Fumio Mizuno

Chinese and African Euphorbiaceae plant extracts: markedly enhancing effect on Epstein-Barr virus-induced transformation

Chinese and African Euphorbiaceae plant extracts were shown to have a markedly enhancing effect on Epstein-Barr virus (EBV)-induced transformation of human lymphocytes. When 5 X 10(5) cord blood lymphocytes were seeded into the semisolid agar medium immediately after EBV exposure, 3-10 times more colonies developed in the presence of the plant extracts at their optimal doses. When a smaller number of 5 X 10(4) cells were seeded, transformed colonies were also observed in the presence of the plant extracts but not in their absence. All of the colonies picked up from the agar medium were EBV-determined nuclear antigen (EBNA)-positive and showed the typical blastoid morphology. There were no colonies detected in the EBV-uninfected cultures with the extracts, indicating that the virus was required for the promotion by these plant extracts of this lymphocyte transformation. Euphorbiaceae plants are known to be employed as local herbal drugs in southern China and tropical Africa, and the possible role as a co-factor of the plant extracts in the development of nasopharyngeal carcinoma (NPC) and African Burkitts lymphoma (BL) is discussed.

Chromosome translocation and c-MY Cactivation by Epstein-Barr virus and Euphorbia tirucalli in B lymphocytes

Dual exposure to Epstein-Barr virus and purified 4-deoxyphorbol ester derived from the plant Euphorbia tirucalli induced a high frequency of chromosomal rearrangements in human B lymphocytes in vitro. Rearrangements most commonly affected chromosome 8, the chromosome most often showing structural changes in Burkitts lymphoma (BL) cells. E tirucalli is indigenous in parts of Africa where BL is endemic and may be an important risk factor for the disease.

Production of human monoclonal antibodies against Epstein-Barr virus-specific antigens by the virus-immortalized lymphoblastoid cell lines

The possible production of human monoclonal antibodies against Epstein-Barr virus (EBV) was assessed through the EBV immortalization technique. When individual lymphocyte samples from 50 clinical patients and healthy donors were immortalized by EBV, 4 lymphoblastoid lines yielded antibodies to EBV antigens. These positive lines were cloned and each line yielded cultures that secreted monoclonal antibodies against either viral capsid antigen (VCA) or membrane antigen (MA) component. Above all, a clonal line TAKA-SP-8 produced 5 micrograms MA antibody/10(6) cells/ml for more than 12 months. The culture fluid specifically immunoprecipitated a single polypeptide with a size of 93K from both P3HR-1 and B95-8 cell extracts. FUKA-SP-3, on the other hand, secreted 5 micrograms VCA antibody/10(6) cells/ml for at least 8 months. This antibody recognized two polypeptides with sizes of 123K and 120K, from P3HR-1 and B95-8 cell extracts, respectively. When B95-8 and P3HR-1 EBV were treated with the human MA monoclonal, both nuclear antigen (EBNA) synthesis and early antigen (EA) induction were strongly inhibited. All EBV antibody-producing cultures were exclusively achieved from splenic lymphocytes of patients with autoimmune diseases, but not from other donors.

Mass spectrometric identification of a phorbol diester, 12-O-hexadecanoylphorbol-13-acetate, an Epstein-Barr virus-activating substance, in the soil collected from under Sapium sebiferum

Soil-extracts collected from the ground from under several Euphorbiaceae plants have been known to possess Epstein-Barr virus (EBV)-activating substances which are thought to be one of the environmental co-factors causing nasopharyngeal carcinoma (NPC) in southern part of China and Burkitts lymphoma (BL) in tropical Africa. Then, a model experiment aimed at chemical characterization of such active substances was carried out using a soil-extract around Sapium sebiferum, a Japanese representative Euphorbiaceae plant. Chromatographic separation guided by the EBV early antigen (EA) inducing activity gave a highly active fraction. Application of this fraction to desorption chemical ionization mass spectrometry identified a major active substance to be 12-O-hexadecanoylphorbol-13-acetate (HPA), which originally occurs in this plant. The method in this model experiment is suggested to be applicable to other samples from the endemic areas of NPC and BL.

Epstein-Barr Virus, Burkitt’s Lymphoma, and an African Tumor Promoter

The Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that immortalizes in vitro primary human B lymphocytes. EBV is clinically associated with certain B-cell malignancies in man, such as Burkitt’s lymphoma (BL) in tropical Africa and opportunistic lymphomas in immunodeficiency (1–3). The vast majority of the African BL cases are positive for EBV genomic DNA and also for specific reciprocal chromosome translocations mostly between chromosomes 8 and 14. EBV exists worldwide, but Burkitt’s lymphoma is endemic in tropical Africa (4). In contrast to BL cells, with their specific chromosome changes, EBV-immortalized B cells have a normal karyotype (5). A cofactor(s) or risk factor(s) may therefore have to be taken into account, in addition to EBV, in the determination of the genesis of African BL. Holoendemic malaria may play a role in lowering EBV-specific cellular immunity and stimulating EBV infection (6, 7). We tried a different approach to the study of the genesis of Burkitt’s lymphoma (8).

Human Epstein-Barr Virus and Cancer

Herpesviruses are ubiquitous in various species, including humans, and some are causally associated with naturally occurring tumors. The Epstein-Barr virus (EBV), which was first discovered in a culture of African Burkitt’s lymphoma in 1964 by Epstein and coworkers Achong and Barr as a previously unknown viral agent, is now considered to be one such oncogenic herpesvirus. EBV can readily convert normal human B lymphocytes in vitro into blast cells with infinite replicative capabilities and is capable of inducing lymphomas in cotton-top marmosets. EBV widely infects humans in early childhood without any serious diseases, followed by lifelong persistence of the virus. EBV is therefore a human ubiquitous viral agent with oncogenic potential. Knowledge of these characteristic features of EBV is essential to understand the functions of this virus in humans.

Epstein-Barr virus (EBV) hypersensitivity of peripheral B lymphocytes in patients with EBV genome-positive Burkitt's lymphoma.

Peripheral blood mononuclear cells (PBMC) from four Japanese patients with Epstein-Barr virus (EBV) genome-positive Burkitts lymphoma (BL) during remission were exposed to the B95-8 strain of EBV. Maximum concentrations of the EBV-determined nuclear antigen (EBNA) before cellular DNA synthesis were similar to those of healthy counterparts. Subsequently, EBV-immortalised cell lines were established. These immortalised lymphoblastoid cells were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) and superinfected with the P3HR-1 strain of EBV. EBV early antigens (EA) and viral capsid antigen (VCA) were expressed in approximately 3-10 fold higher concentrations by these lymphoblastoid cells than by those from patients with other types of malignant neoplasia including EBV genome-negative BL and from healthy counterparts. Moderate to extremely high IgG antibody titres to EBV VCA as well as IgG antibodies to EA were demonstrated in these patients during the study. These results suggest that defective underlying cellular mechanisms for regulating the replication of EBV may be present in patients with EBV genome-positive BL.

An Environmental Plant Factor Enhancing Epstein-Barr Virus-Induced Events in East Africa

Our 3-year-survey in Kenya and Tanzania has shown a geographical coincidence of African Burkitt’s lymphoma (BL) and a particular plant species, Euphorbia tirucalli. The Euphorbia tirucalli had a potent promoter substance, mainly 4-deoxyphorbol ester, of Epstein-Barr (EBV)-induced events, induction of the latent viral genome and enhancement of the lymphocyte transformation, to grow closely associated with daily life of the local people in highly BL-endemic villages.