Restricted expression of Epstein-Barr virus (EBV)-encoded, growth transformation-associated antigens in an EBV- and human herpesvirus type 8-carrying body cavity lymphoma line.

A body cavity lymphoma-derived cell line (BC1), known to carry both Epstein-Barr virus (EBV) and human herpes virus type 8 (HHV-8; or Kaposi's sarcoma-associated herpesvirus, KSHV), was analysed for the expression of EBV-encoded, growth transformation-associated antigens and cellular phenotype by immunofluorescence staining, Western blotting, RT-PCR and flow cytometry. A similar phenotypic analysis was also performed on another body cavity lymphoma line, BCBL1, that is singly infected with HHV-8. Phenotypically, the two lines were closely similar. Although both lines are known to carry rearranged immunoglobulin genes, they were mostly negative for B-cell surface markers. Both expressed the HHV-8-encoded nuclear antigen (LNA1). Similarly to Epstein-Barr nuclear antigen type 1 (EBNA1), LNA1 was associated with the chromatin in interphase nuclei and the mitotic chromosomes in metaphase. It accumulated in a few well-circumscribed nuclear bodies that did not co-localize with EBNA1. BC1 cells expressed EBNA1, LMP2A and EBV-encoded small RNAs but not EBNA2-6, LMP1 and LMP2B. They were thus similar to type I Burkitt's lymphoma cells and latently infected peripheral B-cells. Analysis of the splicing pattern of the EBNA1-encoding message by RT-PCR showed that BC1 cells used the QUK but not the YUK splice, indicating that the mRNA was initiated from Qp and not from Cp or Wp.

Human/mouse microcell hybrid based elimination test reduces the putative tumor suppressor region at 3p21.3 to 1.6 cM.

We have previously identified an approximately 7 cM long common eliminated region (CER), involving the 3p21.3 markers AP20R, D3S966, D3S3559, D3S1029, WI-7947, D3S2354, AFMb362wb9, and D3S32, in human chromosome 3/A9 mouse fibrosarcoma microcell hybrid (MCH) derived SCID mouse tumors. We now report the results of our more detailed analysis on 24 SCID mouse tumors derived from two MCH lines that originally carried intact human chromosomes 3. They were analyzed by fluorescence in situ hybridization (FISH) painting and PCR, using 24 markers covering the region between D3S1611 and D3S13235 at 3p22-p21.2. D3S32 and D3S2354 were regularly eliminated during in vivo tumor growth, whereas the other 22 markers, D3S1611, ACAA, D3S1260, WI-692, AP20R, D3S3521, D3S966, D3S1029, D3S643, WI-2420, MSTI. GNAI2, D3S1235, D3S1298, GLBI, WI-4193, D3S3658, D3S3559, D3S3678, WI-6400, WI-7947, and WI-10865, were regularly retained. We have defined a common eliminated region of approximately 1.6 cM (designated as CER1) inside the 7 cM CER described earlier. CER1 is flanked distally by D3S1029 and proximally by D3S643.

B-CLL cells with unusual properties.

In studies concerning the interaction of B-CLL cells and Epstein-Barr virus (EBV), we encountered one patient whose cells had several unusual properties. In addition to the B-cell markers, the CLL cells expressed the exclusive T-cell markers CD3 and CD8 and carried a translocation t(18,22)(q21;q11), involving the bcl-2 and Ig lambda loci. The patient represents the 4th reported CLL case with this translocation. The CLL cells could be infected and immortalized by the indigenous and by the prototype B958 virus in vitro. The T-cell markers were not detectable on the established lines. In all experiments the immortalized lines originated from the CLL cells. Their preferential emergence over virus-infected normal B cells may be coupled to the high expression of the bcl-2 gene due to the translocation. In spite of the sensitivity of CLL cells to EBV infection in vitro, no EBNA-positive cells were detected in the ex vivo population. In vitro, we could generate cytotoxic function in T-lymphocyte cultures which acted on autologous EBV-infected CLL cells. Therefore we assume that if such cells emerged in vivo they were eliminated by the T-cell response.

Genotypic and phenotypic characterization of two newly established renal cell carcinoma cell lines.

Two new cell lines from human renal cell carcinoma are reported. Primary cell cultures from 75 consecutive cases of nephrectomy and metastatic surgery due to different stages of RCC during 4 years were studied. Two cell cultures could be propagated for more than 50 passages in vitro. HN4 was derived from a grade III clear cell carcinoma. HN51 originated from a metastatic brain lesion of a clear cell carcinoma grade III. Karyotype analysis of HN4 revealed triploidy with a clonal aberration, der(10)t(3;10)(q13;p12). HN51 also had a triploid pattern with different marker chromosomes but without any clonal aberration. Loss of heterozygosity studies revealed no loss of heterozygosity on 3p or other chromosomal markers in HN4 but LOH was found on one 3p marker and one 14q marker in addition to all 17 p and q markers in HN51. In vitro light microscopy showed distinctly different morphology in the two cell lines although they both had a typical epithelial growth pattern. Doubling times in vitro were low but slightly higher for HN51. Repeated tumorigenenic experiments in athymic mice only gave rise to subcutaneous tumors with HN51. On characterization by 2-dimensional gel electrophoresis, the two cell lines exhibited different polypeptide patterns with higher expression of proliferating cell nuclear antigen in HN51 and higher expression of glutathione-S-transferase in HN4 constituting the most prominent differences.

Epstein-Barr virus (EBV) can immortalize B-cll cells activated by cytokines.

B-type of chronic lymphocytic leukemia (B-CLL) cells are inert to the potent transforming action of Epstein-Barr virus (EBV). The mitogenic action of Staphylococcus aureus Cowan I (SAC), MP6-thioredoxin, and interleukin 2 (IL-2), agents previously shown to induce proliferation in normal as well as in B-CLL cells, lifted this block, and EBV-positive cell lines could be established. It was not possible to establish cell lines of leukemic origin from cultures that were incubated with EBV alone or cytokine mix alone. CLL-cells infected with EBV only, expressed the viral nuclear antigen complex (EBNA), but not the viral latent membrane protein (LMP). They were not activated as measured by cell size and 3H-thymidine incorporation. In contrast, cells incubated with EBV and cytokine mix expressed both EBNA and LMP in parallel with enlargement and increased 3H-thymidine incorporation. These results emphasize that LMP expression is a prerequisite for growth transformation and immortalization and that cytokine activation signals are required for its expression in B-CLLs. Cells incubated with SAC/MP6-thioredoxin/IL-2 did not express any of the viral antigens, but were activated with regard to the mentioned parameters. Nine cell lines were established from six patients. From each of the three patients, we obtained 'twin'-pair lines: one corresponding to the malignant cell and the other to a normal B-lymphoblastoid cell. Thus, malignant and normal B-cell counterparts, from the very same donor, are at hand for comparative studies. The cell lines have been carried out for more than 12 months in culture. We conclude that B-CLL that are refractory to EBV-transformation can be rendered susceptible through in vitro cytokine activation.

Surface marker characterization of EBV-carrying cells in blood of healthy seropositive individuals.

We have determined the phenotype of EBV-carrying cells in human blood by establishing spontaneous LCLs from populations selected according to three B cell markers, B1, B2, and 35.1C5. LCLs grew from the B1-positive, B2-positive, B2-negative and 35.1C5-positive population. Thus, EBV-carrying cells in blood of seropositive individuals are restricted to the B cell lineage. These B cells may or may not express the B2 epitope, and they may be derived from the lymph node mantle zone or the splenic marginal zone. These in vivo EBV-carrying cells could enter the viral productive cycle and transform coresident B cells during the initial 3 days in vitro. The characteristic of the LCLs with regard to these 3 B cell markers did not correspond to the original B cell population from which they were derived.

Expression of B-cell-specific markers in different Burkitt lymphoma subgroups.

Forty-three Burkitt lymphoma (BL) lines were examined for the expression of 5 monoclonal antibody (MAb)-identified B-cell-specific markers and immunoglobulin production. All (13) EBV-negative BL lines were CALLA+ LB-1-, whereas 30 EBV-carrying lines showed a more heterogeneous pattern. In the EBV-negative lines, the follicle mantle zone markers BA-1 and 35.1C5 were expressed concordantly, at a different level in each line. This coordination was disrupted in EBV-carrying lines. In the EBV-negative lines, there was also an inverted correlation between the expression of 35.1C5 and the germinal center marker BLA, suggesting that some etiologically important event, perhaps the translocation, had fixed the cells at different stages of their transition from one zone to the other. This inverted relationship was also disrupted in the EBV-carrying lines, suggesting that EBV can interfere with the maturation program of the BL cell. This conclusion was also supported by a comparison between 5 EBV-negative BL lines and their EBV-converted sublines. All converted lines have undergone marker changes, but the degree and nature of these changes was different for each EBV-BL line. Both the coordinated expression of BA-1 and 35.1C5 and the inverted relationship between CALLA and LB-1 were disrupted in several other convertants. We have reexamined our previous finding (Ehlin-Henriksson and Klein, 1984) that the majority of the variant translocation-carrying BL lines were CALLA- LB-1+, in contrast to the majority of the typical translocation carriers that were mostly CALLA+ LB-1-. All II EBV-negative lines were CALLA+ LB-1-, irrespective of the type of translocation. Among the EBV-carrying lines, 4 of 17 typical (8;14) translocation carriers were CALLA- LB-1+, whereas 7 of the 12 variant translocation-carrying lines were CALLA- LB-1+. The remaining two expressed both antigens to some extent. The difference is statistically significant at the 0.03 level.

Expression of the BLA antigen, defined by the monoclonal 38.13 antibody, on Burkitt lymphoma lines, lymphoblastoid cell lines, their hybrids and other B-cell lymphomas and leukemias.

The BLA expression of eight Burkitt lymphoma lines was high, whereas it was negative in four, including the two IgG producers tested. Most lymphoblastoid cell lines (LCL) of normal origin had only a low percentage of positive cells, not significantly above background, although a few had up to 30% positives. EBV conversion of the EBV-negative Burkitt lymphoma line Ramos destabilized the high BLA expression, leading to a decrease in the average number of positive cells in the majority of the converted sublines in parallel with considerable fluctuation in antigen expression within each subline. Our group has previously shown that EBV-conversion of Ramos cells can induce certain differentiation steps (Spira et al., 1981 a). EBV-converted sublines of another EBV-negative Burkitt lymphoma, BJAB, showed a much greater stability previously and remained unchanged with regard to BLA expression in our present experiments. Eight T-cell leukemias, three myeloid leukemia lines and two diffuse histiocytic lymphomas were negative for BLA, whereas two myeloma lines were 30-40% positive. A histiocytic tumor had marginal reactions. Hybrids derived from the fusion of high with low BLA-reactive parental lines showed all three possible patterns (high, intermediate and low), provided that B-cell lines were fused with each other. Fusion of two Burkitt lymphoma lines with the K562 erythroleukemia line led to the extinction of BLA expression, as well as to the eclipse of other B-cell markers. B-lymphoma and leukemia (CLL) cells harvested directly from the patient showed a heterogeneous reactivity pattern. Strong to intermediate BLA expression was found among CLL cells and in most histological groups of B-CLL lymphomas except the centroblastic group (3/3 negatives). IgG-expressing follicular lymphomas were less reactive than IgM +/- IgD lymphomas of the same group. Immunocytomas were also low-reactive. BLA can be thus expressed on a variety of B-cell neoplasms; the degree of its expression appears to be related to the stage of differentiation.