A human hematopoietic cell line (U-937) with exceptional characteristics was derived from a patient with generalized histiocytic lymphoma. The morphology of the cell line was identical to that of the tumor cells in the pleural effusion from which the line was derived. Since Epstein-Barr virus (EBV) carrying diploid lymphoblastoid cell lines unrelated to the tumor population often become established in vitro from non-Burkitt lymphoma explants, several parameters were studied to discriminate the U-937 from such lines: morphology in vitro, growth characteristics, cytochemistry, surface receptor pattern, Ig production, lysozyme production, beta2-microglobulin production, presence of EBV genome and karyotype. In all these respects U-937 differed from prototype lymphoblastoid cell lines. The histiocytic origin of the cell line was shown by its capacity for lysozyme production and the strong esterase activity (naphtol AS-D acetate esterase inhibited by NaF) of the cells. It is therefore concluded that the U-937 is a neoplastic, histiocytic cell line.

Establishment and characterization of a human histiocytic lymphoma cell line (U‐937)

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.

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

Marmoset blood leukocytes transformed in vitro by Epstein-Barr virus regularly release extracellular infectious Epstein-Barr virus with high titers of transforming activity. By comparison, human umbilical cord leukocytes and adult human leukocytes transformed by Epstein-Barr virus release either no extracellular infectious virus or small amounts, irregularly.

https://www.pnas.org/content/pnas/70/1/190.full.pdf

Release of Infectious Epstein-Barr Virus by Transformed Marmoset Leukocytes

The karyotypes of cells from 10 Burkitt lymphoma (BL) biopsies, eight cell lines established from BL and nine cell lines from non-BL sources were studied by chromosome banding techniques. With the exception of the BL-derived cell lines BJAB, GC-BJAB, Maku and U-8691 all biopsies and lines of Burkitt origin contained an extra band at the distal region of the long arm of one chromosome 14. An extra band on chromosome 14 was also found in cells of one non-BL biopsy, in cells from a lymphosarcoma-derived cell line and in a long-established cell line derived from the pleural exudate of a patient with Hodgkin's disease. A distal region at the long arm of one chromosome 8 was missing in all metaphase figures of good technical quality in the same material. The size, morphology and stain-ability of the missing region corresponded fairly well to the extra region at chromosome 14. We therefore suggest that the chromosome 14 marker represents a translocation between chromosomes 8 and 14,t (8q-; 14q+). The translocation was present neither in lymphocytes of the peripheral blood of five Burkitt patients nor in five lymphoblastoid cell lines of non-BL origin. Trisomy 7 was found in two of the 10 BL biopsies, in two BL-derived cell lines, in one non-BL biopsy, in two lymphosarcoma-derived cell lines and in one cell line derived from a patient with Hodgkin's disease.

Characteristic chromosomal abnormalities in biopsies and lymphoid‐cell lines from patients with burkitt and non‐burkitt lymphomas

PERMISSIVE cell systems for the replication of the Epstein–Barr virus (EBV) and some of the related EBV-like agents (for example, Herpesvirus papio and herpesvirus pan) have not yet been established1. EBV is regularly demonstrated in a small number of cells from lymphoblastoid lines of B-cell origin which have been derived from certain lymphoma patients, most frequently from Burkitt's lymphoma or patients with infectious mononucleosis, but also from healthy EBV-reactive individuals. EBV DNA usually persists in cells of these lines in multiple copies2–4, but spontaneous induction of viral antigens and particle synthesis occurs in a majority of the lines at a low rate. To some extent, the percentage of induced cells seems to be cell line-specific1, and two lines which are rather high producers of EBV are the P3HR-I line of BL origin5 and the marmoset B95-8 line isolated from lymphocytes transformed with EBV of infectious mononucleosis origin6. At any time during cultivation, 2–10% of the cells reveal viral capsid antigen synthesis as determined by indirect immunofluorescence7. All attempts to increase the virus yield in these lines by temperature shifts or chemical or physical inducers (IUdR, mitomycin C, X rays) have usually resulted in only barely significant enhancement of viral replication8–10. We now report on a very efficient induction of EBV and other oncogenic herpesviruses by the well established cocarcinogen and tumour promoter 12-O-tetra-decanoylphorbol-13-acetate (TPA)11. In addition some TPA-treated cells show cytopathogenic changes, including polyploidisation. These observations may enable a more complete investigation of the virus-cell-gene balance hypothesis12.

Persisting oncogenic herpesvirus induced by the tumour promoter TPA

DURING the past two years, work in this and other laboratories has demonstrated viral genomes characteristic of Epstein–Barr (EB) virus in cell lines and biopsy cells derived from Burkitt lymphomas1–4, and anaplastic carcinomas of the nasopharynx5. Our technique consists of hybridizing DNA from the cells concerned with the complementary RNA (cRNA) obtained by the transcription of EB virus DNA in vitro with E. coli RNA polymerase. We have now applied the technique to various lymphoblastoid cell lines which lack virus particle and structural viral antigens and found that they all seem to carry EB virus genomes. DNA from human umbilical cord leucocytes, however, seems to be free of such genomes.

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Occurrence of Epstein-Barr virus genomes in human lymphoblastoid cell lines.

autoradiography, andelectron microscopy inaneffort toidentify thestainable cells withthose harboring herpes-type virus particles Immediately after a2-hrpulse of3H-thymidine, from30to60%ofthecells revealed heavynuclear labeling Inmostcases the grains wereevenly dispersed, butinabout3to5% thegrains showed afocal distribution andoccasionally theyextended intothecytoplasm Suchnuclear foci wererarely seenat8hrafter thepulse Whentheanalysis wasrestricted topreselected immunofluorescent cells, upto80%showed label at8hrandcytoplasmic grains wereprominent Toreduce cellular deoxyribonucleic acid(DNA)synthesis, cells wereX-irradiated with3,000 to6,000 R,andtheisotope pulse wasapplied 1, 4,or7dayslater Whereas thetotal numberoflabeled cells decreased inroughly twofold steps attherespective intervals (from 40to10%), theincorporation of3Hthymidine intofluorescent cells wasnotaffected byX irradiation Ineachseries, about 70%ofthefluorescent cells contained label whentheywereexamined at24 and48hrafter thepulse, whereas at8and72hrfewerwerepositive Attheearlier intervals, unlabeled fluorescent cells mostlikely represented cells whichhad completed viral DNA synthesis prior tothepulse; atthelater intervals, unlabeled fluorescent cells wereprobably cells whichcommenced viral replication after the pulse Thesedatasupport theconclusion thattheimmunofluorescent cells arethe oneswhichharbor virus, andalsoconfirm theexpectation that thevirus isaDNA virus fromamemberoftheherpes group Thisconclusion wasfirmly established bysectioning andelectron microscopic examination ofindividual fluorescent cells, all ofwhichcontained numerous virus particles, whereas thenonstained cells prepared inasimilar mannerwerefree ofthem Themajority ofcontinuous cultures derived fromBurkitt lymphomas werefoundonelectron microscopic examination toharbor herpeslike virus particles inasmall proportion ofthecells (3,16,20) Itwasfurther suggested that thevirusproducing cells canalsobedetected byimmunofluorescence techniques Boththeindirect techniquewithserafromAfrican patients with Burkitt tumors, aswellasserafrommany African andAmerican control individuals, and thedirect technique withfluorescein-conjugated

Volker Diehl

Papers

Occurrence of Epstein-Barr virus genomes in human lymphoblastoid cell lines.

Comparative StudyofCultured Burkitt TumorCells byImmunofluorescence, Autoradiography, andElectron Microscopy