Biomedical subjects

Shannon C Kenney

Publications and source records attributed to Shannon C Kenney.

16 recordsLinked to original sources

PubMed2006

Valproic acid enhances the efficacy of chemotherapy in EBV-positive tumors by increasing lytic viral gene expression.

EBV infection in tumor cells is generally restricted to the latent forms of viral infection. Switching the latent form of viral infection into the lytic form may induce tumor cell death. We have previously reported that certain chemotherapy agents can increase the amount of lytic viral gene expression in EBV-positive tumor cells. In this report, we have explored the potential utility of valproic acid (VPA), an anti-seizure drug that also has strong histone deacetylase inhibitory activity, for activating lytic viral gene expression in EBV-positive tumors. Although VPA treatment alone induced only a modest increase in the level of lytic viral gene expression, it strongly enhanced the ability of chemotherapeutic agents to induce lytic EBV gene expression in EBV-positive epithelial and lymphoid cells in vitro. Furthermore, VPA enhanced cell killing in vitro by chemotherapeutic agents in lymphoblastoid cells and gastric cells (AGS) containing wild-type EBV. In contrast, VPA did not enhance the cytotoxicity of chemotherapy in lymphoblastoid cells containing a lytic-defective (BZLF1-knockout) form of EBV or in EBV-negative AGS cells. Finally, we found that the combination of VPA and chemotherapy was significantly more effective in inhibiting EBV-driven lymphoproliferative disease in severe combined immunodeficient mice than chemotherapy alone. These results suggest that VPA could potentiate the efficacy of chemotherapy for EBV-positive tumors in patients.

Animals↗

PubMed2005

BZLF1 activation of the methylated form of the BRLF1 immediate-early promoter is regulated by BZLF1 residue 186.

The Epstein-Barr virus (EBV) genome is highly methylated in latently infected cells. We recently reported that the EBV immediate-early (IE) protein BZLF1 (Z) preferentially binds to and activates transcription from the methylated form of the BRLF1 IE gene promoter (Rp). We now report that serine residue 186 in the Z DNA-binding domain plays an important role in the ability of Z to bind to and activate methylated Rp. A Z mutant containing an alanine residue at position 186 [Z(S186A)] was significantly defective in binding to methylated, as well as unmethylated, ZREs (Z-responsive elements) in Rp and was unable to activate lytic EBV gene transcription from the methylated or demethylated form of the viral genome. A Z mutant containing threonine at residue 186 [Z(S186T)] bound only to the methylated form of the ZRE-2 site in Rp and induced lytic EBV gene transcription from the methylated, but not demethylated, form of the viral genome. The defect in both of these mutants was primarily due to an inability to activate the Rp in the context of the viral genome. Finally, a Z mutant containing an aspartic acid at position 186 [Z(S186D)] did not bind to either the consensus AP-1 site or to the methylated or unmethylated Rp ZRE-2 site and did not induce lytic gene transcription. These results indicate that replacement of serine with threonine at residue 186 in the Z DNA-binding domain differentially affects its ability to reactivate the unmethylated, versus methylated, viral genome.

Cell Line↗

PubMed2005

The Epstein-Barr virus protein BMRF1 activates gastrin transcription.

The Epstein-Barr virus (EBV) BMRF1 gene encodes an early lytic protein that functions not only as the viral DNA polymerase processivity factor but also as a transcriptional activator. BMRF1 has been previously shown to activate transcription of an EBV early promoter, BHLF1, though a GC-rich motif which binds to SP1 and ZBP-89, although the exact mechanism for this effect is not known (D. J. Law, S. A. Tarle, and J. L. Merchant, Mamm. Genome 9:165-167, 1998). Here we demonstrate that BMRF1 activates transcription of the cellular gastrin gene in telomerase-immortalized keratinocytes. Furthermore, BMRF1 activated a reporter gene construct driven by the gastrin promoter in a variety of cell types, and this effect was mediated by two SP1/ZBP-89 binding sites in the gastrin promoter. ZBP-89 has been previously shown to negatively regulate the gastrin promoter. However, ZBP-89 can function as either a negative or positive regulator of transcription, depending upon the promoter and perhaps other, as-yet-unidentified factors. BMRF1 increased the binding of ZBP-89 to the gastrin promoter, and a ZBP-89-GAL4 fusion protein was converted into a positive transcriptional regulator by cotransfection with BMRF1. BMRF1 also enhanced the transcriptional activity of an SP1-GAL4 fusion protein. These results suggest that BMRF1 activates target promoters through its effect on both the SP1 and ZBP-89 transcription factors. Furthermore, as the EBV genome is present in up to 10% of gastric cancers, and the different forms of gastrin are growth factors for gastrointestinal epithelium, our results suggest a mechanism by which lytic EBV infection could promote the growth of gastric cells.

Antigens, Viral↗

PubMed2005

Epstein-Barr virus lytic infection is required for efficient production of the angiogenesis factor vascular endothelial growth factor in lymphoblastoid cell lines.

Although Epstein-Barr virus (EBV)-associated malignancies are primarily composed of cells with one of the latent forms of EBV infection, a small subset of tumor cells containing the lytic form of infection is often observed. Whether the rare lytically infected tumor cells contribute to the growth of the latently infected tumor cells is unclear. Here we have investigated whether the lytically infected subset of early-passage lymphoblastoid cell lines (LCLs) could potentially contribute to tumor growth through the production of angiogenesis factors. We demonstrate that supernatants from early-passage LCLs infected with BZLF1-deleted virus (Z-KO LCLs) are highly impaired in promoting endothelial cell tube formation in vitro compared to wild-type (WT) LCL supernatants. Furthermore, expression of the BZLF1 gene product in trans in Z-KO LCLs restored angiogenic capacity. The supernatants of Z-KO LCLs, as well as supernatants from LCLs derived with a BRLF1-deleted virus (R-KO LCLs), contained much less vascular endothelial growth factor (VEGF) in comparison to WT LCLs. BZLF1 gene expression in Z-KO LCLs restored the VEGF level in the supernatant. However, the cellular level of VEGF mRNA was similar in Z-KO, R-KO, and WT LCLs, suggesting that lytic infection may enhance VEGF translation or secretion. Interestingly, a portion of the vasculature in LCL tumors in SCID mice was derived from the human LCLs. These results suggest that lytically infected cells may contribute to the growth of EBV-associated malignancies by enhancing angiogenesis. In addition, as VEGF is a pleiotropic factor with effects other than angiogenesis, lytically induced VEGF secretion may potentially contribute to viral pathogenesis.

Cell Line↗

PubMed2005

Epstein-Barr virus lytic infection contributes to lymphoproliferative disease in a SCID mouse model.

Most Epstein-Barr virus (EBV)-positive tumor cells contain one of the latent forms of viral infection. The role of lytic viral gene expression in EBV-associated malignancies is unknown. Here we show that EBV mutants that cannot undergo lytic viral replication are defective in promoting EBV-mediated lymphoproliferative disease (LPD). Early-passage lymphoblastoid cell lines (LCLs) derived from EBV mutants with a deletion of either viral immediate-early gene grew similarly to wild-type (WT) virus LCLs in vitro but were deficient in producing LPD when inoculated into SCID mice. Restoration of lytic EBV gene expression enhanced growth in SCID mice. Acyclovir, which prevents lytic viral replication but not expression of early lytic viral genes, did not inhibit the growth of WT LCLs in SCID mice. Early-passage LCLs derived from the lytic-defective viruses had substantially decreased expression of the cytokine interleukin-6 (IL-6), and restoration of lytic gene expression reversed this defect. Expression of cellular IL-10 and viral IL-10 was also diminished in lytic-defective LCLs. These results suggest that lytic EBV gene expression contributes to EBV-associated lymphoproliferative disease, potentially through induction of paracrine B-cell growth factors.

Animals↗

PubMed2005

Anti-CD70 antibodies: a potential treatment for EBV+ CD70-expressing lymphomas.

A monoclonal antibody (Rituximab) directed against the B-cell surface antigen, CD20, is increasingly used as a therapy for B-cell lymphomas. However, CD20 is expressed on all normal mature B cells and hence is not a specific tumor target. In contrast, CD70 is expressed on highly activated lymphocytes as well as on many B-cell and T-cell lymphomas but is not expressed on the great majority of B cells and T cells. In this report, we have explored the potential utility of anti-CD70 monoclonal antibodies for treatment of CD70+ EBV+ B-cell lymphomas. Using two Burkitt's lymphoma lines (Raji and Jijoye) that express surface CD70 and a CD70- Burkitt's lymphoma line (Akata), we show that two different monoclonal antibodies directed against human CD70 allow rabbit and human complement to kill EBV+ B cells in a CD70-dependent manner in vitro. In the absence of complement, neither anti-CD70 antibody induced in vitro killing of CD70+ cell lines. Importantly, i.p. injection of anti-CD70 antibodies also inhibited the growth of CD70+ Burkitt's lymphoma cells in severe combined immunodeficient mice but did not inhibit the growth of CD70- Burkitt's lymphoma cells. These results suggest that anti-CD70 antibodies may be useful for the treatment of CD70+ B-cell lymphomas.

Animals↗

PubMed2004

Reactivation of latent Epstein-Barr virus by methotrexate: a potential contributor to methotrexate-associated lymphomas.

BACKGROUND: Patients with rheumatoid arthritis or polymyositis treated with methotrexate (MTX) develop Epstein-Barr virus (EBV)-positive lymphomas more frequently than patients treated with other, equally immunosuppressive regimens. Here we determined whether MTX, in contrast to other commonly used medications for rheumatoid arthritis or polymyositis, is unique in its ability to induce the release of infectious EBV from latently infected cells. METHODS: The effect of MTX and other immunosuppressant drugs on EBV replication in vitro was assessed using latently infected EBV-positive lymphoblastoid and gastric carcinoma cell lines. Inhibitors of signal transduction pathways were used to define requirements for induction of lytic infection. Drug effects on transcription of the two EBV immediate-early promoters (BRLF1 and BZLF1) and on promoter constructs lacking cis-acting sequences required for activation by other effectors was examined using reporter gene assays. EBV viral load in rheumatoid arthritis and polymyositis patients receiving MTX was compared with that in patients receiving other immunosuppressive medications. Statistical tests were two-sided. RESULTS: MTX activated the release of infectious EBV from latently infected cell lines in vitro, and MTX treatment was associated with activation of the two viral immediate-early promoters in reporter gene assays. Induction of lytic EBV infection by MTX required the p38 MAP kinase, PI3 kinase, and MEK pathways and specific cis-acting motifs in the two viral immediate-early promoters. Patients treated with MTX-containing regimens had statistically significantly higher mean EBV loads in their blood than patients treated with immunosuppressing regimens that did not include MTX (40 EBV copies per 10(6) cellular genomes versus 5.1 copies; geometric mean fold difference in copies = 10.8, 95%, confidence interval = 3.0 to 38; P = .011). CONCLUSION: MTX may promote EBV-positive lymphomas in rheumatoid arthritis and polymyositis patients by its immunosuppressive properties as well as by reactivating latent EBV.

Arthritis, Rheumatoid↗

PubMed2004

BZLF1, an Epstein-Barr virus immediate-early protein, induces p65 nuclear translocation while inhibiting p65 transcriptional function.

We have previously demonstrated that the Epstein-Barr virus immediate-early BZLF1 protein interacts with, and is inhibited by, the NF-kappaB family member p65. However, the effects of BZLF1 on NF-kappaB activity have not been intensively studied. Here we show that BZLF1 inhibits p65-dependent gene expression. BZLF1 inhibited the ability of IL-1, as well as transfected p65, to activate the expression of two different NF-kappaB-responsive genes, ICAM-1 and IkappaB-alpha. BZLF1 also reduced the constitutive level of IkappaB-alpha protein in HeLa and A549 cells, and increased the amount of nuclear NF-kappaB to a similar extent as tumor necrosis factor-alpha (TNF-alpha) treatment. In spite of this BZLF1-associated increase in the nuclear form of NF-kappaB, BZLF1 did not induce binding of NF-kappaB to NF-kappaB responsive promoters (as determined by chromatin immunoprecipitation assay) in vivo, although TNF-alpha treatment induced NF-kappaB binding as expected. Overexpression of p65 dramatically inhibited the lytic replication cycle of EBV in 293-EBV cells, confirming that NF-kappaB also inhibits BZLF1 transcriptional function. Our results are consistent with a model in which BZLF1 inhibits the transcriptional function of p65, resulting in decreased transcription of IkappaB-alpha, decreased expression of IkappaB-alpha protein, and subsequent translocation of NF-kappaB to the nucleus. This nuclear translocation of NF-kappaB may promote viral latency by negatively regulating BZLF1 transcriptional activity. In situations where p65 activity is limiting in comparison to BZLF1, the ability of BZLF1 to inhibit p65 transcriptional function may protect the virus from the host immune system during the lytic form of infection.

Active Transport, Cell Nucleus↗

PubMed2004

The EBV lytic switch protein, Z, preferentially binds to and activates the methylated viral genome.

DNA methylation promotes gene silencing, yet the Epstein-Barr virus immediate-early protein, BZLF1 (Z), converts the virus from the latent to the lytic form of infection even when the viral genome is highly methylated. Here we show that methylation of CpG motifs in Z-responsive elements of the viral BRLF1 immediate-early promoter enhances Z binding to, and activation of, this promoter. Demethylation of the viral genome impairs Z activation of lytic viral genes. Z is the first transcription factor that preferentially binds to, and activates, a methylated promoter. These results identify an unexpected mechanism by which Epstein-Barr virus circumvents the inhibitory effects of viral genome methylation.

Binding Sites↗

PubMed2004

The Epstein-Barr virus polymerase accessory factor BMRF1 adopts a ring-shaped structure as visualized by electron microscopy.

Epstein-Barr virus (EBV) encodes a set of core replication factors used during lytic infection in human cells that parallels the factors used in many other systems. These include a DNA polymerase and its accessory factor, a helicase/primase, and a single strand binding protein. The EBV polymerase accessory factor has been identified as the product of the BMRF1 gene and has been shown by functional assays to increase the activity and processivity of the polymerase. Unlike other members of this class of factors, BMRF1 is also a transcription factor regulating certain EBV genes. Although several polymerase accessory factors, including eukaryotic proliferating cell nuclear antigen, Escherichia coli beta protein, and T4 gene 45 protein have been shown to form oligomeric rings termed sliding clamps, nothing is known about the oligomeric state of BMRF1 or whether it forms a ring. In this work, BMRF1 was purified directly from human cells infected with an adenovirus vector expressing the BMRF1 gene product. The protein was purified to near homogeneity, and examination by negative staining electron microscopy revealed large, flat, ring-shaped molecules with a diameter of 15.5 +/- 0.8 nm and a distinct 5.3-nm diameter hole in the center. The size of these rings is consistent with an oligomer of 6 monomers, nearly twice as large as the trimeric proliferating cell nuclear antigen ring. Unlike the herpes simplex virus UL42 homologue, BMRF1 was found to self-associate in solution. These findings extend the theme of polymerase accessory factors adopting ring-shaped structures and provide an example in which the ring is significantly larger than any previously described sliding clamp.

Adenoviridae↗

PubMed2004

Epstein-Barr virus immediate-early protein BZLF1 inhibits tumor necrosis factor alpha-induced signaling and apoptosis by downregulating tumor necrosis factor receptor 1.

Tumor necrosis factor alpha (TNF-alpha) is a key mediator of host immune and inflammatory responses and inhibits herpesvirus replication by cytolytic and noncytolytic mechanisms. TNF-alpha effects are primarily mediated through the major TNF-alpha receptor, TNF-R1, which is constitutively expressed in most cell types. Here we show that the Epstein-Barr virus (EBV) immediate-early protein BZLF1 prevents TNF-alpha activation of target genes and TNF-alpha-induced cell death. These effects are mediated by down-regulation of the promoter for TNF-R1. Additionally, we demonstrate that expression of TNF-R1 is downregulated during the EBV lytic replication cycle. Thus, EBV has developed a novel mechanism for evading TNF-alpha antiviral effects during lytic reactivation or primary infection.

Apoptosis↗

PubMed2004

The BRRF1 early gene of Epstein-Barr virus encodes a transcription factor that enhances induction of lytic infection by BRLF1.

The switch from the latent to the lytic form of Epstein-Barr virus (EBV) infection is mediated by expression of the viral immediate-early (IE) proteins, BZLF1 (Z) and BRLF1 (R). An EBV early protein, BRRF1 (Na), is encoded by the opposite strand of the BRLF1 intron, but the function of this nuclear protein in the viral life cycle is unknown. Here we demonstrate that Na enhances the R-mediated induction of lytic EBV infection in 293 cells latently infected with a recombinant EBV (R-KO) defective for the expression of both R and Na. Na also enhances R-induced lytic infections in a gastric carcinoma line (AGS) carrying the R-KO virus, although it has no effect in a Burkitt lymphoma line (BL-30) stably infected with the same mutant virus. We show that Na is a transcription factor that increases the ability of R to activate Z expression from the R-KO viral genome in 293 cells and that Na by itself activates the Z promoter (Zp) in EBV-negative cells. Na activation of Zp requires a CRE motif (ZII), and a consensus CRE motif is sufficient to transfer Na responsiveness to the heterologous E1b promoter. Furthermore, we show that Na enhances the transactivator function of a Gal4-c-Jun fusion protein but does not increase the transactivator function of other transcription factors (including ATF-1, ATF-2, and CREB) known to bind CRE motifs. Na expression in cells results in increased levels of a hyperphosphorylated form of c-Jun, suggesting a mechanism by which Na activates c-Jun. Our results indicate that Na is a transcription factor that activates the EBV Zp IE promoter through its effects on c-Jun and suggest that Na cooperates with BRLF1 to induce the lytic form of EBV infection in certain cell types.

Genes, Immediate-Early↗

PubMed2004

Lytic induction therapy for Epstein-Barr virus-positive B-cell lymphomas.

A novel therapy for Epstein-Barr virus (EBV)-positive tumors involves the intentional induction of the lytic form of EBV infection combined with ganciclovir (GCV) treatment. Virally encoded kinases (thymidine kinase and BGLF4) which are expressed only during the lytic form of infection convert GCV (a nucleoside analogue) into its active, cytotoxic form. However, tightly latent EBV infection in B cells has made it difficult to identify drugs that can be used clinically to induce lytic viral infection in B-cell lymphomas. Here we demonstrate that gemcitabine and doxorubicin (but not 5-azacytidine, cis-platinum, or 5-fluorouracil) induce lytic EBV infection in EBV-transformed B cells in vitro and in vivo. Gemcitabine and doxorubicin both activated transcription from the promoters of the two viral immediate-early genes, BZLF1 and BRLF1, in EBV-negative B cells. This effect required the EGR-1 motif in the BRLF1 promoter and the CRE (ZII) and MEF-2D (ZI) binding sites in the BZLF1 promoter. GCV enhanced cell killing by gemcitabine or doxorubicin in lymphoblastoid cells transformed with wild-type EBV, but not in lymphoblastoid cells transformed by a mutant virus (with a deletion in the BZLF1 immediate-early gene) that is unable to enter the lytic form of infection. Most importantly, the combination of gemcitabine or doxorubicin and GCV was significantly more effective for the inhibition of EBV-driven lymphoproliferative disease in SCID mice than chemotherapy alone. In contrast, the combination of zidovudine and gemcitabine was no more effective than gemcitabine alone. These results suggest that the addition of GCV to either gemcitabine- or doxorubicin-containing chemotherapy regimens may enhance the therapeutic efficacy of these drugs for EBV-driven lymphoproliferative disease in patients.

Animals↗

PubMed2003

Virally targeted therapies for EBV-associated malignancies.

In Epstein-Barr virus (EBV)-positive lymphomas, the presence of the EBV genome in virtually all tumor cells, but very few normal cells, suggests that novel, EBV-targeted therapies could be used to treat these malignancies. In this paper, we review a variety of different approaches currently under development that specifically target EBV-infected cells for destruction. EBV-based strategies for treating cancer include prevention of viral oncogene expression, inducing loss of the EBV episome, the purposeful induction of the lytic form of EBV infection, and enhancing the host immune response to virally encoded antigens.

Antigens↗

PubMed2002

Chemotherapy induces lytic EBV replication and confers ganciclovir susceptibility to EBV-positive epithelial cell tumors.

EBV is an oncogenic herpesvirus associated with a number of human malignancies. The consistent presence of the EBV genome in certain tumors offers the potential for novel EBV-targeted therapies. EBV can infect cells in either a latent or lytic form. Here we demonstrate that a variety of chemotherapeutic agents, including cis-platinum, 5-fluorouracil (5-FU), and taxol, induce the switch from the latent to lytic form of EBV infection in tumor cells. This effect requires the protein kinase C delta, phosphatidylinositol 3'-kinase, and p38 stress mitogen-activated protein kinase signaling pathways but not caspase 3 activation. Because the lytic but not latent form of EBV infection converts the cytotoxic prodrug, ganciclovir (GCV), into its active form, we examined whether the combination of GCV and chemotherapy is more effective than chemotherapy alone for killing EBV-positive tumor cells. GCV significantly enhanced the ability of 5-FU and cis-platinum to kill EBV-positive, but not EBV-negative, gastric carcinoma cells in vitro. Most importantly, the combination of GCV and 5-FU (or GCV and cis-platinum) was much more effective in the treatment of EBV-positive nasopharyngeal carcinomas passaged in nude mice than either agent alone. These data suggest that GCV enhances the efficacy of conventional chemotherapy for the treatment of EBV-positive epithelial cell tumors.

Animals↗

PubMed2002

Use of adenovirus vectors expressing Epstein-Barr virus (EBV) immediate-early protein BZLF1 or BRLF1 to treat EBV-positive tumors.

The Epstein-Barr virus (EBV) genome is present in a variety of tumor types, including virtually all undifferentiated nasopharyngeal carcinomas (NPC) and a portion of gastric carcinomas. The uniform presence of the EBV genome in certain tumors (versus only a very small number of normal B cells) suggests that novel therapies which specifically target EBV-positive cells for destruction might be effective for treating such tumors. Although the great majority of EBV-positive tumor cells are infected with one of the latent forms of EBV infection, expression of either viral immediate-early protein (BZLF1 or BRLF1) is sufficient to convert the virus to the lytic form of infection. Induction of the lytic form of EBV infection could potentially result in death of the tumor cell. Here we have examined the efficacy of adenovirus vectors expressing the BZLF1 or BRLF1 proteins for treatment of EBV-positive epithelial tumors. The BZLF1 and BRLF1 vectors induced preferential killing of EBV-positive, versus EBV-negative, gastric carcinoma cells in vitro. Infection of C18 NPC tumors (grown in nude mice) with either the BZLF1 or BRLF1 vector, but not a control adenovirus vector, induced expression of early lytic EBV genes in tumor cells. Injection of C18 tumors with the BZLF1 or BRLF1 adenovirus vector, but not the control vector, also significantly inhibited growth of the tumors in nude mice. The addition of ganciclovir did not significantly affect the antitumor effect of the BZLF1 and BRLF1 adenovirus vectors. These results suggest a potential cancer therapy against EBV-related tumors.

Adenoviruses, Human↗