Dimer-size DNA circles in a leukemic cell immortalized with the Epstein-Barr virus.

Abstract

The intracellular state of the 30 viral genome equivalents of Epstein-Barr virus (EBV) DNA carried in latent form by the CII cell line, established from a chronic lymphocytic leukemia patient, has been partially characterized. The CHI line, which has markers confirming its tumor origin, extends the analysis of the intracellular state of EBV DNA to include other, nonBurkitt lymphoid tumor cells. Monomer-size, free, circular EBV genomes, the major intracellular viral DNA species in other EBVtransformed cells, were absent or present in only minor amounts. Instead, EBV DNA sequences were found associated with a cir- population (8, 9). However, the infection aborts soon after the expression of EBNA and neither blast transformation nor DNA synthesis is observed (9). The culture ultimately degenerates in the absence of EBV-associated cytopathic effects.There have been reports of'the establishment of lymphoid cell lines from CLL patients. However, only in a few instances has it been possible to establish that the line is of tumor origin rather than the outgrowth of a more readily transformable normal B lymphocyte present in the initial cell population (8, 10). (14). In an attempt to reduce nuclease action during DNA isolation, some lysates were prepared in the presence of 1 M salt. Thus, washed cells, at a concentration of 2 X 107 cells per ml in 2 X concentrated sodium phosphate-buffered saline (pH 7.5) were mixed with an equal volume of 1.7 M NaCl just prior to lysis of the cell suspension by the addition of 1/2 vol of 3% (wt/vol) Sarkosyl in 1 M NaCI/0.075 M Tris HCI/0.025 -M EDTA (pH 9.0). One-tenth volume of 0.5% proteinase K (Sigma, protease P0390, in 0.05 M TrisHCI, pH 7.5) was added and the lysate was incubated for 30 min at 37°C. The lysate, diluted to 13 times the initial volume with 1 M CsCl'in 0.01 M Tris HCI (pH 9.0), was gently mixed until homogeneous. The density was then adjusted to 1.718 g/cm3 with solid CsCI, Klebsiella pneumoniae [3H]DNA was added to a final concentration of 200 cpm/ml, and the material was centrifuged as 20-ml aliquots for 65 hr at 33,000 rpm and 20°C in a Spinco Ti6O rotor. Fractions (0.4 ml) were collected through a large (2-4 mm) hole in the bottom of the tube and material banding near the 1.717 g/cm3 Kiebsiella DNA marker was pooled for further analysis. The procedure used to localize EBV DNA sequences by filter hybridization with [32P]RNA complementary to the EBV DNA ([32P]cRNA) was as previously published (15).The method of purifying covalently closed circular DNA molecules by alkaline denaturation and renaturation was according to Casse et aL (16) with the modification that after neutralization, the lysate was digested with proteinase K prior to the phenol extraction (17). Briefly, a loosely packed pellet of cells, washed in sodium phosphate-buffered saline, was lysed by the addition of 1 ml 6f alkaline NaDodSO4 (1%, wt/vol, NaDodSO4 in 0.05 M Tris.HCy0.02 M EDTA, adjusted to pH 12.45 with 2 M NaOH) per 10 cells in the pellet. The mixture was rapidly stirred in a la...