EBV-encoded microRNAs (miRNAs) have been identified and their functions are being studied. The expression pattern of these miRNAs in clinical samples of EBV-associated nonHodgkin's lymphomas is unknown. We analyzed five primary ''endemic'' pediatric Burkitt's lymphomas (BL), two acquired immunodeficiency syndrome (AIDS)-related type I latency BL lines, a type III latency line, three EBV + primary effusion lymphomas (PEL), and three AIDS-related diffuse large B-cell lymphomas (DLBCL) for expression of EBV-encoded miRNAs.

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Primary effusion lymphoma (PEL) is an aggressive B-cell lymphoma most commonly diagnosed in HIV-positive patients and universally associated with Kaposi's sarcoma-associated herpesvirus (KSHV). Chemotherapy treatment of PEL yields only short-term remissions in the vast majority of patients, but efforts to develop superior therapeutic approaches have been impeded by lack of animal models that accurately mimic human disease. To address this issue, we developed a direct xenograft model, UM-PEL-1, by transferring freshly isolated human PEL cells into the peritoneal cavities of NOD/SCID mice without in vitro cell growth to avoid the changes in KSHV gene expression evident in cultured cells. We used this model to show that bortezomib induces PEL remission and extends overall survival of mice bearing lymphomatous effusions. The proapoptotic effects of bortezomib are not mediated by inhibition of the prosurvival NF-κB pathway or by induction of a terminal unfolded protein response. Transcriptome analysis by genomic arrays revealed that bortezomib down-regulated cell-cycle progression, DNA replication, and Myc-target genes. Furthermore, we demonstrate that in vivo treatment with either bortezomib or doxorubicin induces KSHV lytic reactivation. These reactivations were temporally distinct, and this difference may help elucidate the therapeutic window for use of antivirals concurrently with chemotherapy. Our findings show that this direct xenograft model can be used for testing novel PEL therapeutic strategies and also can provide a rational basis for evaluation of bortezomib in clinical trials.Kaposi's sarcoma-associated herpesvirus | Herpesvirus 8

MicroRNAs (miRNAs), like transcription factors, function as regulators of gene expression and are expressed in several eukaryotes as well as in some viruses, particularly in the family of herpesviruses (17). Study of miRNAs has exploded since their relatively recent discovery, and nearly 700 miRNA genes have been identified in humans to date (17). miRNAs have been shown to be key regulators of genes involved in innate immunity, cell growth, differentiation, tumorigenesis, and development acting at the posttranscriptional level (7,11,17,20,43,45,49). Different from small interfering RNA (siRNA), miRNAs inhibit the translation of select groups of mRNA transcripts containing imperfect annealing sequences in their 3Ј-untranslated regions (3Ј-UTRs) and less frequently through other regions of the transcript. Since miRNA profiles are different between normal and cancer cells, miRNA signatures can be used for diagnosis as well as prognosis of human malignancies (3). miR-155 is an evolutionarily conserved miRNA which plays important roles in innate immunity (49,72), and is the first oncogenic miRNA (oncomiR) shown to have increased expression in various types of cancers including lymphomas such as Hodgkin lymphoma and posttransplant lymphoproliferative disease (PTLD) (9,28,64,70), breast cancer, leukemia, pancreatic cancer, and lung cancer (7,15). miR-155 has also been shown to play a critical role in lymphocyte activation in vivo (53,67). Accumulating evidence has revealed high levels of miR-155 in Epstein-Barr virus (EBV) latency 3, but not in latency 1 (4,26,28,77), indicating that miR-155 expression is associated with EBV latency. The importance of miR-155 in cancers is underscored by the fact that at least two oncogenic herpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV) (19, 59) and Marek's disease virus (81), encode functional orthologs of miR-155. The KSHV ortholog miR-K12-11 also shares 100% seed sequence (first 8 nucleotides [nt]) homology with human miR-155 (59). miR-155 is processed from a primary transcript, B-cell integration cluster (BIC), which can be processed via the intermediate precursor miR-155 (pre-miR-155) to the mature 22-nt miR-155 (63). BIC cDNAs from human, mouse, and chicken have 78% identity over 138 nucleotides. miR-155 preferentially targets SHIP1 (42), and also targets

The identity of DNA replication proteins and cell cycle regulatory proteins which can be found in complexes involving PCNA were investigated by the use of PCNA immobilized on Sepharose 4B. A column containing bovine serum albumin (BSA) bound to Sepharose was used as a control. Fetal calf thymus extracts were chromatographed on PCNA-Sepharose and BSA-Sepharose. The columns were washed and then eluted with 0.5 M KCl. The salt eluates were examined for the presence of both DNA replication proteins (Pol alpha, delta, straightepsilon, PCNA, RFC, RFA, DNA ligase I, NDH II, Topo I and Topo II) and cell cycle proteins (Cyclins A, B1, D1, D2, D3, E, CDK2, CDK4, CDK5 and p21) by western blotting with specific antibodies. The DNA replication proteins which bound to PCNA-Sepharose included DNA polymerase delta and straightepsilon, PCNA, the 37 and 40 kDa subunits of RFC, the 70 kDa subunit of RPA, NDH II and topoisomerase I. No evidence for the binding of DNA polymerase alpha, DNA ligase I or topoisomerase II was obtained. Of the cell cycle proteins investigated, CDK2, CDK4 and CDK5 were bound. This study presents strong evidence that PCNA is a component of protein complexes containing DNA replication, repair and cell cycle regulatory proteins.