Gene expression profiling of Epstein-Barr virus-positive diffuse large B-cell lymphoma of the elderly reveals alterations of characteristic oncogenetic pathways.

Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (DLBCL) of the elderly (EBV[+]DLBCL-E) is classified as a subtype of DLBCL. Until now, its molecular pathogenesis has remained unknown. To identify pathways characteristic of EBV(+)DLBCL-E, gene expression profiling of five EBV(+)DLBCL-E and seven EBV-negative DLBCL (EBV[-]DLBCL) cases was undertaken using human oligonucleotide microarray analysis. Gene set enrichment analysis and gene ontology analysis showed that gene sets of the Janus kinase-signal transducer and activator of transcription (JAK-STAT) and nuclear factor kappa B (NF-κB) pathways were enriched in EBV(+)DLBCL-E cases. To confirm the results of the expression profiles, in vitro analysis was performed. Expression profiling analysis showed that high activation of the JAK-STAT and NF-κB pathways was induced by EBV infection into DLBCL cell lines. Activation of the NF-κB pathway was confirmed in EBV-infected cell lines using an electrophoretic mobility shift assay. Western blot analysis revealed an increased protein expression level of phosphorylated signal transducer and activator of transcription 3 (STAT3) in an EBV-infected cell line. Protein expression of phosphorylated STAT3 was frequently observed in lymphoma cells of EBV(+)DLBCL-E clinical samples using immunohistochemistry (EBV[+]DLBCL-E: 80.0% [n = 20/25] versus EBV[-]DLBCL: 38.9% [n = 14/36]; P = 0.001). The results of the present study suggest that activation of the JAK-STAT and NF-κB pathways was characteristic of EBV(+)DLBCL-E, which may reflect the nature of EBV-positive tumor cells. Targeting these pathways as therapies might improve clinical outcomes of EBV(+)DLBCL-E.

Generation of mouse models of lymphoid neoplasm using retroviral gene transduction of in vitro-induced germinal center B and T cells.

Evidence is accumulating that hematologic malignancies develop following acquisition of multiple genetic changes. Despite providing many insights into the way by which given genetic changes contribute to the development of disease, the generation of animal models is often laborious. We show a simplified method that allows the retroviral transduction of genes of interest into mouse B or T cells, thus leading to the rapid generation of models of lymphoid neoplasm in mice. Specifically, germinal center B cells induced in vitro from naive mouse B cells and infected with retroviruses for Myc and Bcl2 rapidly developed a neoplasm of immunoglobulin-expressing mature B cells in transplanted mice. Likewise, T cells induced in vitro from immature hematopoietic cells and infected with retroviruses for Myc, Bcl2, and Ccnd1 rapidly developed CD4(+)CD8(-) and CD4(+)CD8(+) T cell neoplasm in transplanted mice. These findings support the use of our simplified method as a versatile tool for lymphoma research.

Comprehensive gene expression profiles of NK cell neoplasms identify vorinostat as an effective drug candidate.

NK cell neoplasms are lymphoid malignancies with an aggressive clinical course. In the present study, we analyzed gene expression profiling of NK cell neoplasms and attempted to identify important molecular pathways and new effective drugs. Pathway analysis of gene expression profiles suggested the important roles of the JAK-STAT pathway, NF-κB pathway or Wnt pathways in NK cell neoplasms. Notably, western blot analysis revealed that STAT3 was expressed and phosphorylated at a higher level in NK cell lines than in normal NK cells or other cell lines. These findings indicate the occurrence of JAK-STAT activation in NK cell neoplasms. Connectivity Map (CMAP) analysis of gene expression profiles identified candidate drugs against NK cell neoplasms. Among the drugs suggested by CMAP analysis, we focused on puromycin, phenoxybenzamine, LY294002, wortmannin, vorinostat and trichostatin A because they exhibited high enrichment scores. We added these drugs to NK cell lines and other cell lines. Among the drugs, vorinostat suppressed NK cell line proliferation at a significantly lower concentration compared to other cell lines. Suppression of the JAK-STAT pathway appeared to contribute to this effect. Vorinostat may be a good candidate for use in the therapy against NK cell neoplasms.

Identification of a novel amino acid deletion mutation and a very rare single nucleotide variant in a Japanese family with type I antithrombin deficiency.

We studied a Japanese family with type I antithrombin (AT) deficiency and identified a novel in-frame deletion mutation (-ATG at nucleotide position of 2771-2773) in the AT gene, which predicted loss of a methionine (Met) at amino acid number of 103. In addition, we found a single base replacement of G to A at nucleotide position of 67 (4 base upstream to the initial codon) in the mutant allele. Since the G67A substitution in the AT gene was very rare, this family was the second case, in which the nucleotide change was transmitted. To elucidate the mechanism of AT deficiency, we transiently expressed wild type and the mutant AT (DeltaM103) in HuH-7 human hepatoma cells and performed pulse-chase studies. The experiments revealed that the mutant AT (DeltaM103) hardly secreted into the medium and underwent partial intracellular degradation. In addition, we performed luciferase reporter assay to examine the effect of G67A substitution on the AT gene expression, and found that the substitution did not reduce the luciferase activity. These results suggested that secretion defect and intracellular degradation of the variant molecule with the deletion of Met 103 were responsible for AT deficiency in this family.