association with EBV were non-reactive. These included foetal lymphocytes transformed by phytohaemagglutinin, cell lines derived from breast cancer, glioma, normal glia, pleuritis maligna and myeloma, and two marmoset lymphoid lines carrying Herpesvirus saimiri (HVS). In preliminary experiments, the ACIF test was used as a tool to trace the EBV genome at the cellular level. Cells from two Burkitt lymphoma biopsies, one tested after biopsy and one after passaging in nude mice, contained an EBV-specific antigen. Three clones of cells derived from hybrids of mouse somatic cells and a human lymphoblastoid cell line also contained such an antigen, but the number of reactive cells varied from clone to clone. A fourth clone was non-reactive.     

Characterization of lymphoid cell lines established from multiple Epstein-Barr virus (EBV)-induced lymphomas in a cotton-topped marmoset.

Inoculation of a cotton-topped marmoset with cell-free EBV, strain B95-8, resulted in the induction of lymphomas in liver, spleen, and mesenteric lymph nodes. Continous suspension cell lines were established from tumor tissue from each of these three sites. Cells of each line were positive for intracellular EBV antigens and EB nuclear antigen and released low levels of transforming virus. Early after initial culturing each line had a high frequency of cells (84-85%) with receptors for activated complement but these receptors were no longer evident on retesting several months later. All three lines initially showed high levels of cells which stained for surface lambda light chain and gamma and mu heavy chains. Cells of all three lines showed a decrease in gamma chain staining and the lymph-node tumor line showed reductions in surface staining for mu and lambda chains as well after prolonged cultivation. Clonal cells of the three lines resembled their respective parental lines in surface markers. Cytogenetically, all the lines were hypodiploid, 2n = 45, and several karyotypes from all three lines showed the loss of a medium-sized metacentric chromosome. A marker chromosome with an extra band in the centromeric region was apparent in the lymph-node tumor line. These results indicate that cells of individual tumors in a cotton-topped marmoset with multiple EBV-induced lymphomas are similar to each other in a number of characteristics although a certain degree of divergence was observed. Thus, it is possible that EBV-induced lymphomas in cotton-topped marmosets may have a common cell origin as is the case with Burkitt lymphoma in humans.     

Childhood sarcomas and lymphomas. Characterization of new cell lines and search for type-C virus.

Four cell lines were derived from childhood malignancies: rhabdomyosarcoma, sarcoma, lymphosarcoma and an American Burkitt's lymphoma. Cells of the four lines formed tumors in immunosuppressed newborn hamsters. The tumors had a microscopic appearance like that of the tumors from which the cell lines were derived. No virus-like particles were detected by electron microscopy in the cell lines after treatment with bromodeoxyuridine; no hamster type-C virus expression was present in cell lines derived from the hamster tumors. Chromosome constitution and in vitro growth properties of the cell lines were studied as was the fibrinolytic function of the sarcoma lines.     

Cell-density-dependence for growth in agarose of two human lymphoma lines and its decrease after Epstein-Barr virus conversion.

We have compared the growth in agarose medium of two EBV-negative Burkitt lymphoma lines, BJAB and Ramos, and that of their EBV-converted derivatives. Optimal conditions for growth in agarose medium are described. The original EBV-negative lines can grow in agarose to a high efficiency, provided a critical level of cell concentration is attained. Below this level, there is no growth at all. EBV-converted sublines form colonies at a much lower cell concentration, and their maximal plating efficiency is higher. These differences may be related to a super-transformation state induced in these cells by EBV.     

Heterogeneity of Epstein-Barr virus. IV. Induction of a specific antigen by EBV from two transformed marmoset cell lines in Ramos cells.

Infection of cells of the EBV-genome-negative human B-lymphoma Ramos line with viral isolates obtained from two EBV-transformed marmoset cell lines (B95/8; Nyevu) resulted in the induction of a nuclear antigen (RAM-ag) apparently different from other EBV-associated antigen complexes. This antigen is revealed by indirect immunofluorescence and shows no detectable cross-antigenicity with EBNA or any other known EBV-associated antigen. EBV-isolates from P3HR-1 cells fail to induce a similar antigen in Ramos cells although they induce EBNA. No RAM-ag was expressed, either after infection of cells of another EBV-genome-negative human B-lymphoma line BJAB with B95-8 EBV or in a series of EBV-harbouring cell lines. Thus the antigen appears to be cell-line-specific for Ramos cells. It is also induced upon infection of either B95-8 or P3HR-1 converted Ramos sublines with EBV from B95-8 cells. All human sera with RAM-ag-reactivity revealed antibodies against VCA. However, sera from patients with acute infectious mononucleosis containing high anti-VCA-antibodies did not react with RAM-ag. Seroconversion for this antigen apparently more closely coincides with the appearance of EBNA-directed antibodies.     

Purification and characterization of the Epstein-Barr virus nuclear antigen 2 using monoclonal antipeptide antibodies

The Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) is the only one of the EBNA proteins to have been implicated as an EBV-encoded transforming protein. More detailed studies of this protein have been hampered by the lack of EBNA-2-specific monoclonal antibodies (MAbs) and of purified protein. To overcome these problems, we isolated 5 hybridomas producing MAbs reactive with an 18 residue synthetic peptide corresponding to the carboxyterminus of EBNA-2. Four of the 5 MAbs were specifically reactive with EBNA-2 in its denatured form on immunoblots. The 5th antibody (115E) was reactive with the native form of EBNA-2. By using a one-step immunoaffinity purification method with 115E cross-linked to protein-A-Sepharose, we purified EBNA-2 to homogeneity, i.e., more than 1,200-fold, from Burkitt lymphoma cell extracts. A major 32-kDa associated protein and a less abundant 17-kDa protein were co-purified with EBNA-2. Immunoprecipitation with 115E from 35S-methionine-labelled cell extracts showed that the 32-kDa protein co-precipitated with EBNA-2 from EBV-positive cells, but was not detectable in immunoprecipitates of EBV-negative cells. When the immunoprecipitates or the purified proteins were immunoblotted with EBV-immune sera, only EBNA-2 was reactive, indicating that the associated proteins are of cellular origin. Immunoprecipitation of cells labelled with 32P-orthophosphate showed that EBNA-2, but not the associated proteins, is a phosphoprotein. The expression level of EBNA-2 varied between different EBV-carrying cell lines, as measured by a 2-site ELISA based on antibody 115E. In indirect immunofluorescence, the 115E MAb gave an EBNA-2-specific characteristic granular staining pattern. These characteristics of EBNA-2 resemble those of other viral transforming proteins.