Search PubMed⌕ Search

Biomedical subjects

J K Yee

Publications and source records attributed to J K Yee.

At least 37 records · Page 2Linked to original sources
PubMed1993

Mutational analysis of hepatitis B virus enhancer 2.

Hepatitis B virus (HBV) infection is associated with the development of hepatocellular carcinoma in humans. HBV infection is primarily restricted to hepatocytes, and replication and gene expression of HBV require liver-specific transcription factors. Regulation of HBV gene expression has been shown to be controlled by two enhancers, and liver-specific gene expression of HBV can be attributed largely to the activity of enhancer 2. In this study, we have used mutational analysis to identify a 20-base-pair sequence motif essential for the liver-specific enhancer 2 activity. Analysis of the sequence reveals that this motif is similar to the regulatory region of several other liver-specific cellular genes, suggesting that common transcription factors may be involved in the activation of cellular as well as HBV gene expression in hepatocytes.

Base Sequence↗
PubMed1993

Transduction of a drug-sensitive toxic gene into human leukemia cell lines with a novel retroviral vector.

To investigate the possibility of killing tumor cells by the expression of an exogenously introduced toxic gene, we have constructed a novel retroviral vector (LTRNL) which has the polyA signal deleted herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene. The vector becomes toxic by treating cells expressing HSV1-tk with the antiherpetic drugs acyclovir or ganciclovir (GCV). Cells of the human leukemia lines (K562, MEG-01) were infected with this vector and two transduced cell lines (K562/LTRNL, MEG-01/LTRNL) were established. Southern blot analysis confirmed the integration of the HSV1-tk transgene in these cells and Northern blot analysis exhibited the expression of 4.8-kb viral mRNA containing the HSV1-tk gene. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay for the in vitro cytotoxic effects of GCV to these cells demonstrated that concentrations of about 2.5 microM for K562/LTRNL and 1.25 microM for MEG-01/LTRNL cells resulted in 50% inhibition of cell growth after 72 hr. Subcutaneous tumors of MEG-01/LTRNL in KSN nude mice, but not those of uninfected MEG-01 cells, showed durable regressions after exposure of the mice to 40 mg/kg of GCV given subcutaneously once a day for 15 days. This study indicates that the LTRNL-infected human leukemia cells exhibit inducible susceptibility to GCV.

Acyclovir↗
PubMed1992

Regulation of hepatitis B virus gene expression by its two enhancers.

Hepatitis B virus (HBV) infection causes acute and chronic hepatitis and is closely associated with the development of hepatocellular carcinoma. The principal site of HBV infection is liver, and HBV actively replicates in hepatocytes. Two regions of the HBV genome have been shown previously to display properties of a transcriptional enhancer. In this study, we show that either of the two HBV enhancers can activate all three major HBV promoters in several human hepatoma lines, and the cooperative action of the two enhancers ultimately affects overall activity of the three promoters. In addition, our data suggest that HBV gene expression may be differentially regulated by its enhancers. HBV infection causes chronic liver inflammation and hepatocyte regeneration. It has been proposed that progressive accumulation of mutations during the regenerative hyperplasia may lead to alterations in the differentiation state of hepatocytes. Thus, the development of two differentially regulated enhancers may reflect a strategy of HBV to replicate efficiently in less differentiated hepatocytes during hepatocyte regeneration or hepatocarcinogenesis.

Carcinoma, Hepatocellular↗
PubMed1992

Suppression of acute lymphoblastic leukemia by the human wild-type p53 gene.

Independent mutations in both alleles of the p53 tumor suppressor gene are a frequent finding in human T-cell acute lymphoblastic leukemia (T-ALL) cell lines and in the cells of some T-ALL patients in relapse. One major goal of studying the status of p53 (and other tumor suppressor genes) in human cancer is to facilitate the suppression of the tumorigenic phenotype through the restoration of the expression of the wild-type allele. While the efficient insertion of a suppressor into all cells of solid/metastatic human tumors may at present be impossible, insertion into leukemia cells may be feasible due to the accessibility of the leukemia cells in the body. To examine the feasibility of suppressing the tumorigenicity of human T-leukemia cells, the human T-ALL cell line Be-13, which lacks endogenous p53 protein, was infected with a recombinant retrovirus encoding the wild-type allele of human p53 (hwtp53). Expression of p53 reduced the growth rate of infected Be-13 cells in vitro, suppressed colony formation in methylcellulose cultures, and abrogated their tumorigenic phenotype in nude mice in vivo. These results suggest that suppression of the leukemic phenotype of relapse T-ALL-derived Be-13 cells is feasible. Acute leukemia cell suppression via high-efficiency infection with retroviruses encoding wtp53 may be feasible and beneficial in T-ALL cases as part of a bone marrow transplantation regimen in an effort to reduce the frequency of posttransplantation relapse.

Animals↗
PubMed1992

Repression of liver-specific hepatitis B virus enhancer 2 activity by adenovirus E1A proteins.

Two regions of the hepatitis B virus (HBV) genome have been shown to display properties of a transcriptional enhancer. Enhancer 1 is active in most hepatoma lines examined as well as in some non-hepatocyte-derived cell lines. In contrast, enhancer 2 activity is strictly liver specific. In this study, we show that adenovirus E1A expression in the highly differentiated human hepatoma line Huh6 strongly inhibits HBV enhancer 2-stimulated transcription while having no effect on HBV enhancer 1 activity. A sequence motif in HBV enhancer 2 which is essential for its enhancer function is the target for E1A-mediated repression. The repression of HBV enhancer 2 activity is mediated through the N-terminal region of the E1A proteins known to bind a 300-kDa cellular protein. Our results suggest that HBV enhancer function may be modulated by a cellular mechanism similar to E1A-mediated transcriptional repression.

Adenovirus E1A Proteins↗
PubMed1991

Efficient gene expression in mammalian cells from a dicistronic transcriptional unit in an improved retroviral vector.

We have studied the properties of dicistronic transcriptional units in retroviral vectors. In these vectors, the promoter in the 5' retroviral long terminal repeat (LTR) controls expression of both an upstream cistron (luc) encoding firefly luciferase and a downstream cistron (neo), a selectable marker encoding neomycin phosphotransferase (NPTII). By assaying for simultaneous expression of luc and neo after transfection or infection of hamster BHK, rat 208F, and mouse retroviral packaging cell lines, we have identified important factors that affect expression from the downstream cistron, including the presence of intercistronic ATG sequences, the length of the intercistronic sequence and conformity of the sequence surrounding the downstream start codon to the eukaryotic consensus sequence. Optimized dicistronic vectors produced amounts of NPTII comparable to a vector in which neo was driven by a strong internal promoter consisting of a modified Rous sarcoma virus LTR. Additionally, they produced higher virus titers and demonstrated improved stability of gene expression in the absence of selection. By virtue of their physical compactness and elimination of the need for a separate promoter for every gene, dicistronic transcriptional units allow the introduction of larger genes into retroviral vectors and may allow for more than two genes to be placed in a single vector.

Avian Sarcoma Viruses↗
PubMed1991

Pseudotype formation of murine leukemia virus with the G protein of vesicular stomatitis virus.

Mixed infection of a cell by vesicular stomatitis virus (VSV) and retroviruses results in the production of progeny virions bearing the genome of one virus encapsidated by the envelope proteins of the other. The mechanism for the phenomenon of pseudotype formation is not clear, although specific recognition of a viral envelope protein by the nucleocapsid of an unrelated virus is presumably involved. In this study, we used Moloney murine leukemia virus (MoMLV)-based retroviral vectors encoding the gene for neomycin phosphotransferase to investigate the interaction between the VSV G protein and the retroviral nucleocapsid during the formation of MoMLV(VSV) pseudotypes. Our results show that VSV G protein can be incorporated into the virions of retrovirus in the absence of other VSV-encoded proteins or of retroviral envelope protein. Infection of hamster cells by MoMLV(VSV) pseudotypes gave rise to neomycin phosphotransferase-resistant colonies, and addition of anti-VSV serum to the virus preparations completely abolished the infectivity of MoMLV(VSV) pseudotypes. It should be possible to use existing mutants of VSV G protein in the system described here to identify the signals that are important for the formation of MoMLV(VSV) pseudotypes.

Animals↗
PubMed1989

A liver-specific enhancer in the core promoter region of human hepatitis B virus.

An 88-base pair fragment in the core promoter of the human hepatitis B virus (HBV) contains a functional promoter and a strong liver-specific enhancer. This enhancer functions in human hepatoma cells, where it is much more active than the previously described HBV enhancer in stimulating expression of the linked bacterial chloramphenicol acetyltransferase gene expressed from heterologous promoters. Studies of the role of this enhancer-promoter in HBV may help to clarify mechanisms of gene expression in cells infected with HBV and the role of the virus in the pathogenesis of hepatitis and hepatocellular carcinoma.

Animals↗
PubMed1989

Factors affecting long-term stability of Moloney murine leukemia virus-based vectors.

We have examined the long-term functional and structural stability of retroviral vectors in infected murine cells. We have used Moloney murine leukemia virus-based vectors expressing human HPRT, firefly luciferase (luc), and Escherichia coli beta-galactosidase (lacZ) as reporter genes, and the human HPRT and the transposon Tn5 neomycin resistance (neo) gene as selectable markers. All vectors, whether single or double gene, yielded both stable and unstable clones. Stability of the proviruses was dependent on a number of factors, including the nature of the infected cell, the reporter gene, the integration site of the provirus, the relative positions of the component genes in multigene vectors, and the presence or absence of selection pressure. Selection pressure was helpful, but not universally effective, in maintaining provirus structural and functional integrity. Reporter gene expression from an internal promoter was likely to be unstable with or without selection for an upstream, LTR-driven neo gene. In some clones, loss of proviral gene expression was accompanied by deletions, while other inactive clones retained an apparently intact provirus. In the latter clones, treatment with 5-azacytidine failed to reactivate the reporter genes, but superinfection with helper virus resulted in the reappearance of transmissible vector, indicating a reversible epigenetic mechanism for proviral shutdown. The design of effective retroviral vectors and their possible use in vivo will require further characterization of these determinants of provirus stability.

Cell Line↗
PubMed1989

Retrovirus vector-mediated gene transfer into hepatocytes.

The introduction and stable expression of foreign genes in mammalian hepatocytes have recently been demonstrated by several techniques, including the use of physical approaches such as direct injection of a DNA calcium phosphate precipitate, electroporation of plasmid DNA and the exposure to liposome-erythrocyte ghost complexes as well as the biological approach of infection of primary hepatocyte cultures with retrovirus vectors. Retrovirus-mediated transduction has proven to be highly advantageous in many in vitro gene transfer studies of mammalian cells, and recent results with primary rat liver cultures have begun to define the conditions under which foreign genes can be transduced into hepatocytes in vitro. Fully differentiated hepatocytes are poorly susceptible, if at all, to infection with retroviruses, a phenomenon due at least in part to the fact that cells must undergo replication in order to retroviral integration and gene expression to occur. Hepatocytes are largely resting cells, arrested in G0. Nevertheless, primary cultures of hepatocytes are known to demonstrate a partial de-differentiation in vitro and undergo several rounds of replication. During a narrow period of time early in primary culture correlated roughly with the de-differentiation, adult hepatocytes do become susceptible to efficient infection with retrovirus vectors. In infected cells, gene expression remains relatively stable for the several week duration of the primary culture. It is not known if the restriction of virus infection in hepatocytes is a function of the state of cellular differentiation, of the availability of viral receptors or of other factors.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Suppression of the neoplastic phenotype by replacement of the RB gene in human cancer cells.

Mutational inactivation of the retinoblastoma susceptibility (RB) gene has been proposed as a crucial step in the formation of retinoblastoma and other types of human cancer. This hypothesis was tested by introducing, via retroviral-mediated gene transfer, a cloned RB gene into retinoblastoma or osteosarcoma cells that had inactivated endogenous RB genes. Expression of the exogenous RB gene affected cell morphology, growth rate, soft agar colony formation, and tumorigenicity in nude mice. This demonstration of suppression of the neoplastic phenotype by a single gene provides direct evidence for an essential role of the RB gene in tumorigenesis.

Cell Division↗
PubMed1987

Grafting genetically modified cells to the brain: possibilities for the future.

Diagnostic and therapeutic approaches to disorders of the central nervous system (CNS) are particularly difficult to develop because of the relative inaccessibility of the mammalian brain to study and chemical treatment, the complexity and interconnectedness of CNS subsystems, and the profound and continued lack of fundamental understanding of the relationship between structure and function in the CNS. Neural grafting in the CNS has recently suggested a potential approach to CNS therapy through the selective replacement of cells lost as a result of disease or damage. Independently, studies aimed at direct genetic therapy in model systems have recently begun to suggest conceptually new approaches to the treatment of several kinds of human genetic disease, especially those caused by single-gene enzyme deficiencies. We suggest that a combination of these two approaches, namely the grafting into the CNS of genetically modified cells, may provide a new approach toward the restoration of some functions in the damaged or diseased CNS. We present evidence for the feasibility of this approach, including a description of some current techniques for mammalian cell gene transfer and CNS grafting, and several possible approaches to clinical applications.

Brain↗
PubMed1987

Epitope insertion into the human hypoxanthine phosphoribosyltransferase protein and detection of the mutant protein by an anti-peptide antibody.

The translational stop codon TAA of the human hypoxanthine phosphoribosyltransferase (HPRT) cDNA has been changed to GAA by site-specific mutagenesis. This modification extends the open reading frame to a downstream stop codon and results in the addition of a unique negatively charged hexapeptide to the C terminus of human HPRT protein. The mutated cDNA was transferred into HPRT-deficient rodent cells by retroviral vector infection, and the expressed enzyme was found to be fully active. An antibody against a synthetic octapeptide corresponding to the mutated HPRT C terminus precipitated the HPRT protein specifically from cells infected with the mutant virus and not infected with the wild-type HPRT virus. The technique of inserting a novel epitope into a protein by site-directed mutagenesis should be generally applicable in studies of the regulation of gene expression in vitro and in vivo.

Epitopes↗
PubMed1987

Expression of retrovirally transduced genes in primary cultures of adult rat hepatocytes.

Differentiated primary rat hepatocyte cultures have been infected with retroviral vectors expressing human hypoxanthine/guanine phosphoribosyltransferase or the transposon Tn5 neomycin-resistance gene. Expression of the markers was detected only after infection of the cells during a short period of cell replication and transient dedifferentiation from days 1 to 5 of culture. Provirus integrated during that period remains fully expressed during the entire subsequent stationary period of culture up to at least 25 days. Selection with the neomycin analogue G418 of cells infected with the neomycin vector led to the appearance of cells with hepatocyte morphology in which newly synthesized albumin was detectable by immunoprecipitation, indicating successful infection of hepatocytes.

Animals↗
PubMed1987

Gene expression from transcriptionally disabled retroviral vectors.

Retroviral vectors are used for the efficient transfer of foreign genes into mammalian cells. We report here the construction of murine retrovirus-based vectors carrying the full-length cDNA for human hypoxanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8) and from which the enhancer sequences, the "CAAT box," and the "TATA box" in the long terminal repeats (LTRs) have been deleted. After infection of HPRT-deficient rat cells by the vectors, transcriptional activity from the 5' LTR was undetectable and expression of the HPRT cDNA was dependent on an internal promoter. Removal of the LTR regulatory elements increased HPRT gene expression from an internal promoter, indicating interference between the two sets of transcriptional signals. Such disabled vectors may reduce the likelihood of undesirable genetic changes through insertional mutagenesis in cells infected with retroviral vectors.

Base Sequence↗