Stephen D. Jenson

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-Hodgkins 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 p38β-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.

Validation of cDNA microarray gene expression data obtained from linearly amplified RNA.

Background: DNA microarray technology has permitted the analysis of global gene expression profiles for several diseases, including cancer. However, standard hybridisation and detection protocols require micrograms of mRNA for microarray analysis, limiting broader application of this technology to small excisional biopsies, needle biopsies, and/or microdissected tissue samples. Therefore, linear amplification protocols to increase the amount of RNA have been developed. The correlation between the results of microarray experiments derived from non-amplified RNA and amplified samples needs to be evaluated in detail. Methods: Total RNA was amplified and replicate hybridisation experiments were performed with linearly amplified (aRNA) and non-amplified mRNA from tonsillar B cells and the SUDHL-6 cell line using cDNA microarrays containing approximately 4500 genes. The results of microarray differential expression using either source of RNA (mRNA or aRNA) were also compared with those found using real time quantitative reverse transcription polymerase chain reaction (QRT-PCR). Results: Microarray experiments using aRNA generated reproducible data displaying only small differences to data obtained from non-amplified mRNA. The quality of the starting total RNA template and the concentration of the promoter primer used to synthesise cDNA were crucial components of the linear amplification reaction. Approximately 80% of selected upregulated and downregulated genes identified by microarray analysis using linearly amplified RNA were confirmed by QRT-PCR using non-amplified mRNA as the starting template. Conclusions: Linear RNA amplification methods can be used to generate high fidelity microarray expression data of comparable quality to data generated by microarray methods that use non-amplified mRNA samples.

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 κB. 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.

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.

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.

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.

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

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 p38γ and p38β isoforms being predominant. IL-4 stimulation increased the expression of β, γ, and δ isoforms but not α 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.

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.

5'-(RACE) identification of rare ALK fusion partner in anaplastic large cell lymphoma

Anaplastic large-cell lymphoma (ALCL) is a subtype of aggressive non-Hodgkin’s lymphoma first described in 1985, 1 and characterized by the expression of CD30/Ki-1 antigen. By current definition, ALCLs exhibit a T-cell or null phenotype and the majority of cases demonstrate expression of the anaplastic lymphoma kinase (ALK) protein. 2 Most ALK-expressing ALCLs harbor the t(2;5)(p23;q35) chromosomal aberration 3, 4, 5 that involves the 5′-oligomerization motif region of nucleophosmin (NPM) gene on chromosome 5, and the 3′-cytoplasmic tyrosine kinase catalytic domain of anaplastic lymphoma kinase (ALK) gene of chromosome 2 to form the NPM-ALK fusion gene. 6 This rearrangement places the ALK gene under the control of the NPM promoter and results in deregulated expression of the ALK protein. Normal expression of ALK appears to be stringently controlled and limited to the cytoplasm of the testis, ganglion cells of the intestine, and neural tissues. 6 The NPM-ALK fusion protein has been shown by immunohistochemistry to localize in the cytoplasm and the nucleus of the neoplastic cells, thereby providing a distinctive marker for t(2;5)-positive ALCL. The t(2;5)-positive ALCLs account for 80 to 85% of the ALK-positive lymphomas. 7 The remaining 15 to 20% of ALK-positive ALCLs harbor variant fusion partners and exhibit immunohistological patterns different from that observed for t(2;5). 8 In these cases, ALK expression is predominantly cytoplasmic and is not present in a nuclear or nucleolar localization. 8 This is because of ALK forming fusion partners with genes other than NPM. Interestingly, a number of non-t(2;5) translocations have also been identified in a nonhematopoietic soft-tissue neoplasm known as inflammatory myofibroblastic tumor (IMT). 9, 10, 11 Remarkably, IMT may bear a histological resemblance to morphological variants of ALCL such as the lymphohistiocytic, small cell, and sarcomatoid variants. 8, 12 The propensity of ALK for partnering with a diverse variety of genes in both ALCLs and IMTs raises the possibility that other yet unidentified genes that partner with ALK may be involved in the pathogenesis of these tumors. In a quest to identify the ALK fusion partner in a non-t(2,5) ALCL with cytoplasmic expression of the ALK protein, we used the 5′-rapid amplification of cDNA ends (RACE) technique that revealed a fusion between tropomyosin 3 (TPM3) and the ALK genes. Molecular analysis for T-cell clonality also complemented immunohistochemical studies in the definitive assignment of T-cell lineage to the tumor.