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B-cell receptor (BCR) signaling is essential for the development of B cells and has a critical role in B-cell neoplasia. Increasing evidence indicates an association between chronic hepatitis C virus (HCV) infection and B-cell lymphoma, however, the mechanisms by which HCV causes B-cell lymphoproliferative disorder are still unclear. Herein, we demonstrate the expression of HCV viral proteins in B cells of HCV-infected patients and show that HCV upregulates BCR signaling in human primary B cells. HCV nonstructural protein NS3/4A interacts with CHK2 and downregulates its activity, modulating HuR posttranscriptional regulation of a network of target mRNAs associated with B-cell lymphoproliferative disorders. Interestingly, the BCR signaling pathway was found to have the largest number of transcripts with increased association with HuR and was upregulated by NS3/4A. Our study reveals a previously unidentified role of NS3/4A in regulation of host BCR signaling during HCV infection, contributing to a better understanding of the molecular mechanisms underlying HCV-associated B-cell lymphoproliferative disorders.

The molecular mechanism(s) linking tumorigenesis and morphological alterations in the nucleolus are presently coming into focus. The nucleolus is the cellular organelle in which the formation of ribosomal subunits occurs. Ribosomal biogenesis occurs through the transcription of ribosomal RNA (rRNA), rRNA processing and production of ribosomal proteins. An error in any of these processes may lead to deregulated cellular translation, evident in multiple cancers and 'ribosomopathies'. Deregulated protein synthesis may be achieved through the overexpression of ribosomal proteins as seen in primary leukemic blasts with elevated levels of ribosomal proteins S11 and S14. In this study, we demonstrate that ribosomal protein S6 (RPS6) is highly expressed in primary diffuse large B-cell lymphoma (DLBCL) samples. Genetic modulation of RPS6 protein levels with specifically targeted short hairpin RNA (shRNA) lentiviruses led to a decrease in the actively proliferating population of cells compared with control shRNA. Low-dose rapamycin treatments have been shown to affect the translation of 5' terminal oligopyrimidine (5' TOP) tract mRNA, which encodes the translational machinery, implicating RPS6 in 5' TOP translation. Recently, it was shown that disruption of 40S ribosomal biogenesis through specific small inhibitory RNA knockdown of RPS6 defined RPS6 as a critical regulator of 5' TOP translation. For the first time, we show that RPS6 associates with multiple mRNAs containing a 5' TOP tract. These findings expand our understanding of the mechanism(s) involved in ribosomal biogenesis and deregulated protein synthesis in DLBCL.

PBK/TOPK (PDZ-binding kinase, T-LAK-cell-originated protein kinase) is a serine-threonine kinase that is overexpressed in a variety of tumor cells but its role in oncogenesis remains unclear. Here we show, by co-immunoprecipitation experiments and yeast two-hybrid analysis, that PBK/TOPK physically interacts with the tumor suppressor p53 through its DNA-binding (DBD) domain in HCT116 colorectal carcinoma cells that express wild-type p53. PBK also binds to p53 mutants carrying five common point mutations in the DBD domain. The PBK-p53 interaction appears to downmodulate p53 transactivation function as indicated by PBK/TOPK knockdown experiments, which show upregulated expression of the key p53 target gene and cyclin-dependent kinase inhibitor p21 in HCT116 cells, particularly after genotoxic damage from doxorubicin. Furthermore, stable PBK/TOPK knockdown cell lines (derived from HCT116 and MCF-7 cells) showed increased apoptosis, G(2)/M arrest and slower growth as compared to stable empty vector-transfected control cell lines. Gene microarray studies identified additional p53 target genes involved in apoptosis or cell cycling, which were differentially regulated by PBK knockdown. Together, these data suggest that increased levels of PBK/TOPK may contribute to tumor cell development and progression through suppression of p53 function and consequent reductions in the cell-cycle regulatory proteins such as p21. PBK/TOPK may therefore be a valid target for antineoplastic kinase inhibitors to sensitize tumor cells to chemotherapy-induced apoptosis and growth suppression.

In a breast tumor xenograft model, the MCT-1 oncogene increases the in vivo tumorgenicity of MCF7 cells by promoting angiogenesis and inhibiting apoptosis. Increases in the tumor microvascular density are accompanied by a strong reduction in the levels of the angiogenesis inhibitor thrombospondin-1 (TSP1), but the mechanisms underlying this process are unknown. We show that TSP1 expression is controlled, at least in part, by post-transcriptional events. Using RNA interference to knock down the expression of the RNA-binding protein HuR in MCF7 cells as well as HuR overexpression, we demonstrate that HuR plays an important role in translation of the TSP1 mRNA. Furthermore, employing the RIP-Chip assay yielded 595 transcripts with significantly altered binding to HuR in the more tumorigenic breast cancer clones compared with the weakly tumorigenic clones. These mRNAs clustered in several pathways implicated in the transformed phenotype, such as the RAS pathway (involved in mitogenesis), the PI3K pathway (evasion of apoptosis) and pathways mediating angiogenesis and the cellular response to hypoxia. These findings demonstrate for the first time that global changes in HuR-bound mRNAs are implicated in the evolution to a more tumorigenic phenotype in an in vivo tumor model and underscore the role of global mRNA-protein interactions toward tumor progression.

We discovered a novel oncogene in a T-cell lymphoma cell line, multiple copies in T-cell lymphoma-1 (MCT-1), that has been shown to decrease cell-doubling time, shorten the duration of G(1) transit time and/or G(1)-S transition, and transform NIH3T3 fibroblasts. We subsequently demonstrated that there were significantly increased levels of MCT-1 protein in a subset of primary diffuse large B-cell lymphomas. Levels of MCT-1 protein were shown to be increased after exposure to DNA damaging agents. This increase did not require new protein synthesis, suggesting that post-translational mechanisms were involved. Phosphorylation is one potential mechanism by which the activity of molecules involved in cell cycle/survival is rapidly modulated. The RAS/mitogen-activated/extracellular-regulated kinase (MEK)/extracellular signal-regulated kinases (ERK) pathway plays a prominent role in the regulation of cell growth and proliferation through phosphorylation-dependent regulation of several substrates. The MCT-1 protein is predicted to have numerous putative phosphorylation sites. Using a combination of genetic and pharmacological approaches, we established that phosphorylation of MCT-1 protein by p44/p42 mitogen-activated protein kinases is critical for stabilization of MCT-1 protein and for its ability to promote cell proliferation. Our data suggests that targeting the RAS/MEK/ERK signal transduction cascade may provide a potential therapeutic approach in lymphomas and related malignancies that exhibit high levels of MCT-1 protein.

The contribution of oncogene amplification and/or overexpression to T-cell lymphoid neoplasms has only of late been established with the implication of the TCL1 and MTCP1 genes in T-cell malignancies. Our laboratory has recently discovered a novel oncogene, MCT-1, amplified in a T-cell lymphoma and mapped to chromosome Xq22-24. MCT-1 has been shown to decrease cell-doubling time, dramatically shortening the duration of G(1) transit time and/or G1-S transition, and transforms NIH3T3 fibroblasts. Constitutive expression of MCT-1 results in a strong proliferative signal and is associated with deregulation of protein kinase-mediated G1/S phase checkpoints. In this study we analysed the level and subcellular localization of this novel cell cycle regulatory molecule as a function of cell cycle phase. In human lymphoid tumors expression of MCT-1 is constant throughout the cell cycle and remains cytoplasmic. Cells overexpressing MCT-1 have increased expression of cyclin D1 with dysregulation of the G(1)-S checkpoint. Both cyclin D1 and MCT-1 are involved in regulating passage of cells through the G1 phase of the cell cycle. Since prior work has shown that gamma irradiation induces cyclin D1 expression we investigated the induction of MCT-1 to DNA damaging agents. We demonstrate that increases in MCT-1 protein in irradiated human lymphoid cells do not occur at the mRNA level and do not require new protein synthesis.

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the western world. Details of the molecular mechanisms involved in the pathogenesis of this leukemia are unclear at present. In other malignancies tumorigenesis has been shown to proceed via a multistep progression model with a causal relation between the accumulation of genetic abnormalities and more aggressive clinical behavior. The loss of chromosomal segments in malignant cells has proven useful in mapping regions of DNA that contain candidate tumor suppressor genes. The detection of loss of heterozygosity (LOH) has been greatly facilitated by the analysis of highly polymorphic microsatellite markers enabling the identification of submicroscopic losses. Utilizing immunomagnetic bead sorting to separate leukemic from normal cells we identified at least one allelic losses in 8/29 (28%) of analysed CLL cases with a PCR-based assay. On chromosomal arm 3p we have identified homozygous deletions in 3/29 cases at locus D3S1284 residing in the vicinity of the newly described FHIT gene. Several cases of CLL manifested LOH at a locus telomeric to the p15 and p16 tumor suppressor genes, all being early to intermediate stage disease. Our findings suggest that losses at these loci may contribute to the development and/or progression of CLL in at least a subset of cases.