Differentially expressed cytosolic proteins in human leukemia and lymphoma cell lines correlate with lineages and functions

Identification of cytosolic proteins differentially expressed between types of leukemia and lymphoma may provide a molecular basis for classification and understanding their cellular properties. Two-dimensional fluorescence difference gel electrophoresis (DIGE) and mass spectrometry have been used to identify proteins that are differentially expressed in cytosolic extracts from four human leukemia and lymphoma cell lines: HL-60 (acute promyelocytic leukemia), MEC1 (B-cell chronic lymphocytic leukemia), CCRF-CEM (T-cell acute lymphoblastic leukemia) and Raji (B-cell Burkitt's lymphoma). A total of 247 differentially expressed proteins were identified between the four cell lines. Analysis of the data by principal component analysis identified 22 protein spots (17 different protein species) differentially expressed at more than a 95% variance level between these cell lines. Several of these proteins were differentially expressed in only one cell line: HL-60 (myeloperoxidase, phosphoprotein 32 family member A, ras related protein Rab-11B, protein disulfide-isomerase, ran-specific GTPase-activating protein, nucleophosmin and S-100 calcium binding protein A4), and Raji (ezrin). Several of these proteins were differentially expressed in two cell lines: Raji and MEC1 (C-1-tetrahydrofolate synthase, elongation factor 2, alpha- and beta-tubulin, transgelin-2 and stathmin). MEC1 and CCRF-CEM (gamma-enolase), HL-60 and CCRF-CEM (ubiquitin-conjugating enzyme E2 N). The differentially expressed proteins identified in these four cell lines correlate with cellular properties and provide insights into the molecular basis of these malignancies.     

Identification of rat cell lines that preferentially express insulin-like growth factor binding proteins rlGFBP-1, 2, or 3.

The bioavailability and action of the insulin-like growth factors (IGFs) are determined by specific IGF-binding proteins (IGFBP) to which they are complexed. Complementary DNA clones have been isolated that encode three related IGFBPs: human IGFBP-1 (hIGFBP-1), human IGFBP-3 (hIGFBP-3), and rat IGFBP-2 (rIGFBP-2). IGFBP-1 and IGFBP-3 are regulated differently in human plasma, suggesting that they have different functions. In order to study the molecular basis of the regulation of the different IGFBPs, we have identified a panel of rat cell lines that express a single predominant binding protein and developed an assay strategy to distinguish the different binding proteins. Proteins in conditioned medium were examined by ligand blotting, and by immunoprecipitation and immunoblotting using antibodies to rIGFBP-2 and hIGFBP-1; RNAs were hybridized to cDNA probes for rIGFBP-2 and hIGFBP-1. 1) C6 glial cells and B104 neuroblastoma cells express an approximately 40 kilodalton (kDa) glycosylated binding protein that most likely represents rIGFBP-3, the binding subunit of the 150 kDa IGF: binding protein complex in adult rat serum. The C6 and B104 binding proteins do not react with antibodies to rIGFBP-2, and RNAs from C6 and B104 cells do not hybridize to cDNA probes for rIGFBP-2 or hIGFBP-1. 2) BRL-3A, Clone 9, and TRL 12-15 cell lines derived from normal rat liver express rIGFBP-2, a 30 kDa nonglycosylated IGF-binding protein that is recognized by antibodies to rIGFBP-2 but not by antibodies to hIGFBP-1. RNAs from these cells hybridize to a rIGFBP-2 cDNA probe, but not to a hIGFBP-1 probe. 3) H35 rat hepatoma cells express a 30 kDa nonglycosylated IGFBP that is presumptively identified as rIGFBP-1. It does not react with antibodies to rIGFBP-2, but is recognized by polyclonal and monoclonal antibodies to hIGFBP-1. RNA from H35 cells hybridizes to a hIGFBP-1 cDNA probe, but not to a rIGFBP-2 probe. Expression of rIGFBP-1 by the H35 cell line has enabled us to establish and validate specific assays for this protein that allow us to study its regulation in intact rats. Identification of a panel of rat cell lines expressing specific IGFBPs should be useful in elucidating the molecular mechanisms of IGFBP regulation.     

Qualitative and quantitative analyses of Epstein-Barr virus early antigen diffuse component by western blotting enzyme-linked immunosorbent assay with a monoclonal antibody.

We report the use of monoclonal antibody against the early antigen diffuse component (anti-EA-D) of Epstein-Barr virus (EBV) to analyze, both qualitatively and quantitatively, the expression of EA-D in various human lymphoblastoid cell lines activated by chemical inducers. The kinetics of synthesis of EA-D in P3HR-1, B95-8, and Ramos/AW cells were similar in that they all reached the peak of synthesis on day 5 after induction. Surprisingly, no expression of EA-D was found in induced BJAB/GC, an EBV-genome-containing cell line. EBV-negative cell lines, BJAB and Ramos, were negative for EA-D. Raji cells had no detectable EA-D but responded rapidly to induction, reaching a peak on day 3. Superinfection of Raji cells also resulted in marked induction of EA-D, which reached a plateau between 8 to 12 h postinfection. Western blotting coupled with the enzyme-linked immunosorbent assay was employed to identify polypeptides representing EA-D. A family of four polypeptides with molecular weights of 46,000 (46K protein), 49,000, 52,000, and 55,000 were identified to be reactive with monoclonal anti-EA-D antiserum. The pattern of EA-D polypeptides expressed in each cell line was different. Of particular interest was the expression of a large quantity of 46K protein both in induced Raji and P3HR-1 cells, but not in superinfected Raji cells. A 49K doublet was expressed in activated p3HR-1, B95-8, and Ramos/AW cells and in superinfected Raji cells. In addition, two distinct 52K and 55K polypeptides were expressed in induced Ramos/AW and superinfected Raji cells. However, none of these EA-D polypeptides was detectable in BJAB/GC, BJAB, Ramos, and mock-infected Raji cells. To approximate relative concentrations of EA-D in cell extracts, we employed the enzyme-linked immunosorbent assay and immunoblot dot methods by using one of the purified EA-D components to construct a standard curve. Depending upon the cell lines, it was estimated that ca. 1 to 3% (determined by the enzyme-linked immunosorbent assay) and 0.8 to 1.6% (determined by immunoblot dot) of total proteins from maximally induced cells were EA-D. These results suggest that differential expression of EA-D polypeptides could be of importance in the diagnosis of state of EBV infection.     

Host cell-dependent regulation of growth transformation-associated Epstein-Barr virus antigens in somatic cell hybrids.

We have analyzed the expression of the three major known growth transformation-associated Epstein-Barr virus (EBV) proteins, EBNA-1, EBNA-2, and latent membrane protein (LMP), in a series of somatic cell hybrids derived from the fusion of EBV-carrying Burkitt lymphoma (BL) lines with EBV-positive or EBV-negative B-cell lines. Independently of the cell phenotype, EBNA-1 was invariably coexpressed in all EBV-carrying hybrids. In hybrids between EBV-carrying, LMP-positive and LMP-negative Burkitt lymphoma lines, LMP was expressed, indicating positive control. Two EBV-negative lymphoma lines, Ramos and BJAB, differed in their ability to express LMP after B95-8 virus-induced conversion and after hybridization with Raji cells. BJAB was permissive while Ramos was nonpermissive for LMP, although both expressed EBNA-2. The EBNA-2-deleted P3HR-1 virus gave the same pattern of LMP expression in these two cells. Our findings indicate that the expression of EBNA-1, EBNA-2, and LMP is regulated by independent mechanisms.     

NF-kappa B sites function as positive regulators of expression of the translocated c-myc allele in Burkitt''s lymphoma.

An in vivo footprint over a potential NF-kappa B site in the first exon of the c-myc gene has been identified on the translocated allele in the Ramos Burkitt's lymphoma cell line. The potential NF-kappa B site in the 5' flanking sequence of c-myc was found to be occupied on the translocated allele in the Raji Burkitt's cell line. Electrophoretic mobility shift assays with each of these sequences demonstrated complexes with mobilities identical to those of the NF-kappa B site from the kappa light-chain gene. A supershift was obtained with anti-p50 antibody with the exon site. The upstream-site shift complex disappeared with the addition of anti-p50 antibody. Binding of NF-kappa B proteins to the c-myc exon and upstream sites was demonstrated by induction of binding upon differentiation of pre-B 70Z/3 cells to B cells. UV cross-linking experiments revealed that a protein with a molecular mass of 50 kDa bound to the exon and upstream sites. Transfection experiments with Raji cells demonstrated that both sites functioned as positive regulatory regions, with a drop in activity level when either site was mutated. Access to these sites is blocked in the silent normal c-myc allele in Burkitt's lymphoma cells, while Rel family proteins bind to these sites in the translocated allele. We conclude that the two NF-kappa B sites function as positive regulatory regions for the translocated c-myc gene in Burkitt's lymphoma.     

PACE-1, a novel protein that interacts with the C-terminal domain of ezrin

The ERM proteins (ezrin, radixin, moesin) together with merlin comprise a subgroup of the band 4.1 superfamily. These proteins act as membrane cytoskeletal linker proteins mediating interactions between the cytoplasmic domains of transmembrane proteins and actin. To better understand how the ERM proteins function to regulate these junctional complexes, a yeast 2-hybrid screen was undertaken using ezrin as a bait. We describe here the identification and cloning of a novel protein, PACE-1, which binds to the C-terminal domain of ezrin. Characterization of PACE-1 in human breast cancer cell lines demonstrates it to have two distinct intracellular localizations. A proportion of the protein is associated with the cytoplasmic face of the Golgi apparatus. This distribution is dependent upon the presence of the PACE-1 N-terminal myristoylation consensus sequence but is not dependent on an association with ezrin. In contrast, PACE-1 colocalises with ezrin in the lamellipodia, where ezrin has a role in cell spreading and motility. A notable feature of PACE-1 is the presence of a putative N-terminal kinase domain; however, in biochemical assays PACE-1 was shown to have associated rather than intrinsic kinase activity. Together these data suggest that PACE-1 may play a role in regulating cell adhesion/migration complexes in migrating cells.     

Proteomic analysis of the molecular response of Raji cells to maslinic acid treatment

Maslinic acid, a natural pentacyclic triterpene has been shown to inhibit growth and induce apoptosis in some tumour cell lines. We studied the molecular response of Raji cells towards maslinic acid treatment. A proteomics approach was employed to identify the target proteins. Seventeen differentially expressed proteins including those involved in DNA replication, microtubule filament assembly, nucleo-cytoplasmic trafficking, cell signaling, energy metabolism and cytoskeletal organization were identified by MALDI TOF-TOF MS. The down-regulation of stathmin, Ran GTPase activating protein-1 (RanBP1), and microtubule associated protein RP/EB family member 1 (EB1) were confirmed by Western blotting. The study of the effect of maslinic acid on Raji cell cycle regulation showed that it induced a G1 cell cycle arrest. The differential proteomic changes in maslinic acid-treated Raji cells demonstrated that it also inhibited expression of dUTPase and stathmin which are known to induce early S and G2 cell cycle arrests. The mechanism of maslinic acid-induced cell cycle arrest may be mediated by inhibiting cyclin D1 expression and enhancing the levels of cell cycle-dependent kinase (CDK) inhibitor p21 protein. Maslinic acid suppressed nuclear factor-kappa B (NF-κB) activity which is known to stimulate expression of anti-apoptotic and cell cycle regulatory gene products. These results suggest that maslinic acid affects multiple signaling molecules and inhibits fundamental pathways regulating cell growth and survival in Raji cells.     

Andrographolide,a new potential NF-κB inhibitor: docking simulation and evaluation of drug-likeness

Andrographolide is a potent anticancer and anti-inflammatory agent isolated from the plant Andrographis paniculata. It is found to be cytotoxic against various cancer cell lines (in vitro) and also reported to act as an anti-inflammatory agent by interfering with NF-κB protein. Andrographolide induced higher percentage of apoptosis in glutathione-depleted lymphoma cell lines. Andrographolide was also reported to form dehydrated adduct with reduced glutathione at 50^° C. On the basis of these observations, the docking analysis of andrographolide with its target protein (NF-κB/p50) and its proposed anti-target protein (glutathione S-transferase) was carried out. Docking analysis predicted the best putative pose of andrographolide molecule in the active site of NF-κB and glutathione S-transferase proteins. Here we report that the furan ring of andrographolide interacts with cysteine 59 of NF-κB/p50 and thereby inhibiting the protein action. Docking studies showed the andrographolide binding to the H-site of glutathione S-transferase enzyme which resembles the behaviour of susceptible xenobiotics inactivated by glutathione S-transferase enzyme. Andrographolide obeys Pfizer's rule but drug-likeness value for andrographolide is found to be negative as the molecule showed low drug score. Hence, inactivation by glutathione S-transferase and low drug score could possibly be the reasons to make andrographolide inactive in clinical trial.     

Identification of a human protein homologous to the mouse Lyb-2 B cell differentiation antigen and sequence of the corresponding cDNA

We have isolated and sequenced cDNA clones encoding the human homolog of the mouse Lyb-2 B cell differentiation Ag. Previous data suggest that Lyb-2 might represent a growth factor or lymphokine receptor. Human Lyb-2 mRNA is expressed in normal human tonsils and bone marrow cells, in the pre-B cell line REH, in three Burkitt lymphoma cell lines, and in some EBV-transformed B cell lines, but not in antibody-secreting myeloma cell lines, T cell lines, or a promyelocytic leukemia cell line. These data indicate that expression of human Lyb-2 is restricted to B lineage cells and turned off in antibody-secreting plasma cells. A polyclonal mouse antiserum was raised against human Lyb-2 and immunoprecipitates a Mr 42,000 protein from REH, Raji, and Daudi cells and from mouse L(tk) cells transfected with the human Lyb-2 cDNA in an expression vector. The human Lyb-2 protein is related to both the asialoglycoprotein receptor and CD23, the B cell-specific FcR for IgE. These data demonstrate that human B cells express a previously undescribed cell surface protein that is homologous to mouse Lyb-2 and has a similar pattern of expression during B cell development.     

A novel monoclonal antibody, 1B3.1, binds to a new epitope of the VLA-1 molecule

A monoclonal antibody, 1B3.1, was raised against a cloned IL-2-dependent T cell line that expresses the T gamma delta T cell receptor. MoAb 1B3.1 reacted with long-term cultured T cell lines of both T gamma delta and T alpha beta lineage, and with in vivo-stimulated T cells, derived from synovial fluid, but not with resting or short-term activated T cells, B cells, or macrophages. Immunoprecipitation of the 1B3.1 target antigens showed that 1B3.1 recognizes a 200/110 kDa molecule that is identical to the VLA-1 heterodimer precipitated by MoAb TS2/7. 1B3.1, however, binds to an epitope of VLA-1 that is distinct from the TS2/7 binding site. This new MoAb could be useful in further studies of the functions of VLA-1, and of the cells that express this molecule.     

Epoxyquinol B,a Naturally Occurring Pentaketide Dimer,Inhibits NF-κB Signaling by Crosslinking TAK1

Several epoxyquinoids interfere with NF-κB signaling by targeting IKKβ or NF-κB. We report that epoxyquinol B (EPQB), classified as an epoxyquiniod, inhibits NF-κB signaling through inhibition of the TAK1 complex, a factor upstream of IKKβ and NF-κB. cDNA microarray analysis revealed that EPQB decreased TNF-α-induced expression of NF-κB target genes. EPQB covalently bound to a recombinant TAK1-TAB1 fusion protein in vitro, and inhibited its kinase activity. Furthermore, in vitro/in situ treatment with EPQB resulted in a ladder-like hypershift of TAK1 protein bands. We reported recently that EPQB crosslinks proteins via cysteine residues by opening its two epoxides, and our current results suggest that EPQB inhibits NF-κB signaling by crosslinking TAK1 itself or TAK1 through other proteins.     

Rugulactone derivatives act as inhibitors of NF-κB activation and modulates the transcription of NF-κB dependent genes in MDA-MB-231cells

Rugulactone and its analogues were synthesized following Horners–Wadsworth–Emmons and ring-closing metathesis as the key reactions. A library of new rugulactone analogues were designed, synthesized and evaluated for their anticancer activity in breast cancer cells. All analogues have shown anti-proliferative activity, while some of them exhibited significant cytotoxicity. In assays related to cell-cycle distribution, these conjugates induced G1 cell-cycle arrest in MDA-MB-231 cells. The cell cycle arrest nature was further confirmed by examining the effect on Cyclin E and Cdk2 proteins that acts at G1-S phase transition. Immunocytochemistry assay revealed that these compounds inhibited nuclear translocation of NF-κB protein, thereby activation of NF-κB was inhibited. The expression of NF-κB target genes such as Cyclin D1 and Bcl-xL were severely affected. Apart from acting on NF-κB, these compounds also regulate class I Histone deacetylase proteins such as (HDAC-3 and 8) that have a crucial and regulatory role in cell-proliferation. Simultaneously, the apoptotic inducing nature of these compounds was confirmed by activation of PARP protein, a protein that plays a key role in DNA damage and repair pathways. Among all compounds of this series 3g is the most potent compound and can be used for further studies.