Pegah Johansson

Nuclear Factor-κB Binds to the Epstein-Barr Virus LMP1 Promoter and Upregulates Its Expression

ABSTRACT The latent membrane protein 1 (LMP1) oncogene carried by Epstein-Barr virus (EBV) is essential for transformation and maintenance of EBV-immortalized B cells in vitro, and it is expressed in most EBV-associated tumor types. The activation of the NF-κB pathway by LMP1 plays a critical role in the upregulation of antiapoptotic proteins. The EBV-encoded EBNA2 transactivator is required for LMP1 activation in latency III, while LMP1 itself appears to be critical for its activation in the latency II gene expression program. In both cases, additional viral and cellular transcription factors are required in mediating transcription activation of the LMP1 promoter. Using DNA affinity purification and chromatin immunoprecipitation assay, we showed here that members of the NF-κB transcription factor family bound to the LMP1 promoter in vitro and in vivo. Electrophoretic mobility shift assay analyses indicated the binding of the p50-p50 homodimer and the p65-p50 heterodimer to an NF-κB site in the LMP1 promoter. Transient transfections and reporter assays showed that the LMP1 promoter is activated by exogenous expression of NF-κB factors in both B cells and epithelial cells. Exogenous expression of NF-κB factors in the EBNA2-deficient P3HR1 cell line induced LMP1 protein expression. Overall, our data are consistent with the presence of a positive regulatory circuit between NF-κB activation and LMP1 expression.

SCF-FBXO31 E3 Ligase Targets DNA Replication Factor Cdt1 for Proteolysis in the G2 Phase of Cell Cycle to Prevent Re-replication

Background: SCF E3 ligases regulate degradation of proteins involved in many processes including cell cycle progression and DNA repair. Results: SCF-FBXO31 interacts with and regulates the degradation of Cdt1 in G2 phase. Conclusion: FBXO31 regulates Cdt1 proteolysis during the G2 phase to prevent re-replication. Significance: SCF-FBXO31 regulation of Cdt1 is a novel pathway involved in the precise regulation of DNA replication. FBXO31 was originally identified as a putative tumor suppressor gene in breast, ovarian, hepatocellular, and prostate cancers. By screening a set of cell cycle-regulated proteins as potential FBXO31 interaction partners, we have now identified Cdt1 as a novel substrate. Cdt1 DNA replication licensing factor is part of the pre-replication complex and essential for the maintenance of genomic integrity. We show that FBXO31 specifically interacts with Cdt1 and regulates its abundance by ubiquitylation leading to subsequent degradation. We also show that Cdt1 regulation by FBXO31 is limited to the G2 phase of the cell cycle and is independent of the pathways previously described for Cdt1 proteolysis in S and G2 phase. FBXO31 targeting of Cdt1 is mediated through the N terminus of Cdt1, a region previously shown to be responsible for its cell cycle regulation. Finally, we show that Cdt1 stabilization due to FBXO31 depletion results in re-replication. Our data present an additional pathway that contributes to the FBXO31 function as a tumor suppressor.

The Sp1 transcription factor binds to the G-allele of the –1087 IL-10 gene polymorphism and enhances transcriptional activation

The objectives of this study were to evaluate the influence of the −1087 single nucleotide polymorphism (SNP) on the gene expression of interleukin (IL)-10 and to identify transcription factors binding to this site in B cells. Using electrophoretic mobility-shift assays and nuclear extract from the DG75 B-cell line, we demonstrated that the Sp1 transcription factor bound to the −1087 G-allele of the IL-10 promoter and that the transcription factors PU.1 and Spi-B bound to both the G- and A-alleles. Transient transfections showed that lipopolysaccharide stimulation resulted in a 15-fold increase in promoter activity for the G-allele as compared to a 6-fold increase for the A-allele. Co-transfection with Sp1 expression vector in Sp1-deficient SL2 cells leading to Sp1 binding to the G-allele of the −1087 SNP resulted in increased IL-10 promoter activity. The results suggest a role for Sp1 transcription factor in the activation of IL-10 through the G-allele of the −1087 SNP in response to inflammatory signals.

Role of a consensus AP-2 regulatory sequence within the Epstein-Barr Virus LMP1 promoter in EBNA2 mediated transactivation

The Epstein-Barr virus (EBV) tumor-associated latent membrane protein 1 (LMP1) gene expression is transactivated by EBV nuclear antigen 2 (EBNA2) in human B cells. We previously reported that an E-box element at the LMP1 regulatory sequence (LRS) represses transcription of the LMP1 gene through the recruitment of a Max-Mad1-mSin3A complex. In the present study, using deletion/mutation analysis, and electrophoretic mobility shift assays, we show that the promoter region adjacent to the E-box (−59/−67) is required for the full repression conferred by E-box binding proteins. The repressive effect of these factors was overcome by an inhibitor of histone deacetylation, Trichostatin A (TSA), concurring with the reports that histone deacetylation plays an important role in repression mediated by Max-Mad1-mSin3A complex. Furthermore, ChIP analyses showed that histones at the transcriptionally active LMP1 promoter were hyperacetylated, whereas in the absence of transcription they were hypoacetylated. EBNA2 activation of the promoter required a consensus AP-2 sequence in the −103/−95 LRS region. While EMSA results and the low level of AP-2 factors expression in B cells argue against known AP-2 factors binding to this site, several pieces of evidence point to a similar mechanism of promoter activation as seen by AP-2 factors. We conclude that an AP-2 site-binding factor and EBNA2 act in concert to overcome the repression of the LMP1 promoter via the consensus AP-2 site. This activation showed strong correlation with histone hyperacetylation at the promoter, indicating this to be a major mechanism for the EBNA2 mediated LMP1 transactivation.

The p38 Signaling Pathway Upregulates Expression of the Epstein-Barr Virus LMP1 Oncogene

ABSTRACT The Epstein-Barr virus (EBV)-encoded LMP1 oncogene has a role in transformation, proliferation, and metastasis of several EBV-associated tumors. Furthermore, LMP1 is critically involved in transformation and growth of EBV-immortalized B cells in vitro. The oncogenic properties of LMP1 are attributed to its ability to upregulate anti-apoptotic proteins and growth signals. The transcriptional regulation of LMP1 is dependent on the context of cellular and viral proteins present in the cell. Here, we investigated the effect of several signaling pathways on the regulation of LMP1 expression. Inhibition of p38 signaling, using p38-specific inhibitors SB203580 and SB202190, downregulated LMP1 in estrogen-induced EREB2.5 cells. Similarly, p38 inhibition decreased trichostatin A-induced LMP1 expression in P3HR1 cells. Exogenous expression of p38 in lymphoblastoid cell lines (LCLs) led to an increase in LMP1 promoter activity in reporter assays, and this activation was mediated by the previously identified CRE site in the promoter. Inhibition of p38 by SB203580 and p38-specific small interfering RNA (siRNA) also led to a modest decrease in endogenous LMP1 expression in LCLs. Chromatin immunoprecipitation indicated decreased binding of CREB-ATF1 to the CRE site in the LMP1 promoter after inhibition of the p38 pathway in EREB2.5 cells. Taken together, our results suggest that an increase in p38 activation upregulates LMP1 expression. Since p38 is activated in response to stimuli such as stress or possibly primary infection, a transient upregulation of LMP1 in response to p38 may allow the cells to escape apoptosis. Since the p38 pathway itself is activated by LMP1, our results also suggest the presence of an autoregulatory loop in LMP1 upregulation.

In-solution staining and arraying method for the immunofluorescence detection of γH2AX foci optimized for clinical applications.

Immunofluorescence quantification of γH2AX foci is a powerful approach to quantify DNA double-strand breaks induced by cancer therapy or accidental exposure to ionizing radiation. Here we report a modification to the γH2AX immunofluorescence labeling method, whereby cells are stained in-solution before being spotted and fixed onto microscope slides. Our modified method allows arraying of 16 patient samples/slide ready for foci counting in 2 h and demonstrated reliably detection of γH2AX foci in mononuclear cells prepared from patients who had undergone radiation therapy.

Measurement of H2AX Phosphorylation as a Marker of Ionizing Radiation Induced Cell Damage

Cancer therapy often involves drugs or radiation, which induce DNA double-strand breaks (DSBs) and cause death of tumor cells. A dilemma with radiation and chemotherapy is the narrow therapeutic window and the fact that the sensitivity to DNA damage varies greatly in the human population, resulting in severe side effects and therapy-related deaths. Radiation and chemotherapy are usually planned during 1-3 weeks and delivered over a period of weeks to months. DNA damage testing of the patient could therefore be performed as part of the therapy planning. The level of DNA damage and DNA repair capacity could be measured after the first dose of chemotherapy or radiation, to allow personalized dosing during the following weeks of treatment. Induction of DSBs is followed by rapid formation of gamma-H2AX foci, with thousands of H2AX molecules being phosphorylated in the chromatin flanking the DSB site. Gamma-H2AX foci, each representing one radiation-induced DSB, can be measured by flow cytometry or counted in cell nuclei by fluorescence microscopy. In this chapter we will focus on radiation-induced DNA damage and its biological effects. We will also describe available methods for gammaH2AX detection and discuss its possible use in clinical practice.

Validation of a flow cytometry‐based detection of γ‐H2AX, to measure DNA damage for clinical applications

The nucleosomal histone protein H2AX is specifically phosphorylated (γ‐H2AX) adjacent to DNA double‐strand breaks (DSBs) and is used for quantifying DSBs. Many chemotherapies and ionizing radiation (IR) used in cancer treatment result in DSBs. Therefore, γ‐H2AX has a significant potential as a biomarker in evaluating patient sensitivity and responsiveness to IR and chemotherapy.

Calibrators for Clinical Measurements of Phosphorylated H2AX in Patient Cells by Flow Cytometry

Agents that induce DNA double-strand breaks (DSBs), such as ionizing radiation, are frequently used in cancer therapy. H2AX is rapidly phosphorylated in response to DSBs and serves as a way to measure the extent of DSBs induced in patient cells. We have previously reported a flow cytometry-based method for measuring H2AX phosphorylation in pa- tient cells undergoing radiotherapy. To be able to implement measurement of H2AX phosphorylation in clinical practice, we have characterized calibrators for the flow cytometry analysis based on phosphopeptide-coated beads and fixed cells. The calibrator beads and fixed cells lost less than 11% of the signal after storage for 40 days under optimal conditions and were able to correct for day-to-day variation in method performance.

The Endothelin receptor type A is a downstream target of Hoxa9 and Meis1 in Acute Myeloid Leukemia

Endothelin receptor type A (EDNRA) is known as a mediator of cell proliferation and survival. Aberrant regulation of EDNRA has been shown to play a role in tumor growth and metastasis. Using a global gene expression screen, we found that expression of Ednra was upregulated in murine leukemia inducing cells co-expressing Hoxa9 and Meis1 compared to cells only expressing Hoxa9. The aim of this study was to explore the role of Ednra in leukemogenesis further. In a murine bone marrow transplantation model, mice transplanted with cells overexpressing Ednra and Hoxa9 succumbed to leukemia significantly earlier than mice transplanted with cells overexpressing Hoxa9 only. Furthermore, overexpression of Ednra led to increased proliferation and resistance to apoptosis of bone marrow cells in vitro. We could also show that Meis1 binds to the Ednra promoter region, suggesting a regulatory role for Meis1 in Ednra expression. Taken together, our results suggest a role for Ednra in Hoxa9/Meis1-driven leukemogenesis.