Hybridization between a human epithelial line, infectable by Epstein-Barr virus, and Burkitt lymphoma lines: membrane properties, superinfectability, inducibility and tumorigenicity

The human epithelial line U, which is partially infectable with EBV, was hybridized with the EBV-genome carrying Burkitt lymphoma lines P3HR-1 and Daudi. Authenticity of the hybrids U-Put and U-Dut was established by isoenzyme studies. Although the two hybrids carried the EBV genome derived from the lymphoma parent, being 100% positive for Epstein-Barr-virus-associated nuclear antigen (EBNA), they resembled the U parent in many respects: they were deficient for membrane immunoglobulins and Fc receptors, and had a lower concentration of EBV-C3 receptors than either parent. Unlike the P3HR-1 parent, U-Put hybrid was nonpermissive for both the EBV cycle antigens, early antigen (EA) and viral capsid antigen (VCA). The inducing agent 12-O-tetra-decanoyl-phorbol-13-acetate (TPA) caused distinct viral early antigen synthesis (EA) in U-Put, lower, however, than that of the parental P3HR-1. U-Dut was completely nonpermissive and noninducible for early and viral capsid antigens. Thus, even an epithelial parent infectable by EBV restricted, although not completely, expression of EBV antigens, with the exception of EBNA. It has been suggested that EBNA is an autonomous function of the viral genome, independent of host cell control; the latter regulates expression of antigens related to viral cycle. The hybrids U-Put and U-Dut resembled the U parent also in regard to growth in soft agar and tumorigenicity in nude mice, although in this respect the lymphoma parental properties were not completely eclipsed.     

EBV-transformed lymphoblastoid cell lines down-regulate ebna in parallel with secretory differentiation

Four monoclonal and one polyclonal lymphoblastoid cell line (LCL) were studied with regard to cytoplasmic immunoglobulin (clg) expression, presence of the Epstein-Barr virus (EBV)-determined nuclear antigen (EBNA) and DNA synthesis. Each line was found to consist of two subpopulations, with only minimal overlap. Proliferating, EBNA-positive, clg-negative cells formed the majority. The minority were EBNA-negative, contained abundant clg and were largely non-proliferating. This suggests the continuous occurrence of a maturation process within each LCL. The concomitant downregulation of EBNA raises the interesting question whether continued synthesis of the nuclear antigen is incompatible with differentiation for epigenetic reasons, or, alternatively, whether differentiation takes place when the viral genomes are suppressed or lost.     

Characterization of a second Epstein-Barr virus-determined nuclear antigen associated with the BamHI WYH region of EBV DNA

The Epstein-Barr virus-determined nuclear antigen (EBNA) is the only known virally-determined component that is regularly associated with EBV-transformed cells. A main component of EBNA, herein designated EBNA-1, has been conclusively localized to the BamHI K fragment of the viral genome. EBNA-1 is present in all EBV-carrying cell lines so far studied. Our current study deals with a second component. We have found that the EBNA reaction detected by anti-complement immunofluorescence (ACIF) in Burkitt lymphoma lines Daudi, Jijoye, and P3HR-1 could be completely removed by preabsorption of sera with any one of these 3 lines, when tested against any other of them. The same absorbed sera still gave a brilliant nuclear staining against other EBV-carrying lines, e.g. Raji or B95-8. The 3 lines in the first category carry EBV genomes that have deletions in the BamHI WYH region of the EBV genome. This region is intact in the second group of lines. This result is interpreted as showing the existence of 2 different ACIF-stainable EBV-determined nuclear antigens, one of which is associated with the BamHI WYH region. We designate this antigen as EBNA-2. We found that the two different EBNAs are different with regard to their association with metaphase chromosomes. In lines positive for both EBNA subtypes, metaphase chromosomes gave brilliant EBNA-1 staining, but could not be stained for EBNA-2, indicating differences in chromatin association of the two EBNAs. An 86 kd polypeptide was identified by immunoblotting of DNA-binding proteins from EBV-transformed lymphoid cell lines. EBV-specificity of the polypeptide was demonstrated by the presence of antibodies against this polypeptide in antisera from a population of EBV-seropositive donors, but not from seronegative donors, by the presence of the polypeptide itself in EBV-carrying but not in EBV-negative cell lines and by the appearance of antibodies against this polypeptide during the course of infectious mononucleosis (IM). The polypeptide was absent from the EBV-carrying P3HR-1, Daudi and Jijoye cell lines, which suggested that it may be encoded by the BamHI WYH region that is deleted from the viral substrains carried by these lines.     

Hybridization of a myeloid leukemia-derived human cell line (K562) with a human Burkitt's lymphoma line (P3HR-1)

The myeloid leukemia-derived Epstein-Barr virus (EBV)-negative human lymphoid cell line K562 was successfully hybridized with the EBV-carrying Burkitt's lymphoma line P3HR-1. Authenticity of the hybrid PUTKO-1 was established by chromosome and isoenzyme studies. A virtually complete hybrid PUTKO-1 carried the EBV genome derived from the lymphoma parent. It averaged 26 EBV DNA copies per cell and was 100% positive for Epstein-Barr virus-associated nuclear antigen (EBNA). In most respects, the hybrid resembled the K562 parent: It had a high Fc receptor concentration, high sensitivity to natural killer cells, absence of EBV C3 receptors, and deficiency of membrane-associated beta 2-microglobulin (beta 2M) and HLA, in parallel with intracellular synthesis and secretion of beta 2M to the medium. Unlike the P3HR-1 parent, the hybrid was completely nonpermissive for antigens of the EBV cycle, early antigen, and viral capsid antigen. None of the 3 inducing agents, 5-lodo-2'-deoxyuridine, 12-O-tetradecanoyl-phorbol 13-acetate, or sodium butyrate, caused any viral antigen synthesis in PUTKO-1 in contrast to the good inducibility of the parental P3HR-1 subline. Thus the myeloid parent restricted expression of EBV antigens except EBNA. This exception further supports the concept that EBNA is an autonomous function of the viral genome, independent of host cell control that regulates expression of antigens related to the viral cycle. On the contrary, extinction of viral antigens in this hybrid between 2 cell lineages supports our previous concept that the ability to produce viral antigens is similar to a differentiated B-cell property.     

Expression of Epstein-Barr virus-encoded proteins in nasopharyngeal carcinoma

Expression of the Epstein-Barr virus (EBV) encoded nuclear antigens (EBNA 1 to 6) and membrane-associated protein (LMP) was investigated by immunoblotting in 83 nasopharyngeal carcinoma (NPC) biopsies and 25 other tumor and normal tissue specimens from the head and neck region. Fifty-eight of the 83 NPC biopsies were large enough to yield parallel data on virus DNA and viral expression. All 16 cases of clinically diagnosed and histologically confirmed NPCs from North Africa contained EBV DNA and expressed EBNA-1. Of 31 clinically diagnosed NPCs from China, 29 contained EBV DNA and 25 of these expressed EBNA-1. One control tissue biopsy from the oropharynx of NPC patients contained EBV DNA, but none expressed EBNA-1. The latent membrane protein (LMP) was detected in 22/31 of the Chinese and in 10/16 of the North African NPC biopsies. None of the NPC biopsies or control tissues expressed detectable amounts of EBNA 2 or any of the other 4 nuclear antigens which are invariably expressed in EBV-transformed B cells. A smaller number of tumors from Malaysia and East Africa exhibited a similar pattern of expression. EBV was rescued from a nude-mouse-passaged North African NPC tumor by co-cultivation of the tumor cells with umbilical cord blood lymphocytes. The tumor expressed EBNA 1 and LMP, but not EBNA 2 or the other 4 EBNAs. The resulting LCLs expressed all 6 nuclear antigens, EBNA 1 to 6 and LMP. Our data suggest that expression of the EBV genome is regulated in a tissue-specific fashion.     

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.     

Relationship between Epstein-Barr virus (EBV) DNA and the EBV-determined nuclear antigen (EBNA) in Burkitt lymphoma biopsies and other lymphoproliferative malignancies

Twenty-six of 27 African Burkitt lymphomas with histologically confirmed diagnosis contained relatively large amounts of EBV DNA (10–101 viral genomes per cell), as determined by nucleic acid hybridization. Twenty-five of the 26 EBV DNA-positive lymphomas contained the EBV-determined nuclear antigen, EBNA, in the majority of the nuclei. Technical reasons may have accounted for the apparent EBNA-negativity of one EBV DNA-positive biopsy. Four African lymphoma biopsies, one with a definite diagnosis of Burkitt's lymphoma and three with a questionable diagnosis of the same disease, were all EBV DNA- and EBNA-negative. The same was true for a collection of Swedish cases of Hodgkin's disease, lymphocytic lymphoma, chronic lymphatic leukemia and some other lymphoproliferative malignancies. Thus, there is excellent agreement between the presence of EBV DNA and of EBNA in tumor biopsies. The EBNA antigen test therefore appears a relatively simple way of testing for the presence of the virus genome, provided it is carried out with appropriate controls. Several of the EBV-genome and EBNA-negative cases came from patients with high serum titers of EBV antibodies. It is concluded that the virus does not really travel along with malignant lymphomas as a passenger in the seropositive patients. In comparison with other lymphomas, African Burkitt's lymphoma of the high endemic areas is unique in that the tumors (with rare exceptions) represent the proliferation of an EBV-genome carrying clone. These findings stress the necessity to distinguish between EBV-seropositive status and evidence for EBV-genome-carrying neoplastic cells.     

Somatic cell hybrids between human lymphoma cell lines. V. IdUrd inducibility and P3HR-1 superinfectability of Daudi/HeLa (DAD) and Daudi/P3HR-1 (DIP-1) cell lines.

We have studied two types of somatic cell hybrid with regard to expression of the Epstein-Barr virus (EBV) cycle and its regulation. The first, DIP-1, a hybrid formed between two human lymphoma EBV producers (Daudi and P3HR-1), contained EBV DNA, expressed the virus-determined nuclear antigen (EBNA), andwas a producer of the EBV-associated antigens EA (early antigen) and VCA (viral capsid antigen). The second, DAD, a hybrid series of clones formed between Daudi and a HeLa cell derivative (D98), differed with regard to the expression of EBNA, EA, VCA and the content of EBV DNA. EA was regularly induced in the EBV DNA-containing hybrids following treatment with iododeoxyuridine (IdUrd). This induction was greater in lines spontaneously expressing EA. In two hybrids, DIP-1 and DAD10, VCA and virus DNA synthesis were also induced in the presence of IdUrd, the latter being detected by in situ hybridization with P3HR-1 EBV complementary RNA. Finally, while DIP-1 was superinfectable by the P3HR-1 EBV strain, the DAD series of hybrids were refractory to P3HR-1 superinfection and lacked EBV receptors.     

Contribution of viral recombinants to the study of the immune response against the Epstein-Barr virus

Over the past two decades, Epstein-Barr virus (EBV) mutants have become valuable tools for the analysis of viral functions. Several experimental strategies are currently used to generate recombinant mutant genomes that carry alterations in one or several viral genes. The probably most versatile approach utilizes bacterial artificial chromosomes (BAC) carrying parts or the whole EBV genome, which permits extensive genetic manipulations in Escherichia coli cells. The 'mini-EBVs', for example, which contain roughly half of the wild type viral information, efficiently transform primary B cells and have been used as gene vectors for foreign antigens. After expression in lymphoblastoid cell lines (LCLs), these antigens are efficiently presented on MHC molecules and recognized by antigen-specific T cells. These vectors, however, cannot undergo lytic replication and require a helper cell line for efficient replication and DNA packaging. Further experimental systems include the complete viral genome cloned onto a BAC. These mutants can typically be complemented by expression plasmids, some of which are expressed on EBV-derived vectors and can be propagated without requirement of a helper cell line. Over the last years, these viral recombinants have been utilized increasingly to analyse different aspects of the immune response against EBV. Immunological applications are manifold and steadily growing and include crude screening of T cell clones for their specificity towards latent versus lytic antigens, or more detailed analyses in which the exact specificity of T cells is determined using EBV mutants that lack a single viral antigen. Other applications include detailed analysis of protein domains important for immune recognition, e.g. Gly-Ala repeats in the EBV nuclear antigen 1 (EBNA1) protein, expansion of T cell clones directed against virion structures using virus-like particles and phenotypic analysis of virus mutants defective in infection. Future developments might include the genetic identification and characterization of viral proteins involved in the modulation of the immune response and, in particular, immune evasion. Recombinant viral strains are already being used experimentally for the expansion of T cells in vitro prior to in vivo cellular therapy and have been proposed as potential prophylactic vaccines.     

Establishment of a cell line (NPC/HK1) from a differentiated squamous carcinoma of the nasopharynx

A long-term cell culture epithelioid cell line was established from a recurrent squamous carcinoma of the nasopharynx of a Chinese male 17 1/2 years after radiation therapy. The cell line, designated NPC/HK1, has been passed 72 times over a period 1 year. The cells have been shown by light and electron microscopies to be of the squamous epithelial type. When they were transplanted subcutaneously into the back of athymic nude BALB/c (nu/nu) mice, tumors developed at the sites of inoculation, which on histological examination were shown to be well-differentiated squamous carcinomas, similar in morphology to the recurrent human tumor from which they were derived. Karyotypic analysis of cells from the cell line demonstrates an aneuploid human type with a modal chromosome number of 74 with both numerical and structural aberrations. Viral particles or Epstein-Barr viral nuclear antigen (EBNA) has not been demonstrated in the cells from the primary culture or several of the subcultures tested. The presence of EBNA in touch smears prepared from the biopsy tissue was inconclusive. Infection of the subcultured cells with EBV from P3HR1 and B95-8 cells was unsuccessful.     

Absence of human papillomavirus-16 and -18 DNA and Epstein-Barr virus DNA in esophageal squamous cell carcinoma

To elucidate the association of human papillomavirus (HPV) and Epstein-Barr virus (EBV) with carcinogenesis of the esophagus, 41 surgically resected specimens and 12 cell lines of esophageal squamous cell carcinoma were examined for the presence of HPV DNA and EBV DNA by polymerase chain reaction using primers for the E6 regions of HPV-16 and -18 and for the EBNA 1 region of EBV. We designed the reaction condition to amplify HPV and EBV DNA specifically and detected by gel electrophoresis. In ethidium bromide staining, no band was detected either for the HPV E6 region of for the EBV EBNA 1 gene in any of the surgically resected specimens and the cell lines, although the HPV sequence was detectable by Southern blot hybridization, which is a more sensitive detection method than staining; three out of 41 surgically resected specimens were positive for HPV-18 by Southern blot hybridization of polymerase chain reaction products. However, the number of viral genomes has been estimated as lower than 1 x 10(-3) copies per cell based on the intensity of the hybridization signals. Moreover, the DNA samples extracted from the corresponding non-cancerous esophageal mucosa were also positive for HPV-18, and the intensities of the hybridization signals were almost the same as those of the tumors. The results of our study indicate that HPV-16, HPV-18 and EBV are not generally associated with esophageal carcinogenesis.      

Antagonistic effects of c-myc and Epstein-Barr virus latent genes on the phenotype of human B cells

Epstein-Barr virus (EBV) immortalized cells and Burkitt lymphoma cells have a completely different growth pattern and phenotype. EBV immortalized cells express a set of 11 viral genes to accommodate B cell activation and proliferation, whereas EBV-positive Burkitt lymphoma cells highly express the c-myc oncogene that is activated through translocation into 1 of the immunoglobulin loci and EBNA1 as the only viral protein. We have developed a primary human B cell line conditionally immortalized by Epstein-Barr virus in which the viral gene program responsible for the induction of proliferation can be switched on and off by the addition or withdrawal of estrogen (cell line EREB2-5). Starting from this cell line we have generated 2 daughter cell lines that proliferate in a c-myc dependent fashion, 1 with a highly active exogenous c-myc gene (cell line A1) and 1 with a regulatable c-myc gene that can be switched on by withdrawal and switched off by addition of tetracycline (cell line P493-6). The comparison of the 3 cell lines has allowed us to dissect the contribution of c-myc and EBV genes to the regulation of the growth pattern and expression of cell surface molecules. We show that MYC and EBNA2 (and their respective target genes) have opposing effects on the expression of several surface markers involved in B cell activation. We show that MYC contributes to the phenotype of Burkitt lymphoma cells by upregulating CD10 and CD38 and downregulating activation markers. The phenotype of the cells is determined on one hand by the absence of the viral gene products EBNA2 and LMP1 that mediate the phenotype of activated lymphoblasts and to a lesser extent by an active contribution of the c-myc gene.     

Interaction between Epstein-Barr virus and type-C virus in human cells.

The interaction between EBV and type-C viruses was studied in our FVNC experimental system, in which EBV and type-C viral genomes are contained in each cell. The data indicate that the human lymphoid FVNC cells are sensitive to both EBV and type-C virus exposure, showing high frequencies of induction of both repressed viral genomes. The two different viral genomes may be associated with different chromosomes in individual cells.     

Heterogeneity of Epstein-Barr virus derived from P3HR-1 cells.

Infection of cells of the EBV-free human B-lymphoma lines BJAB and Ramos resulted in conversion of these cells to EBV-genome carriers expressing EBNA. EBV isolates from P3HR-1 cells induced a heterogeneous EBNA pattern: both a faintly granular pattern and brilliant EBNA-expression were observed. The two types of EBNA-expressing cells could be separated upon cloning. Brilliantly EBNA-expressing cells always segregated varying percentages of EBNA-negative cells. An EBNA-negative subclone derived from these cells was devoid of detectable EBV DNA. Nucleic acid hybridization experiments failed to reveal a correlation between the intensity of EBNA expression and the number of EBV genome equivalents per cell. EBV genome-containing cells had an average of 14-fold more cells showing EA synthesis after superinfection by P3HR-1 virus, when compared with EBNA-negative cells infected under identical conditions. Studies on the kinetics of EA induction in EBNA-positive and EBNA-negative cells indicate that complementation is required for the induction of EA after superinfection.