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.     

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.     

Abortive expression of the Epstein-Barr virus (EBV) cycle in a variety of EBV DNA-containing cell lines, as reflected by nucleic acid hybridization in situ.

A variety of Epstein-Barr virus (EBV) DNA-containing cell lines have been tested for the expression of the EBV-associated antigens EBNA (nuclear antigen), EA (early antigen), and VCA (viral capsid antigen), and for the presence of cells containing disproportionate amounts of EBV DNA. The antigen tests utilized immunofluorescence and 125I-labelled antibodies combined with autoradiography. EBV-DNA was detected by in situ hybridization with 3H-labelled EBV RNA complementary to P3HR-1 EBV DNA (P-EBVcRNA). The P-EBVcRNA has been shown to represent the majority of the P3HR-1 EBV DNA sequences. It was concluded that EBV DNA-containing cell lines can be divided into those that express only EBNA, those that express EBNA and EA and those that express EBNA, EA and VCA and also contain cells that undergo disproportionate EBV DNA synthesis. Consequently, in some cell lines there is an abortive expression of the EBV cycle in that some cells spontaneously express EA but fail to continue further to viral DNA synthesis. A similar pattern can be found after experimental induction of the EBV cycle, suggesting that related mechanisms govern the spontaneous expression of the EBV cycle and the extent of its inducibility.     

Spontaneous and induced patterns of the Epstein-Barr virus (EBV) cycle in a new set of somatic cell hybrids.

A somatic cell hybrid line and its subclones obtained by fusing two Burkitt lymphoma lines (Raji and Namalwa) were examined for the expression of EBV-specific antigens both spontaneously and after induction. The hybrids retained spontaneous early antigen (EA) production at the level characteristic of Raji. Similarly the more permissive Raji pattern dominated the induction of EA by IUDR treatment or P3HR-1 virus superinfection. These findings accord with our previous results on independently derived Raji/Namalwa hybrids. Virus capsid antigen was induced in the hybrids by P3HR-1 virus superinfection though at a lower level than in the Raji parental cell.     

High irradiation sensitivity of epstein-barr-virus (ebv) in lymphoblastoid-cells from patients with ataxia-telangiectasia and ebv genome-positive burkitts-lymphoma

Epstein-Barr virus (EBV)-immortalized lymphoblastoid cell lines derived from the peripheral blood of patients with ataxia telangiectasia (AT) and EBV genome-positive Burkitt's lymphoma (BL) were tested for expression of EBV-related lytic antigens by means of irradiation. We used 1 Gy in each experiment, according to the results of the P3HR-1 (derived from African BL) cell line. Significantly higher expression of early antigens (EA) and viral capsid antigen (VCA) was demonstrated in lymphoblastoid cell lines derived from both patients with AT and those with EBV genome-positive BL, as compared to those derived from healthy individuals. These results suggested that defective regulating mechanisms on B lymphocytes, responsible for EBV infection, may underlie for the pathogenesis of development of lymphoproliferative diseases both in patients with AT and EBV genome-positive BL.     

Somatic cell hybrids between human lymphoma lines. IV. Establishment and characterization of a P3HR-1/Daudi hybrid.

A new human B-lymphoma hybrid line designated DIP-1 was derived from the fusion of the Burkitt lines Daudi and P3HR-1. Cytogenetic analyses proved hybridity and showed that the hybrid was nearly complete from the chromosomal point of view. Hybridity was also confirmed by isozyme marker tests. The Daudi-derived IgM-kappa ring was fully exposed on DIP-1, together with the P3HR-1-derived human beta-2-microglobulin. Expression of C3 receptors was intermediate between the two parents and EBV receptors were some what reduced in comparison with both. The spontaneous low-level EBV antigen (EA and VCA) production of the parents was maintained and amplified in the hybrid. The hybrid, like its Daudi parent, was highly inducible with IdUrd. In P3HR-1 virus super-infection experiments, the Daudi parent was more permissive than the BU-P3HR-1 parent. The hybrid was intermediate, resembling the previously studied Raji/Daudi hybrid and contrasting with Raji/Namalwa and Raji/BJAB hybrids. Virus DNA replication patterns after P3HR-1 virus superinfection resembled the antigen pattern. The implication of these findings for the understanding of virus-host cell relationships and the regulation of the viral cycle is discussed The findings are meant to imply that genetically determined isozyme markers are autonomously expressed, as in other systems. Differentiation-related markers and EBV-cycle-related characteristics are autonomously expressed in some cases (surface immunoglobulin, beta-2-microglobulin, spontaneous EBV-production, IdUrd inducibility) and appear to be under a restrictive control in others (EA inducibility by P3HR-1 virus superinfection, C3 and EBV receptors, viral DNA replication).     

Differences between soluble complement-fixing antigens derived from P3HR-1, RAJI, and Hk-Ly-28 cell lines.

Soluble complement-fixing (CF) antigens of the virus-producing and virus-nonproducing P3HR-1 and RAJI Burkitt lymphoma cell lines and Hk-Ly-28 nasopharyngeal cancer cell line, identified by use of viral capsid antigen-positive human sera, could readily be distinguished by differences in their stability to chemical and physical conditions. The P3HR-1 soluble CF antigen lost titer when heated or exposed to acid perchlorate under conditions in which RAJI and Hk-Ly-28 soluble antigens were stable. Differences in solubility were also found. These results contribute not only to the interpretation of the relationship of Epstein-Barr virus (EBV)-associated components of RAJI to P3HR-1 and Hk-Ly-28 cell lysates but also to the selection of conditions for the development of identification tests and purification procedures for CF antigens and antibodies of Burkitt's lymphoma, nasopharyngeal cancer, and other EBV-associated tumors.     

Differential inhibition of epstein-barr virus induction by the amino acid analogue,L-canavanine

The effect of an amino acid analogue, L-canavanine, on the synthesis of Epstein-Barr virus (EBV) antigens was investigated in lymphoblastoid cells. The analysis revealed that after infection of BJAB and NC-37 cells with P3HR-I EBV synthesis of early antigen (EA) was not affected by canavanine in concentrations up to 8.4 mM. The synthesis of EBV-determined nuclear antigen (EBNA) and of viral capsid antigen (VCA) was significantly inhibited at concentrations higher than 2.8 mM. Spontaneous induction of EA in P3HR-I cells was not affected by canavanine. On the other hand, EA induction by the tumor promoter TPA, by iododeoxyuridine (IdUrd), by antiserum to human IgM and by n-butyric acid was clearly inhibited by this treatment. Application of 0.3 mM canavanine resulted in more than 95% inhibition of EA induction by TPA. Under these conditions cell growth and incorporation of radiolabelled amino acids into an acid-insoluble fraction was significantly impaired. Differential treatment of the cells with canavanine established that EA induction was completely suppressed when the cells were treated concomitantly with canavanine and TPA. Subsequent treatment with canavanine after prior exposure to TPA resulted in some viral antigen induction depending on the time period of TPA exposure. Pretreatment of the cells overnight with canavanine followed by washing and addition of the tumor promoter did not suppress EA induction by TPA. These data support the concept that EA induction by superinfection follows a different pathway from antigen induction by chemical inducers.     

Productive Epstein-Barr viral infection of the human lymphoblastoid cell line 6410 with release of early antigen inducing and transforming virus.

The human lymphoblastoid cell line 6410 was superinfected with P3HR-1 derived Epstein-Barr virus. Subsequent to the first cycle of infection, characterized by early (EA) and virus capsid (VCA) antigen synthesis, the culture, designated 6410-EBV, continued to synthesize VCA. Further immunofluorescence and electron microscopic examination over 18-24 months showed the 6410-EBV culture to be productively infected with EBV. Virus was recovered, in quantity, from the culture fluids and assayed for ability to induce EA synthesis in Raji cells and to transform human umbilical cord lymphocytes. The virus was found to possess both properties, in contrast to P3HR-1 virus which only induces EA. HLA and karyologic analyses of P3HR-1, 6410 and 6410-EBV cultures showed relatedness of 6410-EBV to 6410 cells and dissimilarity to P3HR-1. The biologic activity data coupled with the cytologic analyses confirm a productive infection of the target cells. The reason for differences in biologic activity between the infecting (P3HR-1) and progeny virus is unresolved. It may be speculated that the endogenous EBV genome of 6410 cells was activated and gave rise to a different strain of EBV or to a mixed progeny-parent population as a result of dual infection. Alternatively, the parent strain of virus may have been modfied as a result of interaction with the new host cell.     

Somatic cell hybrids between human lymphoma lines. II. Spontaneous and induced patterns of the Epstein-Barr virus (EBV) cycle.

The regulation of spontaneous, IUDR-induced and P3HR-1 virus-induced EA and VCA production patterns was studied in two new somatic hybrids between human lymphoma lines. The hybrid 8A was derived from the crossing of the non-producer Raji with the spontaneous producer Daudi line. The second hybrid, 83, was produced by the fusion of Raji with the EBV-genome-negative B-lymphoma line, BJAB. The studies suggest the following EBV regulation patterns: (1) the spontaneous production of EA and VCA appears to be regulated by controls that differ from the regulators of P3HR-1 virus-induced or IUDR-induced EA synthesis. While spontaneous producer status was dominant over non-producer status, the level of EA inducibility was set by one of the parental cells, Raji ATG, and could either raise (in the previously studied Raji/Namalwa hybrid, cf Nyormoi et al. 1973) or depress (in Raji/Daudi) the level of relative EA inducibility found in the partner cell. (2) Although EA production is a prerequisite for VCA synthesis, the latter is under its own restriction mechanisms, quite independent of those that regulate the level of EA synthesis. (3) Inducibility of EA synthesis by P3HR-1 virus and by IUDR appear to be under the influence of at least partially identical controls. (4) EBV-negative lymphoma cells, exemplified by BJAB, may exert a "complementation" effect on the EA inducibility of their EBV-positive fusion partner, in spite of their own restrictivity against virus-induced EA synthesis. In more general terms, it is obvious that the EBV cycle is under the influence of multiple regulatory mechanisms in the human lymphoid cell. Depending on the parental cell and viral genomes that are allowed to interact, somatic cell hybrids may display a variety of patterns. At this time, cell hybridization is one of the few pathways that permit an approach to this complex and completely unknown world.     

Differential induction of Epstein-Barr virus-related antigens in heterokaryon cultures.

Different paterns of induction of Epstein-Barr virus (EBV)-related antigens were observed in heterokaryons produced by Sendai virus-mediated fusion of producer and non-producer human lymphoblastoid cells with various other cell types. EBV-related early antigens (EA) and viral capsid antigen (VCA) could obviously be induced in heterokaryons between producer cells (P3HR-1 and QIMR-WIL), normally expressing these natigens at very low frequency, and human FL or HeLa cells. Positive cells were detected as early as 3 h after fusion and there often followed a rapid increase in positive cells. In contrast, in heterokaryons between non-producer cells (Raji and NC-37) and FL or HeLa cells, only EA but not VCA was induced. EA induction was also evident in fusion of human lymphoblastoid cells with monkey cells (Vero) but with mouse cells (L-M(TK-) C11D and MCB-2) no EBV induction occurred. The EBV induction in heterokaryons was significantly enhanced by 5-iododeoxyuridine treatment.     

Properties of a baboon lymphotropic herpesvirus related to Epstein-Barr virus.

Three lymphoblastoid cell lines were established from splenic lymphocytes of a lymphomatous baboon (Papio hamadryas) by co-cultivation of the lymphocytes with X-irradiated cells of marmoset or baboon lymphoblastoid cell cultures; the baboon splenic lymphocytes failed to grow when cultured alone. A herpesvirus, associated with each cell line, was identified by immunofluorescence, molecular hybridization and electron microscopy. Antigenic comparison with Epstein-Barr virus (EBV) showed that the baboon herpesvirus and EBV shared cross-reacting viral capsid antigens (VCA): 20 of 20 (100%) anti-VCA (EBV)-positive human sera and 55 of 62 (89%) baboon sera reacted with the baboon lymphoblastoid cells and baboon sera stained EBV VCA in P3HR-1 and EB-3 cells. No nuclear antigen, as assayed by anti-complement immunofluorescence tests, was detected in baboon lymphoblastoid cells when human or baboon anti-VCA positive sera were used. Baboon anti-VCA-positive sera also failed to stain EBV nuclear antigens (EBNA) in Raji or P3HR-1 cells. Preliminary molecular hybridization studies showed only approximately 40% homology between viral DNA of baboon cell lines and DNA of EBV derived from P3HR-1 cells.     

Comparative study of anti EBV antibodies in non-Hodgkin's lymphomams and angio-immunoblastic lymphadenopathies (author's transl)]

Antibody titers to Epstein-Barr virus (EBV)--related antigens (viral capsid antigen : VCA; early antigen : EA; and EBV associated nuclear antigen : EBNA) were determined in the sera of 86 patients and 150 matched control subjects. The patients belonged to four histological groups : diffuse and nodular non-hodgkin's lymphomas angio-immunoblastic lymphadenopathies and apparented syndroms. The incidence of antibodies to other herpes-viruses (cytomégalovirus, herpes simplex virus, and varicella zoster virus) was compared. There was a significantly higher incidence of anti VCA and anti EA titers in some patients, not associated with an increase in titres of antibodies to other herpes viruses.     

Heterogeneity of Epstein-Barr virus originating from P3HR-1 cells. I. Studies on EBNA induction.

Infection of EBV-negative human B-lymphoma cells of the lines BJAB and Ramos with EBV from P3HR-1 or B95-8 cells resulted in gradual conversion of these cells to EBNA synthesis. Whereas B 95-8 virus-infected cells exhibited a uniform brilliant EBNA fluorescence, two distinct fluorescence patterns were observed in P3HR-1 virus-converted BJAB and Ramos cells, a faint granular and a brilliant fluorescence, with predominance of the faint granular pattern. Cloning of P3HR-1 virus-converted BJAB cells resulted in 20 clones, 11 of them showing the heterogenous parental pattern, six revealing exclusively faint granular EBNA staining, and three with brilliantly stained nuclei, containing also a varying percentage of EBNA-negative cells. Further subcloning of one of the latter clones resulted in 26 subclones with brilliant EBNA expression, always segregating a significant percentage of EBNA-negative cells and one entirely EBNA-negative subclone. Reassociation kinetics did not reveal striking differences in the genome content of clones showing exclusively the faint granular or the brilliant type of EBNA expression. The EBNA-negative clone did not contain detectable amounts of EBV-DNA. Upon superinfection of the converted clones by the parental P3HR-1 virus, a significant increase in EA induction was noted when compared to non-converted BJAB and Ramos cells. This accounted in particular for cells with faint granular EBNA expression. These data support previous interpretations (Fresen and zur Hausen, 1976), suggesting the existence of at least two populations of EBV molecules within P3HR-1 cells. The reason for the apparently labile association of P3HR-1 EBV genomes inducing the brilliant EBNA flourescence in BJAB cells still remains obscure. The possible existence of a "helper" effect, exerted by the faint granular EBNA-inducing virus in stabilizing the persistence of the former, is discussed.     

Effect of retinoic acid (RA) on the Epstein-Barr virus (EBV)-inducing effect of sodium butyrate

Sodium butyrate is a powerful inducer of the Epstein-Barr virus (EBV) cycle in the P3HR-1 cell line. Retinoic acid (RA) was found to reduce the butyrate induction of early antigen (EA) and viral capsid antigen (VCA) by 26-41%. This is similar to the previously reported effect of RA on IUDR and TPA induction, with certain quantitative differences between the systems.     

Elevated antibody levels to Epstein-Bbarr virus antigens in patients with hairy cell leukemia compared to controls in relation to exposure to pesticides, organic solvents, animals, and exhausts

Epstein-Barr virus (EBV) is a B-lymphotropic human herpes virus infecting B-cells, which has been associated with lymphoid malignancies, above all non-Hodgkin lymphomas (NHL). Severe immunosuppression is the best recognized risk factor for NHL. Many factors in the environment that have been described as risk factors for NHL cause measurable changes in immune functions. Hairy cell leukemia (HCL) is a rare, indolent non-Hodgkin lymphoma, originating from B-lymphocytes. This was a case-control study including 111 male cases with HCL and 400 controls. In a subgroup of 57 cases and 65 controls analysis of antibodies to EBV early antigen, viral capsid antigen, and EBNA-1, measured as P107, was performed. In this study, we confirm other studies describing elevated levels of antibodies to the EBV early antigen (EA) in patients with HCL compared to controls. We found only minor differences in the levels of antibodies to the viral capsid antigen (VCA) and EBNA-1. measured as P107. We found a positive association of a titer to EA IgG > or =40 (OR 4.1; CI 1.9-9.5). The ORs were further elevated when subjects with high levels of EA IgG and exposed to environmental agents such as organic solvents, certain pesticides, impregnating agents, animals, and exhausts were compared to those subjects with low levels that were not exposed. Antibody reactivity against the EBV EBNA 1-alanine-glycine repeat (P107 IgG) above the median gave an increased OR for HCL, which further increased in subjects exposed to organic solvents, certain pesticides. impregnating agents, animals, and exhausts. However, the numbers of exposed cases and controls were small in some of the calculations.     

Immunofluorescence and anti-complement immunofluorescence absorption tests for quantitation of Epstein-Barr virus-associated antigens.

Immunofluorescence absorption methods are described which permit quantitative estimation and differentiation of Epstein-Barr virus (EBV)-associated antigens (virus capsid antigen, VCA, early antigen, EA and EBV-determined nuclear antigen, EBNA) in cell extracts. EBNA was present in all cell lines (producer and non-producer) which carried the EBV-genome, while VCA and EA were present in producer lines only. All the antigens were absent from a lymphoid cell line (MOLT-4) which lacked the EBV-genome, as well as from leukemia cells from peripheral blood. The techniques demonstrated antigenic identity of the various antigens when prepared from different cell lines.     

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.     

Activation of Epstein-Barr virus expression in human lymphoblastoid P3HR-1 and Raji cells with propionic acid and with culture fluids of propionic acid-producing anaerobes

Epstein-Barr virus (EBV)-associated early (EA) and virus capsid antigens (VCA) were efficiently induced in the viral genome-carrying human lymphoblastoid cells, P3HR-1 and Raji, by the culture fluids of Propionibacterium acnes, P. avidum, P. lymphophilum and Arachnia propionica, the anaerobes which are commonly seen among the normal flora of man. The active principle for EBV-induction in the 2 cell lines was the propionic acid produced by the microbes and such activity was shown to correlate with the fatty acid content of the culture media.