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.

P38 mitogen activated protein kinase expression and regulation by interleukin-4 in human B cell non-Hodgkin lymphomas.

UNLABELLED: The prevalence and regulation of p38 mitogen activated protein kinase (MAPK) expression in human lymphomas have not been extensively studied. In order to elucidate the role of p38 MAPK in lymphomagenesis, we examined the expression of native and phosphorylated p38 (p-p38) MAPK in cell lines derived from human hematopoietic neoplasms including B cell lymphoma-derived cell lines using Western blot analysis. The p-p38 MAPK protein was also analyzed in 30 B cell non-Hodgkin lymphoma (NHL) tissue biopsies by immunohistochemistry. Our results show that the expression of p38 MAPK was up-regulated in most of the cell lines as compared with peripheral blood lymphocytes, while the expression of p-p38 MAPK was more variable. A subset of B cell NHL biopsies showed increased expression of p-p38 MAPK relative to reactive germinal center cells. Interleukin-4 (IL-4) induced a dose-dependent increase in the expression of p-p38 MAPK (1.6- to 2.8-fold) in cell lines derived from activated B cell-like diffuse large B cell lymphoma (DLBCL) but not those from germinal center-like DLBCL. No change was seen in native p38 MAPK. The in vitro kinase activity of p38 MAPK, however, was induced (1.6- to 3.2-fold) in all five cell lines by IL-4. Quantitative fluorescent RT-PCR demonstrated that all four isoforms of p38 MAPK gene were expressed in the lymphoma cell lines, with p38gamma and p38beta isoforms being predominant. IL-4 stimulation increased the expression of beta, gamma, and delta isoforms but not alpha isoform in two cell lines. In conclusion, there is constitutive expression and activation of p38 MAPK in a large number of B-lymphoma-derived cell lines and primary lymphoma tissues, supportive of its role in lymphomagenesis. The differential IL-4 regulation of p38 MAPK expression in cell lines derived from DLBCL may relate to the cellular origin of these neoplasms. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12308-009-0049-5) contains supplementary material, which is available to authorized users.

Analysis of gene expression profile of TPM3-ALK positive anaplastic large cell lymphoma reveals overlapping and unique patterns with that of NPM-ALK positive anaplastic large cell lymphoma.

Anaplastic large cell lymphoma (ALCL) comprises a group of non-Hodgkin lymphomas characterized by the expression of the CD30/Ki-1 antigen. A subset of ALCL is characterized by chromosomal translocations involving the anaplastic lymphoma kinase (ALK) gene on chromosome 2. While the most common translocation is the t(2;5)(p23;q35) involving the nucleophosmin (NPM) gene on chromosome 5, up to 12 other translocations partners of the ALK gene have been identified. One of these is the t(1;2)(q25;p23) which results in the formation of the chimeric protein TPM3-ALK. While several of the signaling pathways induced by NPM-ALK have been elucidated, those involved in ALCLs harboring TPM3-ALK are largely unknown. In order to investigate the expression profiles of ALCLs carrying the NPM-ALK and TPM3-ALK fusions, we carried out cDNA microarray analysis of two ALCL tissue samples, one expressing the NPM-ALK fusion protein and the other the TPM3-ALK fusion protein. RNA was extracted from snap-frozen tissues, labeled with fluorescent dyes and analyzed using cDNAs microarray containing approximately 9,200 genes and expressed sequence tags (ESTs). Quantitative fluorescence RT-PCR was performed to validate the cDNA microarray data on nine selected gene targets. Our results show a significant overlap of genes deregulated in the NPM-ALK and TPM-ALK positive lymphomas. These deregulated genes are involved in diverse cellular functions, such as cell cycle regulation, apoptosis, proliferation, and adhesion. Interestingly, a subset of the genes was distinct in their expression pattern in the two types of lymphomas. More importantly, many genes that were not previously associated with ALK positive lymphomas were identified. Our results demonstrate the overlapping and unique transcriptional patterns associated with the NPM-ALK and TPM3-ALK fusions in ALCL.

Proteomic identification of oncogenic chromosomal translocation partners encoding chimeric anaplastic lymphoma kinase fusion proteins.

The anaplastic lymphoma kinase (ALK) on 2p23 is a tyrosine kinase that forms chimeric fusions with numerous translocation partners. We describe a mass spectrometry-based approach for the identification of ALK fusion partners. This approach accurately identified the nucleophosmin (NPM)-ALK fusion protein in an anaplastic large cell lymphoma (ALCL)-derived cell line carrying the t(2;5)(p23;q35), and the TPM3-ALK in a clinical biopsy of inflammatory myofibroblastic tumor (IMT) carrying the t(1;2)(q21;p23). This study shows the ability of mass spectrometry to identify oncogenic chimeric proteins resulting from chromosomal rearrangements. This strategy can be adapted for the identification of known and unknown translocation partners of chimeric ALK fusion proteins involved in oncogenesis.

Expression of the Rho-family GTPase gene RHOF in lymphocyte subsets and malignant lymphomas.

We have studied the expression of RHOF, a member of the Rho-GTPase family, in an array of lymphoid cells and tissues. Previous microarray studies demonstrated RHOF upregulation in a subset of transformed follicular lymphomas. Real-time quantitative polymerase chain reaction evaluated RHOF expression in lymphocyte subpopulations, and normal and malignant lymphoid tissue. Cells and tissues of B-cell origin expressed higher RHOF levels than their T-cell counterparts. Neoplastic cells and tissues of B-cell origin expressed higher levels of RHOF than their benign cellular counterparts. Relatively elevated levels of RHOF were seen in lymphomas derived from germinal centre origin.

Quantitative proteomic and transcriptional analysis of the response to the p38 mitogen-activated protein kinase inhibitor SB203580 in transformed follicular lymphoma cells.

The p38 mitogen-activated protein kinase (MAPK) is a key mediator of stress, extracellular-, growth factor-, and cytokine-induced signaling, and has been implicated in the development of cancer. Our previous work showed evidence for p38 MAPK activation in a subset of transformed follicular lymphomas (Elenitoba-Johnson et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 7259). We demonstrated that inhibition of p38 MAPK by SB203580 resulted in dose- and time-dependent caspase-3-mediated apoptosis. In order to further elucidate the basis of the cellular effects of SB203580, we have employed a systems biologic approach involving cDNA microarray and quantitative proteomic analysis of transformed follicular lymphoma derived-cells (OCI Ly-1) treated with SB203580. Gene expression profiling revealed differential expression (>/=1.5-fold) of 374 genes/ESTs in cells treated for 3 h and 515 genes/ESTs in cells treated for 21 h. The majority (52% at 3 h and 91% at 21 h) were down-regulated, including genes encoding growth cytokines, transcriptional regulators and cytoskeletal proteins. Quantitative proteomic analysis using ICAT-LC-MS/MS identified 277 differentially expressed proteins at 3 h and 350 proteins at 21 h of treatment with SB203580, the majority of which were also down-regulated. Analysis of functional groups of the differentially expressed proteins implicated components of diverse overlapping pathways including the IL-6/phosphatidylinositol 3-kinase, insulin-like growth factor 2/Ras/Raf, WNT8d/Frizzled, MAPK-activated protein kinase 2, and nuclear factor kappaB. The differential phosphorylation status of selected kinase-active proteins was validated by Western blotting analysis. Our complementary genomic and proteomic approach reveal the global cellular consequences of SB203580 treatment and provide insights into its growth inhibitory effect on transformed follicular lymphoma cells.

Application of SELDI-TOF mass spectrometry for the identification of differentially expressed proteins in transformed follicular lymphoma.

Completion of the human genome project has focused scientific attention on the development of methods that permit rapid characterization of proteins that are encoded by the genome. Recent improvements in two-dimensional separation techniques in combination with protein identification software/databases and mass spectrometry (MS) now permit rapid comprehensive large-scale analysis of individual proteins within complex protein mixtures. We have performed pairwise comparisons of low-grade and transformed follicular lymphomas (FLs) in order to identify proteins that may be involved in FL progression using surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometer (ProteinChip, Ciphergen Biosystems). This system utilizes preactivated differential binding surfaces to achieve multidimensional chromatography. The protein-bound chips were then analyzed by a SELDI-TOF mass spectrometer to generate protein profiles. In preliminary experiments, we established that the MS data obtained from SELDI-TOF MS were reproducible, and that reduction in sample complexity improved the ability to detect lower abundance proteins. With specific regard to FL transformation, we rapidly identified a number of potential candidate proteins involved in this process. These included an upregulated 32 kDa protein and a down-regulated 11.8 kDa protein. Protein database searches revealed several candidates, among them cyclin D3 (32.5 kDa) and caspase 3 (11.8 kDa) whose differential expression were confirmed by immunoblotting and/or immunohistochemical analysis on the primary tissue specimens. Our studies demonstrate the utility of SELDI-TOF-MS for the rapid discovery of differentially expressed proteins using femtomolar quantities of crude protein derived from biopsy material. The versatility of this methodology supports its application to the rapid discovery of potential biomarkers in a variety of cellular systems.

Utility of linearly amplified RNA for RT-PCR detection of chromosomal translocations: validation using the t(2;5)(p23;q35) NPM-ALK chromosomal translocation.

The requirement for sufficient quantities of starting RNA has limited the ability to evaluate multiple transcripts using reverse transcriptase-polymerase chain reaction (RT-PCR). In this study, we demonstrate the utility of linear RNA amplification for RT-PCR analysis of multiple gene transcripts including a chromosomal translocation, using the t(2;5)(p23;q35) as a model. RNA from the t(2;5)-positive cell line, SU-DHL-1, and the t(2;5)-negative cell line, HUT-78, was extracted and exposed to two rounds of linear amplification. RT-PCR using cDNA from the resultant amplified (a) RNA and total RNA resulted in the 177 bp NPM-ALK fusion gene product from the SU-DHL-1 cell line, but not from aRNA or total RNA from the HUT-78 cell line. DNA sequencing of the RT-PCR products from total and aRNA of SU-DHL-1 cells demonstrated identical sequences corresponding to the NPM-ALK fusion gene. Evaluation of 25 snap-frozen tissue samples, including eight NPM-ALK-positive ALCLs demonstrated 100% concordance of t(2;5) detection between cDNA from total RNA and that from aRNA. Our results show that linear amplification of RNA can enhance starting RNA greater than 200-fold and can be used for rapid and specific detection of multiplex gene expression from a variety of sources. This method can generate a renewable archive of representative cDNA, which can be used for retrospective screening of stored samples as well as positive controls for the clinical molecular diagnostic laboratory.

Involvement of multiple signaling pathways in follicular lymphoma transformation: p38-mitogen-activated protein kinase as a target for therapy.

Follicular lymphoma (FL) is the most common form of low-grade non-Hodgkin's lymphoma. Transformation to diffuse large B cell lymphoma (DLBCL) is an important cause of mortality. Using cDNA microarray analysis we identified 113 transformation-associated genes whose expression differed consistently between serial clonally related samples of FL and DLBCL occurring within the same individual. Quantitative RT-PCR validated the microarray results and assigned blinded independent group of 20 FLs, 20 DLBCLs, and five transformed lymphoma-derived cell lines with 100%, 70%, and 100% accuracy, respectively. Notably, growth factor cytokine receptors and p38beta-mitogen-activated protein kinase (MAPK) were differentially expressed in the DLBCLs. Immunohistochemistry of another blinded set of samples demonstrated expression of phosphorylated p38MAPK in 6/6 DLBCLs and 1/5 FLs, but not in benign germinal centers. SB203580 an inhibitor of p38MAPK specifically induced caspase-3-mediated apoptosis in t(14;18)+/p38MAPK+-transformed FL-derived cell lines. Lymphoma growth was also inhibited in SB203580-treated NOD-SCID mice. Our results implicate p38MAPK dysregulation in FL transformation and suggest that molecular targeting of specific elements within this pathway should be explored for transformed FL therapy.

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.

Fluorescence PCR quantification of cyclin D1 expression.

We have used a continuous fluorescence monitoring method to assess cyclin D1 mRNA expression in a variety of hematological and non-hematological processes. We examined 14 cell lines, 11 reactive lymphoid tissues, and 57 primary hematopoietic neoplasms including mantle cell lymphoma (MCL) (n = 10), chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) (n = 11), acute lymphoblastic leukemia/lymphoma (n = 15), follicular lymphoma (n = 6), peripheral T-cell lymphoma (PTCL) (n = 3), anaplastic large cell lymphoma (n = 3), hairy cell leukemia (n = 3), Burkitt lymphoma (n = 1), Burkitt-like lymphoma (n = 4), and plasmacytoma (n = 1) for the expression of cyclin D1 mRNA using fluorescently labeled sequence-specific hybridization probes. Fluorescence (F) was plotted against cycle (C) number over 45 cycles. The log-linear portion of the F versus C graph identified a fractional cycle number for threshold fluorescence. A beta-globin mRNA transcript with equivalent amplification efficiency to that of cyclin D1 was used for assessment of RNA integrity and normalization. In general, the MCLs demonstrated substantially higher levels of cyclin D1 mRNA than the other lymphoproliferative processes. Moderately high levels of cyclin D1 mRNA were detected in one PTCL. On average, the CLL/SLL cases showed cyclin D1 mRNA levels two to three orders of magnitude lower than observed in the MCLs. Cell lines derived from non-hematopoietic neoplasms such as fibrosarcoma, small cell carcinoma, and neuroblastoma showed comparable or higher levels of cyclin D1 mRNA than the MCLs. Our results indicate that quantitative real-time reverse transcription (RT) polymerase chain reaction is a simple, rapid, and accurate technique for assessing cyclin D1 expression, and while it is not specific, it can reliably be used in the distinction of MCL from CLL/SLL.