Gregory Milman

A cell culture assay for compounds which inhibit hepatitis B virus replication

Infection by hepatitis B virus (HBV) is a major worldwide health problem with over 200 million individuals chronically-infected with HBV (Beasley and Hwang, 1984). In addition to causing both acute and chronic liver disease, HBV infection is epidemiologically linked to the formation of primary hepatocellular carcinoma (HCC) (Beasley and Hwang, 1984; Popper, et al., 1987). Several types of treatment regimens have been reported for individuals with chronic HBV infection, including interferons and nucleoside analogs (see Tabor, 1987 and Thomas, 1987 for reviews). However, these treatments have moderate to serious side effects, are only transiently effective in suppressing HBV, or are effective for only a small percentage of the general population of HBV-infected individuals. A major obstacle in the development of new therapeutic agents for HBV is the lack of a suitable in vitro culture system which accurately models chronic HBV infection in man. Relevant animal models of HBV infection and disease, including HCC, have been developed (e.g. the woodchuck hepatitis virus (WHV) and its natural host, the Eastern woodchuck (Gerin, 1984; Popper et al., 1986)). Several potentially suitable HBV culture systems have been developed by transfection of human liver cell lines with cloned HBV DNA (Sureau et al., 1986; Sells et al., 1987; Tsurimoto et al., 1987). The various HBV genomic forms present in chronically infected cells represent well characterized stages of viral replication (Sum-

Efficient DNA transfection and rapid assay for thymidine kinase activity and viral antigenic determinants

We describe DEAE dextran-mediated DNA transfection in suspension which routinely gives transient gene expression in 0.1–1 % of the transfected cells. We have used normal diploid human skin fibroblasts, monkey BSC cells, and mouse L or 3T6 cells with almost equal efficiency. Gene expression is detected 1–3 days after addition of the DNA. SV40 and polyoma T and V antigen are detected by in situ immunofluorescence and thymidine kinase gene expression is detected by in situ autoradiography. The high efficiency of transfection and the speed of detection together provide a means to study transfecting gene functions that does not rely on selection to obtain stable transformants. It should be possible to screen for the expression of any gene product which can be assayed by in situ immunofluorescence or autoradiographic techniques.

Characterization of human folylpolyglutamate synthetase expressed in Chinese hamster ovary cells

Chinese hamster AUX B1 cells lack the enzyme folylpolyglutamate synthetase (FPGS) responsible for adding polyglutamates to folie acid. The human gene for FPGS was introduced into AUX B1 cells by cotransfection with human genomic DNA and either pSV2neo or pSV2gpt plasmid DNA. Cells were coselected for FPGS expression by growth in the absence of glycine, adenosine, and thymidine, and for drug resistance by growth in geneticin or mycophenolic acid. The presence of human enzyme in transfected cells was identified by relative enzyme activity determinations with ATP or dATP as substrates. The human HeLa cell enzyme displayed a 50% higher activity with dATP, and the hamster enzyme showed a 50% higher activity with ATP. Hamster and human enzymes also differed in the chain length of polyglutamates on cellular folate. Only monoglutamates were detected in AUX B1 cells, while wild-type hamster cells had predominantly hexa- and heptaglutamates, and human HeLa cells had predominantly hepta- and octaglutamates. Transformants with FPGS activity that showed a human enzyme preference for dATP also had folate polyglutamate chain lengths characteristic of the human enzyme.

In situ autoradiographic detection of folylpolyglutamate synthetase activity

The enzyme folylpolyglutamate synthetase (FPGS) catalyzes the conversion of folate (pteroylmonoglutamate) to the polyglutamate forms (pteroylpolyglutamates) that are required for folate retention by mammalian cells. A rapid in situ autoradiographic assay for FPGS was developed which is based on the folate cofactor requirement of thymidylate synthase. Chinese hamster AUX B1 mutant cells lack FPGS activity and are unable to accumulate folate. As a result, the conversion of [6-3H]deoxyuridine to thymidine via the thymidylate synthase reaction is impaired in AUX B1 cells and no detectable label is incorporated into DNA. In contrast, FPGS in wild-type Chinese hamster CHO cells causes folate retention and enables the incorporation of [6-3H]deoxyuridine into DNA. Incorporation may be detected by autoradiography of monolayer cultures or of colonies replica plated onto polyester discs. Introduction of Escherichia coli FPGS into AUX B1 cells restores the activity of the thymidylate synthase pathway and demonstrates that the E. coli FPGS enzyme can provide pteroylpolyglutamates which function in mammalian cells.

Analysis of HGPRT − CRM + human lymphoblast mutants

Three 6-thioguanine-resistant mutants of the human diploid lymphoblast line MGL-8 were studied. The inactivation by heat of both HGPRT activity and antigenicity of the HGPRT immunologically cross-reacting material of the A30 mutant cells were not protected by PRPP, indicating that the HGPRT in A30 cells has an altered PRPP binding site, leading to lack of stabilization and rapid degradation of the enzyme. Two dimensional separations of the immunoprecipitates from extracts of the parental and mutant cell lines showed that the A35 mutant CRM has a more acidic isoelectric pH, while the A30 CRM has a more basic isoelectric pH and that the A30 protein has a faster rate of degradation than the wild-type HGPRT. The A30 CRM also has a smaller molecular size than the wild-type enzyme.

Multiple species of HPRT polypeptides in mouse L cells

Abstract Mouse L cells contain three polypeptide species which react with antibody specific for HPRT; loss or diminution of these polypeptides can occur in association with a heritable deficiency in HPRT enzymatic activity. Although HPRT activity is not detectable in deficient A9 cells, two of the three polypeptide species are detectable. Clonal revertant A9 cells which spontaneously regained HPRT activity also have re-acquired apparently normal amounts of all three polypeptide species; by contrast, HPRT-positive A9 cells which acquired the enzymatic activity via human metaphase chromosome transfer exhibited the human polypeptide species characteristic of that expressed in human cells. The heterogeneity exhibited by the mouse HPRT polypeptides suggests that the active mouse enzyme may be subject to a form of modification and/or regulation which is not shared by human HPRT.

EBNA-1 Binds Specifically to Plasmids Containing a Synthetic 29 BP Binding Site

The carboxyl-third of EBNA-1 encoded in the Epstein-Barr virus (EBV) BamH1 K-restriction fragment was synthesized in bacteria (28K-EBNA). This bacterially synthesized peptide specifically binds to clustered EBV DNA sequence repeats in the Ori-P region required for plasmid maintenance [1]. To elucidate the binding properties, a synthetic DNA binding site 5′ GATCTAGGATAGCAT|ATGCTACCCCGGGG 3′ 3′ ATCCTATCGTA|TACGATGGGGCCCCCTAG 5′ was cloned as a monomer, dimer, or trimer into the BamH1 site of plasmid pUC8 creating Plasmids pR1, pR2, and pR3.

Prognostic Significance of Serial EBV Antibody Titers in Treated Nasopharyngeal Carcinoma Patients

Antibody titers to Epstein-Barr virus (EBV) antigens among nasopharyngeal carcinoma (NPC) patients may fluctuate during the course of disease. An assay pattern that is predictive of treatment effectiveness would be very useful for monitoring patients. Analyses of serial antibody titers are often complicated by censorship of the time to response. All patients will not experience the response of interest, e.g., death or disease recurrence, before the time of analysis. Another problem is that serial antibody titers are only measured at relatively infrequent intervals during follow-up. These problems are accommodated by the proportional hazards model with titer measurements treated as a time-dependent covariate. This model was used to evaluate the prognostic significance of antibody titers to EBV nuclear antigen (EBNA) and early antigen (EA) in treated NPC patients.