Apoptosis of t(14;18)-positive lymphoma cells by a Bcl-2 interacting small molecule.

Overexpression of Bcl-2 protein occurs via both t(14;18)-dependent and independent mechanisms and contributes to the survival and chemoresistance of non-Hodgkin lymphomas. HA14-1 is a nonpeptidic organic small molecule, which has been shown to inhibit the interaction of Bcl-2 with Bax, thereby interfering with the antiapoptotic function of Bcl-2. In this study, we sought to determine the in vitro efficacy of HA14-1 as a therapeutic agent for non-Hodgkin lymphomas expressing Bcl-2. Assessment of cell viability demonstrated that HA14-1 induced a dose- (IC(50) = 10 µM) and time-dependent growth inhibition of a cell line (SudHL-4) derived from a t(14;18)-positive, Bcl-2-positive, non-Hodgkin lymphoma. HA14-1 effectively induced apoptosis via a caspase 3-mediated pathway but did not affect either the p38 MAPK or p44/42 MAPK pathways. Western blot analyses of Bcl-2 family proteins and other cell cycle-associated proteins were performed to determine the molecular sequelae of HA14-1-induced apoptosis. The results show down-regulation of Mcl-1 but up-regulation of p27(kip1), Bad, Bcl-xL, and Bcl-2 proteins, without change in Bax levels during HA14-1-mediated apoptosis. Our findings further elucidate the cellular mechanisms accompanying Bcl-2 inhibition and demonstrate the potential of Bcl-2 inhibitors as therapeutic agents for the treatment of non-Hodgkin lymphomas.

The proteomic signature of NPM/ALK reveals deregulation of multiple cellular pathways.

Constitutive expression of the chimeric NPM/ALK fusion protein encoded by the t(2;5)(p32;q35) is a key oncogenic event in the pathogenesis of most anaplastic large cell lymphomas (ALCLs). The proteomic network alterations produced by this aberration remain largely uncharacterized. Using a mass spectrometry (MS)-driven approach to identify changes in protein expression caused by the NPM/ALK fusion, we identified diverse NPM/ALK-induced changes affecting cell proliferation, ribosome synthesis, survival, apoptosis evasion, angiogenesis, and cytoarchitectural organization. MS-based findings were confirmed using Western blotting and/or immunostaining of NPM/ALK-transfected cells and ALK-deregulated lymphomas. A subset of the proteins distinguished NPM/ALK-positive ALCLs from NPM/ALK-negative ALCLs and Hodgkin lymphoma. The multiple NPM/ALK-deregulated pathways identified by MS analysis also predicted novel biologic effects of NPM/ALK expression. In this regard, we showed loss of cell adhesion as a consequence of NPM/ALK expression in a kinase-dependent manner, and sensitivity of NPM/ALK-positive ALCLs to inhibition of the RAS, p42/44ERK, and FRAP/mTOR signaling pathways. These findings reveal that the NPM/ALK alteration affects diverse cellular pathways, and provide novel insights into NPM/ALK-positive ALCL pathobiology. Our studies carry important implications for the use of MS-driven approaches for the elucidation of neoplastic pathobiology, the identification of novel diagnostic biomarkers, and pathogenetically relevant therapeutic targets.

Expression of Akt (protein kinase B) and its isoforms in malignant lymphomas.

Akt (protein kinase B) is a serine/threonine kinase involved in the regulation of cell survival signals. Akt is expressed in T- and B-lymphocytes and is activated in response to cytokine and antigen-receptor stimulation. Three isoforms of Akt have been identified, Akt-1, -2 and -3, but the expression pattern and specific functions of each have not yet been determined for many cell types. To determine whether Akt signaling is enhanced in human malignant lymphomas and to analyse the expression pattern of Akt isoforms in these neoplasms, Akt-1, -2 and -3 expression was studied in 38 cell lines derived from hematopoietic neoplasms, by RT-PCR and western blot analysis. The level of phosphorylated (active) Akt was also analysed in cell lines as well as in 72 human malignant non-Hodgkin's lymphoma tissues. The results suggest that there is constitutive activation of Akt in the majority of primary human lymphomas and hematopoietic cell lines and support its proposed key role in lymphoma cell survival.

Analysis of phosphatase and tensin homolog tumor suppressor interacting proteins by in vitro and in silico proteomics.

The phosphatase and tensin homolog (PTEN) tumor suppressor is a multifunctional protein deregulated in many types of cancer. To date, a comprehensive documentation of PTEN interacting proteins has not been performed. The goal of our study was to characterize the PTEN interactome using affinity pull-down and tandem mass spectrometry (MS/MS). Wild-type PTEN cDNA was inserted into pTRC-His2 vector to create a 6-His tagged protein, which was expressed in Escherichia coli. Lysate from a human lymphoma cell line was used in pull-down assays, utilizing affinity for nickel-agarose beads. Bound proteins were eluted with imidazole, digested and analyzed on an LCQ DecaXP ion trap mass spectrometer. The nickel affinity pull-down efficiency was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Acquired data were searched against the NCBI nr.fasta nonredundant protein database using the SEQUEST algorithm and screened using INTERACT and ProteinProphet. All experiments were performed in duplicate with 6-His-lacZ serving as control. A total of 79 proteins were identified in the wild-type 6-His-PTEN pull-down by MS/MS. We further validated a subset of the proteins present in the PTEN interactome by performing immunoprecipitation using an anti-PTEN antibody and establishing the presence of the proteins in the immunocomplex by Western blot analysis. A search of published PTEN interactions was also performed using Online Mendelian Inheritance in Man, Human Protein Reference Database, the IntAct Project database, and PubMed. This in silico analysis confirmed 42 out of 79 (53%) of the proteins identified by MS/MS. The remaining 37 proteins represent probable PTEN interactions not previously documented in public databases or reported in the literature. These results highlight the value of combining both in vitro biochemical approaches with in silico analyses for a comprehensive study of protein-protein interactions.

Comparative microarray analysis of gene expression during activation of human peripheral blood T cells and leukemic Jurkat T cells.

Activation of T cells involves a complex cascade of signal transduction pathways linking T-cell receptor engagement at the cell membrane to the transcription of multiple genes within the nucleus. The T-cell leukemia-derived cell line Jurkat has generally been used as a model system for the activation of T cells. However, genome-wide comprehensive studies investigating the activation status, and thus the appropriateness, of this cell line for this purpose have not been performed. We sought to compare the transcriptional profiles of phenotypically purified human CD2(+) T cells with those of Jurkat T cells during T-cell activation, using cDNA microarrays containing 6912 genes. About 300 genes were up-regulated by more than 2-fold during activation of both peripheral blood (PB) T cells and Jurkat T cells. The number of down-regulated genes was significantly lower than that of up-regulated genes. Only 79 genes in PB T cells and 37 genes in Jurkat T cells were down-regulated by more than 2-fold during activation. Comparison of gene expression during activation of Jurkat and PB T cells revealed a common set of genes that were up-regulated, such as Rho GTPase-activating protein 1, SKP2, CDC25A, T-cell specific transcription factor 7, cytoskeletal proteins, and signaling molecules. Genes that were commonly down-regulated in both PB T cells and Jurkat T cells included CDK inhibitors (p16, p19, p27), proapoptotic caspases, and the transcription factors c-fos and jun-B. After activation, 71 genes in PB T cells and only 3 genes in Jurkat T cells were up-regulated 4-fold or more. Of these up-regulated genes and expressed sequence tags, 44 were constitutively expressed at high levels in nonactivated Jurkat cells. Quantitative real-time RT-PCR analysis confirmed our microarray data. Our findings indicate that although there is significant overlap in the activation-associated transcriptional profiles in PB T cells compared with Jurkat T cells, there is a subset of genes showing differential expression patterns during the activation of the two cell types.

Microarray analysis of B-cell lymphoma cell lines with the t(14;18).

The t(14;18) is the most common genetic alteration in follicular lymphoma, and is detectable in a subset of diffuse large B-cell lymphomas (DLBCL), resulting in over-expression of the anti-apoptotic protein BCL-2. Although the t(14;18)-induced over-expression of BCL-2 is an important step in lymphomagenesis, this aberration alone is not sufficient to produce malignant lymphoma. Further analysis of these tumors is needed to identify additional genes that might be involved in the genesis of follicular lymphoma and progression to DLBCL. To address this issue, we analyzed the gene expression profiles of four t(14;18)-positive cell lines and two t(11;14)-positive mantle-cell lymphoma cell lines using cDNA microarrays containing 4364 genes, and compared them to the genetic profile of phenotypically purified B-cells obtained from hyperplastic tonsils. A total of 137 genes were differentially expressed by approximately twofold or more in the t(14;18) cell lines relative to tonsillar B-cells. 68 genes were up-regulated, 69 genes were down-regulated, and approximately 20% of the differentially regulated genes had no known function. The up-regulated genes included a number of genes involved in the promotion of cellular proliferation and survival, as well as cell metabolism. Down-regulated genes included mediators of cell adhesion and negative regulators of cell activation and growth. Hierarchical clustering analysis separated the t(14;18) and mantle-cell lines into distinct groups based on their gene expression profiles. We confirmed the differential expression of approximately 80% of selected up- and down-regulated genes identified by microarray analysis by quantitative real-time fluorescence reverse transcriptase polymerase chain reaction (RT-PCR) analysis and/or immunoblotting. This study demonstrates the utility of cDNA microarray analysis for the assessment of global transcriptional changes that characterize t(14;18)-positive cell lines, and also for the identification of novel genes that could potentially contribute to the genesis and progression of non-Hodgkin's lymphomas with this translocation.

Gene expression profiling of cell lines derived from T-cell malignancies.

The expression profiles of eight cell lines derived from T-cell malignancies were compared to CD4-positive T-cells using cDNA microarray technology. Unsupervised hierarchical clustering of 4364 genes demonstrated substantial heterogeneity resulting in four distinct groups. While no genes were found to be uniformly up- or down-regulated across all cell lines, we observed 111 over-expressed genes (greater than two-fold) and 1118 down-regulated genes (greater than two-fold) in the lymphomas as a group when compared to CD4-positive T-cells. These included genes involved in cytokine signaling, cell adhesion, cytoskeletal elements, nuclear transcription factors, and known oncogenes and tumor suppressor genes. Quantitative fluorescent reverse transcription-polymerase chain reaction analysis demonstrated 70% concordance with the microarray results. While freshly isolated malignant cells may differ in their individual expression patterns relative to established cell lines from the same diagnoses, we feel that the variety of different lymphocytic cell lines that we examined provides a representative picture of the molecular pathogenesis of T-cell malignancies.