Short‐term assays for detection of conditional cancerogens I. Construction of DR‐CAT Raji cells and some of their characteristics as tester cells

Abstract

A number of agents including the tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate (TPA) (TPA) can induce an abortive virus cycle in the EBV-non-producer Burkitt's-lymphoma line Raji. Two distant regions, DL and DR, of the EBV genome with almost complete homology carry strong promoters which are induced in an abortive or lytic cycle and additionally function as lytic origins of viral DNA replication. To set up a system in which the activity of EBV-inducing agents can be measured in a quantitative and reproducible fashion, we generated a cell line which carries multiple copies of a DR-promoter chloramphenicol-acetyltransferase (CAT) construct on an episomal vector. CAT activity is low in untreated cells, but high upon treatment of the cells with various EBV-inducing agents. Combinations of different agents can produce an over-additive effect. The Raji-DR-CAT cell line may provide a simple quantitative and reproducible test system for EBV-inducing agents, especially for tumor promoters which activate protein kinases C.

Tumor Promoters in Commercial Indoor-Plant Cultivars of the Euphorbiaceae

“…er is induced to high levels only in the pres? ence of EBV-inducing agents (14). Figure 3A shows a dose-response curve for TPA, a well-known standard tumor promoter.…”

“…cial cultivars of Euphorbia lomii hybrids, Euphorbia pulcherrima (Poinsettia), Euphorbia leuconeura, and Codiaeum variegatum for tumor-promoting diterpenoids using highperformance liquid chromatography (HPLC). In addition, the tumor-promoting potential of plant extracts was measured by an EBV induction assay that used the reporter gene luciferase under the control of an EBV early antigen promoter in Raji cells (14). Marked differences in tumor promoter contents among the tested commercial indoor-plant cultivars were discovered.…”

“…Cell culture and luciferase assay. Raji cells, which contained a firefly luciferase reporter gene under the control of the EBV-DR promoter in an autoreplicative plasmid, were used as described by Polack et al (14). Cells were grown at 37?C in a humidified atmosphere with 5% C02 in medium RPMI 1640 containing 10% fetal calf serum, 2 mM glutamine, 100 U/mL penicillin, 50 ug/mL streptomycin, and 300 ug/mL hygromycin B.…”

The chemopreventive compound curcumin is an efficient inhibitor of Epstein‐Barr virus BZLF1 transcription in Raji DR‐LUC cells*

“…The presence of these antibodies indicates that the lytic cycle of EBV activation was activated at some (possibly transient) stages of tumorigenesis. Burkitt's lymphoma lines such as Raji have been shown to be effective model systems for studying EBV induction by exogenous factors [34–39]. We previously reported that the chloramphenicol transferase (CAT) gene under the control of the EBV‐derived right duplication (DR) promoter can be used as a surrogate to measure EBV reactivation in Raji DR‐CAT cells [22,37,38].…”

“…Burkitt's lymphoma lines such as Raji have been shown to be effective model systems for studying EBV induction by exogenous factors [34–39]. We previously reported that the chloramphenicol transferase (CAT) gene under the control of the EBV‐derived right duplication (DR) promoter can be used as a surrogate to measure EBV reactivation in Raji DR‐CAT cells [22,37,38]. This induction is inhibited by curcumin [22], which is an effective chemopreventive compound in animal studies and practically nontoxic in humans by the oral route [21,58].…”