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Biomedical subjects

Megan S Lim

Publications and source records attributed to Megan S Lim.

At least 19 recordsLinked to original sources
PubMed2006

Mass spectrometry-based proteomic studies of human anaplastic large cell lymphoma.

Malignant lymphomas are a diverse group of malignant neoplasms that arise as a result of a complex interplay of multiple factors including genetic aberrations, immunosuppression, and exposure to noxious agents such as ionizing radiation and chemical agents. Anaplastic large cell lymphoma (ALCL) is an aggressive T-lineage lymphoma harboring chromosomal translocations involving the anaplastic lymphoma kinase (ALK) tyrosine kinase. The most common translocation in ALCL is the t(2;5)(p23;q35). This results in the formation of a chimeric fusion kinase, nucleophosmin/ALK. Nucleophosmin/ALK activates numerous downstream signaling pathways resulting in enhanced survival and proliferation. Using a variety of mass spectrometry-driven proteomic strategies, we have studied several aspects of the ALCL proteome. In this review, we provide a summary of mass spectrometry-based proteomic studies that expands the current understanding of the molecular pathogenesis of ALCL and provides the basis for the identification of biomarkers and targets for novel therapeutic agents.

Anaplastic Lymphoma Kinase↗
PubMed2006

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.

Amino Acid Sequence↗
PubMed2006

Identification of proteins released by follicular lymphoma-derived cells using a mass spectrometry-based approach.

The recent advent of mass spectrometry-based methodologies for the analysis of complex protein mixtures opens new opportunities for the discovery of biomarkers that may aid in the diagnostic work-up of cancer. Follicular lymphoma (FL) is the most common form of low-grade non-Hodgkin lymphoma in the Western Hemisphere. Identification of tumor markers that facilitate early disease detection would be a significant advance in the management of FL. We have employed a strategy that entailed propagation of a follicular-derived cell line in serum-free media, protein extraction, and reverse-phase liquid chromatography, with subsequent electrospray ionization and tandem mass spectrometry analysis for the identification of proteins that are released by FL. Using a two-peptide minimum per protein and standard criteria, 209 proteins (5.6% maximum predicted error rate) released from the FL cells were identified. The released proteins included several growth factors, cytokines, acute phase reactants and cellular components previously known to be present in FL cells. Importantly, a greater proportion of proteins previously unassociated with FL were identified with high statistical confidence. Our studies provide a list of proteins, which may be candidates for early screening, diagnosis and therapeutic monitoring of patients with a suspected or biopsy-confirmed diagnosis of FL.

Biomarkers, Tumor↗
PubMed2006

Detection of acquired Janus kinase 2 V617F mutation in myeloproliferative disorders by fluorescence melting curve analysis.

BACKGROUND: The genetic lesion underlying the pathogenesis of chronic myeloproliferative disorders (MPDs) has been identified in the Janus kinase 2 (JAK2) gene. A point mutation in codon 617 causes a valine to phenylalanine substitution (V617F) in the JH2 autoinhibitory region of the protein, resulting in constitutive activation of the tyrosine kinase. The high prevalence of this conserved mutation in MPD makes it an excellent candidate as a diagnostic molecular marker. METHODS AND RESULTS: We report here the development and validation of a single oligonucleotide probe-based PCR approach using fluorescence melting curve analysis for point mutation detection in DNA derived from unfractionated peripheral blood samples. Using this assay and serial dilutions of an erythroleukemia cell line harboring the homozygous JAK2 V617F mutation, we successfully detected the mutation within a background of wild type sequences at a sensitivity of 2.5%. Our novel fluorescence probe-based assay was compared with allele-specific PCR-gel assay and sequencing techniques. Using the single probe assay, we examined 70 cases with a presumptive diagnosis of MPD, of which 38 (54%) yielded positive results for the presence of the JAK2 V617F mutation, and 92 follicular lymphoma cases, which were negative for the JAK2 V617F mutation. Additionally, the probe-based assay detected a previously unreported T>C base substitution at nucleotide 2342 (JAK2, codon 616), which was not detected by an allele-specific PCR assay. CONCLUSION: The single fluorescent probe-based assay described here is a rapid, homogeneous, and robust method for the detection of the JAK2 V617F mutation with favorable performance characteristics that make it advantageous for clinical diagnosis.

Alleles↗
PubMed2006

Analytical characteristics of cleavable isotope-coded affinity tag-LC-tandem mass spectrometry for quantitative proteomic studies.

Quantitative proteomic studies using cleavable isotope-coded affinity tags (cICAT) in concert with tandem mass spectrometry (MS/MS) permit unbiased comparisons between biologically distinct samples. We sought to determine the analytical characteristics of cICAT-based studies by examining the cumulative results of multiple, separate cICAT-based experiments involving human lymphoma-derived cells. We found that the number of identified proteins increased with larger numbers of fractions analyzed. The majority of proteins were identified by single peptides. Only 24 to 41% of the peptides contained cysteine residues, but 85% of the cysteine-containing peptides yielded quantification data. Approximately 28% of all identified proteins yielded quantification data, with 57% of these being differentially expressed by at least 1.5-fold. The quantification ratios of peptides for proteins with multiple quantified peptides were concordant in trend in 87% of instances. cICAT-labeled peptides identified proteins in all subcellular compartments without significant bias. Analysis of the flow-through fraction did not increase the number of peptides identified per protein. Our studies indicate that cICAT-LC-MS/MS yields quantifications primarily based on single peptides, and analysis of flow-through peptides does not contribute signifi-cantly to the results. Nevertheless, identifications based on single cICAT-labeled peptides with tryptic ends provide sufficiently reliable protein identifications and quantification information in cICAT-LC-MS/MS-based proteomic studies.

Amino Acid Sequence↗
PubMed2005

State of the Art and Future Needs in Cytogenetic/Molecular Genetics/Arrays in childhood lymphoma: summary report of workshop at the First International Symposium on childhood and adolescent non-Hodgkin lymphoma, April 9, 2003, New York City, NY.

BACKGROUND: A significant number of studies describe the cytogenetics and molecular genetics of adult non-Hodgkin lymphoma (NHL); however, similar knowledge is lacking regarding pediatric NHL. METHODS: A workshop to discuss the "State of the Art and Future Needs in Cytogenetic/Molecular Genetics/Arrays" in pediatric NHL was held in conjunction with the First International Symposium on Childhood and Adolescent Non-Hodgkin Lymphoma on April 9, 2003 in New York City. RESULTS: Cytogenetic characteristics of pediatric NHL include 14q11.2 rearrangements in T-cell lymphoblastic leukemia/lymphomas (LBL), ALK rearrangements in anaplastic large cell lymphomas (ALCL), and CMYC translocations in both Burkitt and Burkitt-like lymphomas (BL/BLL). Pediatric diffuse large B-cell lymphoma (DLBCL) is cytogenetically different from DLBCL in adults, suggesting a different disease in children. Microarray studies demonstrate three types of T-cell leukemia, the leukemic counterpart of LBL, that block T-cell differentiation at different stages of T-cell development, corresponding to LYL, TAL1, and HOX-expressing leukemias. ALCL cell lines have a unique expression profile compared to normal T-cells. Germinal centers of BL have CMYC expression signatures, indicating that CMYC expression is ectopic and does not reflect the physiology of the normal cell counterpart. CONCLUSIONS: Additional cytogenetic, molecular and microarray investigations of NHL in children are vital to better understand these diseases, their etiology, and differences from adult NHL. A greater understanding of pediatric NHL will lead to disease-specific and patient-individualized therapies of these diseases.

Adolescent↗
PubMed2005

Analysis of BCL6-interacting proteins by tandem mass spectrometry.

B-cell lymphoma 6 (BCL6) is a 95-kDa nuclear phosphoprotein and member of the Pox virus zinc finger/bric-a-brac, tramtrack, broad complex (POZ/BTB) family of transcription factors. BCL6 is a transcriptional repressor required for germinal center formation, and the gene encoding it is frequently altered in diffuse large B-cell and follicular lymphomas. The dysregulation of BCL6 has therefore been implicated in lymphomagenesis. A limited number of proteins is known to interact with BCL6 and modulate its activity or participate in its role in transcriptional regulation. Identification of additional BCL6-binding proteins could reveal potential signaling targets and previously undescribed functional roles for BCL6. We used a functional proteomic approach to determine the identity of proteins that interact with BCL6. Proteins were isolated by co-immunoprecipitation with an anti-BCL6 antibody and identified using MS/MS. We identified 61 proteins in the BCL6 immunocomplex from the following Gene Ontology categories: transcription regulator activity (n = 18), binding activity (n = 11), signal transducer activity (n = 10), catalytic activity (n = 8), structural molecule activity (n = 3), enzyme regulator activity (n = 3), transporter activity (n = 2), motor activity (n = 2), chaperone activity (n = 1), and unknown function (n = 3). Importantly we identified BCL6 and several previously reported BCL6-interacting proteins in the BCL6 immunocomplex. The remaining proteins have not been shown previously to be associated with BCL6. MS/MS results were validated on four proteins using immunoprecipitation and Western blotting. Two of these protein interactions were further confirmed by reciprocal immunoprecipitation. This study demonstrates the utility of antibody immunoprecipitation and subsequent peptide identification by MS/MS for the elucidation of BCL6-binding proteins. Many of the novel proteins identified in this study suggest additional functional roles for BCL6 beyond transcriptional repression.

Amino Acid Sequence↗
PubMed2005

Childhood and adolescent non-Hodgkin lymphoma: new insights in biology and critical challenges for the future.

Pediatric non-Hodgkin lymphoma (NHL) is a common and fascinating group of diseases with distinctive underlying genetic events that characterize the major histologic subtypes: diffuse large B-cell lymphoma, Burkitt lymphoma, anaplastic large cell lymphoma and lymphoblastic lymphoma. With systematic improvements in therapy over recent decades, the vast majority of children with NHL of all subtypes are now cured. The similarities and differences between adult and childhood presentations of disease, and whether or not some subtypes of NHL and leukemia are the same or different disease entities, are interesting questions that will be addressed with advances in our understanding of the molecular and genetic bases of these diseases. As is the case with other pediatric malignancies, growing emphasis is now being placed on the development of less toxic, targeted therapeutic approaches, and this review highlights some of the biological discoveries that will potentially open these avenues.

Adolescent↗
PubMed2005

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.

Algorithms↗
PubMed2005

Identification of proteins from formalin-fixed paraffin-embedded cells by LC-MS/MS.

There exists a need for robust approaches for tandem mass spectrometry (MS/MS)-based identification of proteins in formalin-fixed paraffin-embedded (FFPE) material. We demonstrate herein the identification of proteins in FFPE material using enzymatic cleavage for extraction of peptides from the FFPE specimen and liquid chromatography (LC) followed by MS/MS. We identified 324 proteins from a 3-year-old FFPE cell-block of a human lymphoma cell line. The identified proteins were assigned to the membrane, cytosol and nucleus, with diverse cellular functions. The results were comparable to those obtained with lysates from a fresh specimen of the lymphoma cell line. Western blotting analysis and immunofluorescence microscopy confirmed the expression of selected proteins. The functional significance of one protein (PKC eta) was validated using a PKC inhibitory peptide which resulted in lymphoma cell death in vitro. The ability to identify proteins from FFPE specimens has significant implications for MS/MS-based proteomics of vast repositories of archival primary tissue samples for disease-related discovery research.

Cell Line, Tumor↗
PubMed2005

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.

B-Lymphocytes↗
PubMed2005

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.

Analysis of Variance↗
PubMed2005

Annotated proteome of a human T-cell lymphoma.

As the reliable identification of proteins by tandem mass spectrometry becomes increasingly common, the full characterization of large data sets of proteins remains a difficult challenge. Our goal was to survey the proteome of a human T-cell lymphoma-derived cell line in a single set of experiments and present an automated method for the annotation of lists of proteins. A downstream application of these data includes the identification of novel pathogenetic and candidate diagnostic markers of T-cell lymphoma. Total protein isolated from cytoplasmic, membrane, and nuclear fractions of the SUDHL-1 T-cell lymphoma cell line was resolved by SDS-PAGE, and the entire gel lanes digested and analyzed by tandem mass spectrometry. Acquired data files were searched against the UniProt protein database using the SEQUEST algorithm. Search results for each subcellular fraction were analyzed using INTERACT and ProteinProphet. All protein identifications with an error rate of less than 10% were directly exported into excel and analyzed using GOMiner (NIH/NCI). The Gene ontology molecular function and cell location data were summarized for the identified proteins and results exported as user-interactive directed acyclic graphs. A total of 1105 unique proteins were identified and fully annotated, including numerous proteins that had not been previously characterized in lymphoma, in functional categories such as cell adhesion, migration, signaling, and stress response. This study demonstrates the utility of currently available bioinformatics tools for the robust identification and annotation of large numbers of proteins in a batchwise fashion.

Algorithms↗
PubMed2004

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.

Cell Line, Transformed↗
PubMed2004

Identification of NPM-ALK interacting proteins by tandem mass spectrometry.

Constitutive overexpression of nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) is a key oncogenic event in anaplastic large-cell lymphomas with the characteristic chromosomal aberration t(2;5)(p23;q35). Proteins that interact with ALK tyrosine kinase play important roles in mediating downstream cellular signals, and are potential targets for novel therapies. Using a functional proteomic approach, we determined the identity of proteins that interact with the ALK tyrosine kinase by co-immunoprecipitation with anti-ALK antibody, followed by electrospray ionization and tandem mass spectrometry (MS/MS). A total of 46 proteins were identified as unique to the ALK immunocomplex using monoclonal and polyclonal antibodies, while 11 proteins were identified in the NPM immunocomplex. Previously reported proteins in the ALK signal pathway were identified including PI3-K, Jak2, Jak3, Stat3, Grb2, IRS, and PLCgamma1. More importantly, many proteins previously not recognized to be associated with NPM-ALK, but with potential NPM-ALK interacting protein domains, were identified. These include adaptor molecules (SOCS, Rho-GTPase activating protein, RAB35), kinases (MEK kinase 1 and 4, PKC, MLCK, cyclin G-associated kinase, EphA1, JNK kinase, MAP kinase 1), phosphatases (meprin, PTPK, protein phosphatase 2 subunit), and heat shock proteins (Hsp60 precursor). Proteins identified by MS were confirmed by Western blotting and reciprocal immunoprecipitation. This study demonstrates the utility of antibody immunoprecipitation and subsequent peptide identification by tandem mass spectrometry for the elucidation of ALK-binding proteins, and its potential signal transduction pathways.

Amino Acid Sequence↗
PubMed2004

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.

Cell Line, Tumor↗