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K D Robertson

Publications and source records attributed to K D Robertson.

24 records · Page 2Linked to original sources
PubMed1997

Mapping promoter regions that are hypersensitive to methylation-mediated inhibition of transcription: application of the methylation cassette assay to the Epstein-Barr virus major latency promoter.

Methylation-associated transcriptional repression is recognized in many settings and may play a role in normal differentiation and in tumorigenesis. Both sequence-specific and nonspecific mechanisms have been elaborated. Recently, we have presented evidence that methylation-associated inhibition of the Epstein-Barr virus (EBV) major latency promoter (BamHI C promoter or Cp) in Burkitt's lymphoma and Hodgkin's disease may play an important role in the pathogenesis of these tumors by protecting them from CD8+ cytotoxic T-cell immunosurveillance. The mechanism of transcriptional repression may relate to specific inhibition of the binding of a cellular transcription factor by methylation. To dissect the viral promoter with regard to transcriptional sensitivity to methylation, we have devised an assay that allows the methylation of discrete regions of reporter plasmids. During the course of the assay, methylation patterns appeared to be stable; there was no evidence of either spread or reversal of the imposed methylation pattern. Application of the assay to the 3.8-kb region upstream of the major EBV latency promoter with natural Cp reporter plasmids showed that sensitivity to methylation is not homogeneously distributed but is concentrated in two discrete regions. The first of these methylation-hypersensitive regions (MHRI) is the previously identified EBNA-2 response element, which includes the methylation-sensitive CBF2 binding site. The second (MHRII) is a sequence further downstream whose potential role in methylation-mediated transcriptional repression had been previously unsuspected. In chimeric enhancer/promoter plasmids, methylation of this downstream region was sufficient to virtually abolish simian virus 40 enhancer-driven transcription. Further dissection indicated that methylation of the EBNA-2 response element (MHRI) was sufficient to abolish EBNA-2-mediated Cp activity while methylation of a region including the EBNA-2 response element and downstream sequence (MHRI and MHRII) was sufficient to abolish all Cp-mediated reporter activity, including that driven by the EBNA-1-dependent enhancer in the origin of plasmid replication, oriP.

Binding Sites↗
PubMed1996

CpG methylation of the major Epstein-Barr virus latency promoter in Burkitt's lymphoma and Hodgkin's disease.

The Epstein-Barr virus (EBV) latency C promoter drives expression of a family of viral proteins commonly targeted by CD8 cytotoxic T cells. These proteins are not generally expressed in African Burkitt's lymphoma and in EBV-associated Hodgkin's disease. The failure to express these proteins is almost certainly an important factor in the evasion of immunosurveillance by EBV-associated tumors. In a previous study, we have shown that transcriptional activation of the C promoter is inhibited by methylation of a particular CpG site upstream of the promoter that prevents binding of a cellular protein (CBF2), and we have shown that this and adjacent CpG sites are methylated in a Burkitt's lymphoma cell line. In the present study, we show that CpG sites in the CBF2 binding region are predominantly methylated in African Burkitt's lymphoma and in EBV-associated Hodgkin's disease. In addition, we present the first direct evidence that the C promoter is transcriptionally silent in Burkitt's lymphoma. In contrast, we show a complete absence of methylation in the CBF2 binding region in a case of reversible EBV-associated B-cell lymphoma arising in an immunocompromised patient whose tumor shows C promoter transcriptional activity. By inhibiting expression of highly antigenic viral proteins, methylation of transcriptional control sequences may veil the presence of virus in tumor tissue from CD8(+) cytotoxic T-cell immune surveillance and thus facilitate viral tumorigenesis.

5-Methylcytosine↗
PubMed1996

Epstein-Barr virus as a therapeutic target in Hodgkin's disease and nasopharyngeal carcinoma.

Conventional approaches to the treatment of malignancy are often not curative or are associated with serious complications. New approaches to treatment are needed. A variety of specific approaches to the destruction of virus-associated tumor cells are illustrated in the context of EBV-associated Hodgkin's disease and nasopharyngeal carcinoma. Viral antigens expressed by tumors may be targeted by cytotoxic T cells. Other viral antigens not naturally expressed by tumors may be induced by pharmacologic manipulations such as treatment with demethylating agents. Viral enzymes not naturally expressed by tumors such as thymidine kinase may be induced by protein kinase C activators, thus rendering tumor cells sensitive to killing by ganciclovir.

Antigens, Viral↗
PubMed1995

Transcriptional activation of the Epstein-Barr virus latency C promoter after 5-azacytidine treatment: evidence that demethylation at a single CpG site is crucial.

The Epstein-Barr Virus (EBV) latency C promoter (Cp) is the origin of transcripts for six viral proteins. The promoter is active in lymphoblastoid B-cell lines but silent in many EBV-associated tumors and tumor cell lines. In these latter cell lines, the viral episome is hypermethylated in the vicinity of this promoter. We show that in such a cell line (Rael, a Burkitt's lymphoma line), 5-azacytidine inhibits DNA methyltransferase, brings about demethylation of EBV genomes, activates Cp transcription, and induces the expression of EBNA-2. Investigation of the phenomenon demonstrates the importance of the methylation status of a particular CpG site for the regulation of the Cp: (i) genomic sequencing shows that this site is methylated when the Cp is inactive and is not methylated when the promoter is active; (ii) methylation or transition mutation at this site abolishes complex formation with a cellular binding activity (CBF2) as determined by electrophoretic mobility shift analyses, competition binding analyses, and DNase I footprinting; and (iii) a single C --> T transition mutation at this site is associated with a marked reduction (> 50-fold) of transcriptional activity in a reporter plasmid. Thus, the CBF2 binding activity is shown to be methylation sensitive and crucial to EBNA-2-mediated activation of the Cp.

Antigens, Viral↗
PubMedDate not supplied

Kinetic analysis of the reaction of melphalan with water, phosphate, and glutathione.

The reaction kinetics of the hydrolysis, phosphatolysis, glutathionyl conjugation, and alpha-glutathione-S-transferase (GST)-catalyzed glutathione conjugation of [3H-ring]melphalan were investigated at pH 6.5 and 7.4. The distribution of products relative to the initial parent compound radioactivity over time was measured by HPLC and analyzed by nonlinear regression techniques using a system of rate and distribution equations that describe the complete precursor-product pathways applicable to each reaction condition. The kinetic parameters calculated in the analysis were the first- and second-order rate constants of formation and the product ratios of the aziridinium intermediates. The second-order rate constants were normalized to those obtained for the hydroxylation reactions to yield relative rate constants. The first-order rate constants of aziridinium ion formation from melphalan and from all the monosubstituted melphalan species, except chloro, hydroxyl melphalan, were similar under all reaction conditions. The relative second-order rate constant for nonenzymatic glutathionylation of the aziridinium intermediate was 7 times larger at pH 7.4 than at pH 6.5. GST was found to react only with the aziridinium intermediate formed from melphalan and to dissociate slowly from the resultant GST-product complex (dissociation half-life, 1 hr at pH 7.4 and 3.5 hr at pH 6.5). The kinetic parameter estimates found in this study can be used to make preliminary calculations of the impact that cellular phosphate, glutathione, and GST concentrations will have on the intracellular detoxication of melphalan.

Glutathione↗