DNA Synthesis in EB Virus-Containing Burkitt Lymphoma Cultures during a Temperature Cycling Procedure

Abstract

Discussion and Summary

The temperature cycling or temperature reduction procedures applied to EBV-containing Burkitt lymphoma cells usually produce cultures which have a higher incidence of EBV-containing cells than nontreated cultures. The magnitude and kinetics of the response was not predictable. It appears that other unknown factors are involved in this stimulation. Henle (10) has suggested that the level of arginine in serum may have an effect upon the level of virus-containing cells in Burkitt lymphoma cell cultures. Minowada et al. (2) has indicated that an active period of cell proliferation accompanied by the synthesis of immunoglobulin may be prerequisite to the synthesis of EBV antigen.

“EBV-free” Burkitt lymphoma cells exhibited a loss in viability and a reduction in the rate of DNA synthesis when subjected to these procedures. The direct immunofluorescent test using fluorescein-labeled human 7S globulin reacted with both cells and debris in P1R cultures, and the incidence of this fluorescence increased when the cells were subjected to a temperature cycling procedure. When P1R cells from the same culture were examined by the indirect test, the cells did not fluoresce, and in subsequent experiments in which only the indirect test was used, fluorescent P1R cells were rarely seen. In comparison, when HR1K cells were examined by the direct and indirect tests, the incidence of positive cells in noncycled cultures was the same in both tests and, although there was an increase in the incidence of fluorescent cells in the cycled cultures in both tests, the incidence was significantly less with the indirect test. We have compared the incidence of virus-containing cells by the indirect test to that determined by electron microscopy in several Burkitt lymphoma cell lines and have found a good correlation (unpublished data). Minowada et al. (2) has reported that some nonspecific fluorescence was observed with the direct test, especially in old or damaged cultures. Thus, in addition to EBV-containing cells, the direct test used in this report involved the binding of labeled protein to dead cells and debris, and this did not occur or was not detected, when an indirect test was used.

When HR1K and P1R cultures were subjected to a temperature cycling or reduction procedure, both exhibited a reduced rate of DNA synthesis compared to their noncycled counterparts. However, the rate of DNA synthesis was less affected in HR1K cultures in which the incidence of virus-containing cells was increased relative to noncycled HR1K cultures. The larger the proportional increase in the incidence of EBV-containing cells, the less the degree of inhibition (Fig. 3). DNA was synthesized in cycled HR1K cultures at a faster rate than cycled P1R cultures, whereas in noncycled HR1K cultures, DNA synthesis, relative to that in noncycled P1R cultures, was synthesized at similar rates (Fig. 4). These elevated or less inhibited rates of DNA synthesis in cycled HR1K culture (depending upon whether the comparison was to cycled P1R or noncycled HR1K cultures) are not due to a more temperature resistant mechanism of cellular HR1K DNA synthesis because the effects could be related to the incidence of virus-containing cells. The data presented in Table I support this interpretation, because noncycled HR1K cultures synthesized DNA at faster rates than noncycled P1R cultures when there was a higher incidence of EBV-containing cells.

These data have been interpreted to indicate that in HR1K cultures, the cells exist in two forms, a virion-free form which synthesizes DNA at a slower rate than the second form, a virus-producing cell. The virus-producing cell, in addition to virus DNA, may or may not be synthesizing cellular DNA. Cloning experiments (Maurer and Imamura, unpublished data) have indicated that virus-negative cells could give rise to populations of cells which had a similar incidence of virus-containing cells as the parent culture. Also, the recloning of selected negative clones gave rise to populations of cells which had the same incidence of virus-containing cells as the original parent population. These data have been interpreted to indicate that virus-negative cells were “induced” to a virus-productive state. Since such “induced” cells produce virus and cells productively infected with herpes viruses inhibit the synthesis of cellular DNA (11, 12), the DNA synthesized by EBV-containing cells is probably EBV DNA. Attempts, so far, to separate two types of DNA from EBV-containing Burkitt lymphoma cells have not been successful. Such attempts have included equilibrium centrifugation in CsCl density gradients and column chromatography using Sepharose 2B or methylated albumin kieselguhr.