Lysis Of Cell Lines Research Articles

Novel Tumor-associated Antigen of Hepatocellular Carcinoma Defined by Monoclonal Antibody E4-65

A monoclonal antibody, E4-65, produced by immunizing mice with SMMC-7721 cells, a human hepatocellular carcinoma (HCC) cell line, was used to identify and characterize an unreported HCC-associated antigen. Indirect immunofluorescence studies showed that E4-65 antibody reacted with five out of eight HCC cell lines, but not with 10 non-HCC tumor cell lines or a normal liver cell line. Using immunohistochemical examination, E4-65 antigen was detected on the cell membranes and in the cytoplasm of human liver tumor tissues, but was not found in most other tumors, or normal adult or fetal tissues, except for a weakly positive reaction in tissues of the digestive system. Western blot analysis showed that E4-65 antibody bound to a 45 kDa protein in the human HCC cell line and tissue lysates. Enzyme treatment and lectin blotting did not detect the carbohydrate chain in E4-65 antigen. This HCC-associated protein represents a potentially useful target for diagnoses and immunotherapy of human HCC.

An experimental strategy for quantitative analysis of the humoral immune response to prostate cancer antigens using natural protein microarrays

The identification of human tumor antigens has potential utility in the diagnosis and treatment of cancers. We demonstrate here a complete strategy to profile immunoreactivity and identify tumor antigens from proteins derived from tumor cell lines. Microarrays of proteins produced from 2-D LC fractionation of prostate tumor cell-line lysates were used to profile immunoreactivity in the sera of prostate cancer patients and control subjects. Cancer-associated immunoreactivity to distinct groups of chromatography fractions was present in about 50% of the patients, with greater immunoreactivity present in patients with non-organ-confined cancer than in patients with organ-confined cancer. We grouped the immunoreactive fractions by similarities in elution order and patterns of immunoreactivity to guide and interpret the MS analysis of selected fractions, which was used to identify the proteins that may be responsible for the immunoreactivity. As a complementary method to further characterize and validate the immunoreactivity of the proteins identified by mass spectrometry, we demonstrate the use of focused microarrays of recombinant proteins. Disease-associated immunoreactivity was confirmed for one of the identified proteins, human Kallikrein 11. These results demonstrate a practical approach to screening, identifying, and validating immunoreactive proteins that could be applied to diverse studies on humoral immune responses.

Cell Biology Assessment of Glucokinase Mutations V62M and G72R in Pancreatic β-Cells

Mutations in the glucokinase (GK) gene cause defects in blood glucose homeostasis. In some cases (V62M and G72R), the phenotype cannot be explained by altered enzyme kinetics or protein instability. We used transient and stable expression of green fluorescent protein (GFP) GK chimaeras in MIN6 beta-cells to study the phenotype defect of V62M and G72R. GK activity in lysates of MIN6 cell lines stably expressing wild-type or mutant GFP GK showed the expected affinity for glucose and response to pharmacological activators, indicating the expression of catalytically active enzymes. MIN6 cells stably expressing GFP V62M or GFP G72R had a lower GK activity-to-GK immunoreactivity ratio and GK activity-to-GK mRNA ratio but not GK immunoreactivity-to-GK mRNA ratio than wild-type GFP GK. Heterologous expression of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK2/FDP2) in cell lines increased GK activity for wild-type GK and V62M but not for G72R, whereas expression of liver GK regulatory protein (GKRP) increased GK activity for wild type but not V62M or G72R. Lack of interaction of these mutants with GKRP was also evident in hepatocyte transfections from the lack of nuclear accumulation. These results suggest that cellular loss of GK catalytic activity rather than impaired translation or enhanced protein degradation may account for the hyperglycemia in subjects with V62M and G72R mutations.

Evaluation of a Novel Tumor Vaccine in Dogs with Hemangiosarcoma

Hemangiosarcoma (HSA) is a highly metastatic and often rapidly fatal tumor of dogs. At present, adjuvant chemotherapy is the only proven effective treatment for dogs with HSA, though the benefits from chemotherapy are modest. Administration of immunotherapy together with chemotherapy has also been reported to improve survival in dogs with HSA. Therefore, we evaluated safety and immunologic responses to a novel tumor vaccine administered together with doxorubicin chemotherapy in dogs with different stages of HSA. That tumor vaccination could be safely and effectively combined with doxorubicin chemotherapy for treatment of dogs with HSA. Twenty-eight dogs with various stages of HSA were enrolled in the study. The HSA vaccine was prepared with lysates of allogeneic canine HSA cell lines mixed with an adjuvant composed of liposome-DNA complexes. Dogs received a series of 8 immunizations administered over a 22-week period, and most also received chemotherapy. Clinical adverse effects were noted, immune responses were measured by enzyme-linked immunosorbent assay (ELISA) and flow cytometry, and survival times were calculated. The most common adverse effects observed in vaccinated dogs also treated with doxorubicin chemotherapy were diarrhea and anorexia. Vaccinated dogs were found to mount strong humoral immune responses against a control antigen and, most dogs also mounted antibody responses against canine HSA cells. Thirteen dogs with stage II splenic HSA that received the tumor vaccine plus doxorubicin chemotherapy had an overall median survival time of 182 days. We conclude that an allogeneic tumor lysate vaccine is safe in dogs with HSA and can elicit humoral immune responses in dogs that are receiving concurrent doxorubicin chemotherapy.

Evaluation of a Novel Tumor Vaccine in Dogs with Hemangiosarcoma

Background: Hemangiosarcoma (HSA) is a highly metastatic and often rapidly fatal tumor of dogs. At present, adjuvant chemotherapy is the only proven effective treatment for dogs with HSA, though the benefits from chemotherapy are modest. Administration of immunotherapy together with chemotherapy has also been reported to improve survival in dogs with HSA. Therefore, we evaluated safety and immunologic responses to a novel tumor vaccine administered together with doxorubicin chemotherapy in dogs with different stages of HSA. Hypothesis: That tumor vaccination could be safely and effectively combined with doxorubicin chemotherapy for treatment of dogs with HSA. Animals: Twenty‐eight dogs with various stages of HSA were enrolled in the study. Methods: The HSA vaccine was prepared with lysates of allogeneic canine HSA cell lines mixed with an adjuvant composed of liposome‐DNA complexes. Dogs received a series of 8 immunizations administered over a 22‐week period, and most also received chemotherapy. Clinical adverse effects were noted, immune responses were measured by enzyme‐linked immunosorbent assay (ELISA) and flow cytometry, and survival times were calculated. Results: The most common adverse effects observed in vaccinated dogs also treated with doxorubicin chemotherapy were diarrhea and anorexia. Vaccinated dogs were found to mount strong humoral immune responses against a control antigen and, most dogs also mounted antibody responses against canine HSA cells. Thirteen dogs with stage II splenic HSA that received the tumor vaccine plus doxorubicin chemotherapy had an overall median survival time of 182 days. Conclusions: We conclude that an allogeneic tumor lysate vaccine is safe in dogs with HSA and can elicit humoral immune responses in dogs that are receiving concurrent doxorubicin chemotherapy.

Ribosomal Protein S24 Gene Is Mutated in Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA) is a rare congenital red-cell aplasia characterized by anemia, bone-marrow erythroblastopenia, and congenital anomalies and is associated with heterozygous mutations in the ribosomal protein (RP) S19 gene (RPS19) in approximately 25% of probands. We report identification of de novo nonsense and splice-site mutations in another RP, RPS24 (encoded by RPS24 [10q22-q23]) in approximately 2% of RPS19 mutation-negative probands. This finding strongly suggests that DBA is a disorder of ribosome synthesis and that mutations in other RP or associated genes that lead to disrupted ribosomal biogenesis and/or function may also cause DBA.

Ribosomal Protein S24 Gene Is Mutated in Diamond-Blackfan Anemia.

Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia with marked clinical heterogeneity, an increased risk of malignancy and mutations in ribosomal protein (RP) S19 in 25% of probands. To identify other gene(s) mutated in DBA and investigate their expression and function, we performed a genome-wide screen using a 10,000 single nucleotide polymorphism mapping set (Affymetrix) on a large family comprising 10 informative meioses. We found linkage of the DBA phenotype to regions on chromosome 8q, 10 and 6. The RP gene RPS24, is located in the linked region on chromosome 10; we sequenced exons, intron-exon boundaries and the promoter regions in this family, and found a nonsense mutation (316C>T) in exon 4 of RPS24 in five affected individuals, and a wild type sequence in five unaffected family members. This mutation causes the change of Gln106STOP and is predicted to result in formation of a truncated RPS24 protein. Subsequently, we sequenced DNA from 215 unrelated DBA probands, 30 with RPS19 mutations and 185 without. We found another nonsense mutation in exon 2 in a sporadic case, and a splice site deletion resulting in skipped exon 2 in another proband and in his father; over 200 control individuals did not have any of the above sequence changes, indicating that they are pathogenic mutations. To explore the normal role of RPS24 and consider how its dysfunction might result in DBA we performed real time RT-PCR (rt-PCR) and western blotting experiments on 20 normal human tissues and on lymphoblastoid cell lines from diseased and control individuals. Interestingly, rt-PCR of total human RPS24 and RPS19 mRNA revealed a tissue-specific variation in expression level. We found co-ordinate expression of both genes in the majority of studied tissues. Lymphoblastoid cell lines from both probands with nonsense mutations showed a reduced level of RPS24 mRNA, suggesting degradation of mutated transcripts due to nonsense mediated decay, while the RPS19 mRNA level in these patients was normal or elevated. Western blot experiments revealed a reduction of RPS24 protein in lymphoblastoid cell lines from all three mutated probands compared to control samples. Interestingly, co-ordinate expression of RPS24 and RPS19 protein was found in these patients as well as in other patients with RPS19 mutations or without any mutations, suggesting co-regulation of RP expression. To determine whether recruitment of mRNA to polysomes was impaired in DBA patients, we separated lymphoblast cell line lysates from nine diseased and four control individuals on sucrose gradients. We did not detect any significant difference in the RNA ratio of polysome-bound/free ribosomal subunits between diseased and control samples (p<0.3). It is likely that lymphoblasts, which are not defective in DBA, have sufficient ribosomes to mask an abnormality in translation.In summary, our results suggest that in addition to RPS24 and RPS19, which are mutated in ~ 27% of index cases, other DBA genes are also RP genes or genes involved in ribosome biogenesis or translation, and reinforce the notion that DBA is a ribosomal disease. This study opens new avenues for studying and understanding the pathogenesis of DBA.

Unraveling the Complex Proteome for Biomarker Discovery in Gastrointestinal and Liver Diseases

Unraveling the Complex Proteome for Biomarker Discovery in Gastrointestinal and Liver Diseases

Constitutive c-jun N-terminal kinase activity in acute myeloid leukemia derives from Flt3 and affects survival and proliferation

c-jun N-terminal kinase (JNK) has been implicated in proliferation and survival downstream from the tyrosine kinase oncogene, p210 BCR-ABL, in chronic myeloid leukemia. We studied whether a similar relationship between JNK and FMS-like tyrosine kinase 3 (Flt3) describes acute myeloid leukemia (AML). By immunoprecipitation, Flt3 was found to be activated and identified as the potential origin of JNK activity in a heavy majority of JNK+ve AML blasts tested. Often, Flt3 activity is associated with activating mutation of the gene locus. However, statistical linkage tied JNK activity with Flt3 expression levels rather than with mutation. An adaptor network to describe the signal cascade Flt3-to-JNK was uncovered. Active Flt3 was linked to p85 phosphoinositide-3 (PI-3) kinase, and p85 with cbl and CrkII/CrkL by co-immunoprecipitaton assays from lysates of model cell lines and primary AML blasts. JNK1 co-immunoprecipitated from such lysates with p85-cbl-crkII/L and bound to Crk species SH3 domain in pull-down assay. siRNA-mediated depletion of Flt3 or of cbl, the adaptor at the nexus of this signaling group, inhibited JNK activity on substrate c-jun. Within AML blast cells influenced by Flt3 signaling, selective inhibition of JNK by a small molecule inhibitor, led to proliferative inhibition, apoptosis, and sensitizing cells to the anthracycline, daunorubicin. These effects occurred upon JNK inhibition without off-target inhibition of extracellular signal-regulated kinase or AKT pathways, and p38-kinase activation, an effector in the p53/p14 arf tumor suppressor pathway, was also maintained or augmented. JNK is a bonafide signaling pathway from Flt3 in AML whose function for proliferation and survival is required in a significant AML cohort with active Flt3 signaling, by mutation or overexpression of Flt3.

Interrelated Roles for Mcl-1 and BIM in Regulation of TRAIL-mediated Mitochondrial Apoptosis

The current study demonstrates a novel cross-talk mechanism between the TRAIL receptor death signaling pathway and the mitochondria. This newly identified pathway is regulated at the mitochondrial outer membrane by a complex between the prosurvival Bcl-2 member, Mcl-1 and the BH3-only protein, Bim. Under non-apoptotic conditions, Bim is sequestered by Mcl-1. Direct degradation of Mcl-1 by TRAIL-activated caspase-8 or caspase-3 produces Mcl-1-free Bim that mediates a Bax-dependent apoptotic cascade. Using Mcl-1 or Bim RNAi, we demonstrate that a loss in Mcl-1 expression significantly enhances the mitochondrial apoptotic response to TRAIL that is now mediated by freed Bim. Whereas overexpression of Mcl-1 contributes to the preservation of the mitochondrial membrane potential, Mcl-1 knockdown facilitates the Bim-mediated dissipation of this potential. Loss of Mcl-1 contributes to an increased level of caspase activity downstream of the mitochondrial response to TRAIL. Furthermore, the Mcl-1 expression level at the mitochondrial outer membrane determines the release efficiency for the apoptogenic proteins cytochrome c, Smac, and HtrA2 in response to Bim. These are the first findings to demonstrate the involvement of Bim in the TRAIL-mediated mitochondrial cascade. They also suggest that Mcl-1 may serve as a direct substrate for TRAIL-activated caspases implying the existence of a novel TRAIL/caspase-8/Mcl-1/Bim communication mechanism between the extrinsic and the intrinsic apoptotic pathways.

Protein tyrosine phosphatase PRL-3 in malignant cells and endothelial cells: expression and function

Protein tyrosine phosphatase PRL-3 mRNA was found highly expressed in colon cancer endothelium and metastases. We sought to associate a function with PRL-3 expression in both endothelial cells and malignant cells using in vitro models. PRL-3 mRNA levels were determined in several normal human endothelial cells exposed or unexposed to the phorbol ester phorbol 12-myristate 13-acetate (PMA) and in 27 human tumor cell lines. In endothelial cells, PRL-3 mRNA expression was increased in human umbilical vascular endothelial cells and human microvascular endothelial cells (HMVEC) exposed to PMA. An oligonucleotide microarray analysis revealed that PRL-3 was among the 10 genes with the largest increase in expression on PMA stimulation. Phenotypically, PMA-treated HMVEC showed increased invasion, tube formation, and growth factor-stimulated proliferation. A flow cytometric analysis of cell surface markers showed that PMA-treated HMVEC retained endothelial characteristics. Infection of HMVEC with an adenovirus expressing PRL-3 resulted in increased tube formation. In tumor cells, PRL-3 mRNA levels varied markedly with high expression in SKNAS neuroblastoma, MCF-7 and BT474 breast carcinoma, Hep3B hepatocellular carcinoma, and HCT116 colon carcinoma. Western blotting analysis of a subset of cell line lysates showed a positive correlation between PRL-3 mRNA and protein levels. PRL-3 was stably transfected into DLD-1 colon cancer cells. PRL-3-overexpressing DLD-1 subclones were assessed for doubling time and invasion. Although doubling time was similar among parental, empty vector, and PRL-3 subclones, invasion was increased in PRL-3-expressing subclones. In models of endogenous expression, we observed that the MCF-7 cell line, which expresses high levels of PRL-3, was more invasive than the SKBR3 cell line, which expresses low levels of PRL-3. However, the MDA-MB-231 cell line was highly invasive with low levels of PRL-3, suggesting that in some models invasion is PRL-3 independent. Transfection of a PRL-3 small interfering RNA into MCF-7 cells inhibited PRL-3 expression and cell invasion. These results indicate that PRL-3 is functional in both endothelial cells and malignant cells and further validate PRL-3 as a potentially important molecular target for anticancer therapy.

Induction of Myeloma Cell Line-specific Cytotoxic T Lymphocytes using Monocyte-derived Dendritic Cells Pulsed with Myeloma Cell Line Lysates

Background: In multiple myeloma (MM), the idiotype (ID) determinant of the paraprotein has been used for immunotherapy using dendritic cells (DCs). However, ID-specific immune responses showed limited clinical responses after the Id vaccination. Therefore, an alternative approach using DCs pulsed with other tumor antigens is required. Methods: We investigated the possibility of immunotherapy for MM using myeloma cell line-specific cytotoxic T lymphocytes (CTLs), that were stimulated in vitro by monocyte-derived DCs pulsed with the myeloma cell line ysates. CD14+ cells isolated from the peripheral blood of HLA-A0201+ healthy donors were cultured in the presence of GM-CSF and IL-4. On day 6, the immature DCs were pulsed with the myeloma cell line lysates (IM-9: HLA0201+ and ARH-77: HLA0201+), and then maturation of DCs was induced by the addition of TNF-α for 2 days. CTL lines were generated by a 2 time stimulation with DCs to the autologous CD3+ T cells. Results: DCs pulsed with myeloma cell lysates showed the production of IL-12p70, but less than that of unpulsed DCs. CTLs lines stimulated with the DCs pulsing, for the myeloma cell line lysates, showed potent cytotoxic activities against autologous target cells, but not against HLA-A2－ cell lines (RPMI-8226). Mature DCs pulsed with the myeloma cell line lysates showed a higher stimulatory capacity for autologous CTL when compared with mature non-pulsed DCs.

T-Cell Recognition and Cytokine Profile Induced by Melanocyte Epitopes in Patients with HLA-DRB1*0405-Positive and -Negative Vogt-Koyanagi-Harada Uveitis

Vogt-Koyanagi-Harada disease (VKH), an autoimmune disease targeted against melanocytes, is associated with HLA-DRB1*0405. This study was undertaken to analyze T-cell recognition and the cytokine expression profile induced by melanocyte epitopes in HLA-DRB1*0405-positive and -negative patients with VKH uveitis. Peripheral blood mononuclear cells (PBMC) proliferation and Th1 (IFN-gamma) and Th2 (IL-4 and IL-5) cytokine production were analyzed in HLA-DRB1*0405-positive (n = 12) and -negative (n = 22) patients with VKH and HLA-DRB1*0405-positive (n = 9) and -negative (n = 8) control subjects in response to human melanoma cell line lysate (HMCLL) and 28 synthetic peptides derived from the human melanocyte differentiation proteins TYR, TRP1, TRP2, and Pmel17. The peptides were selected using the TEPITOPE algorithm, based on their predicted binding to HLA-DRB1*0405 and to the non-disease-related HLA-DRB1*15. HMCLL was recognized exclusively by the patients' PBMC (44%) but not by those of the control subjects (P < 0.01). PBMC from patients with VKH recognized an increased breadth of melanocyte-derived peptides at lower peptide concentrations than in the control subjects (68% vs. 25%; P < 0.01, at 1 microM) and did not produce the Th2 cytokine IL-4 in response to disease-specific peptides (0% vs. 50%, P < 0.001). Five peptides were exclusively recognized in patients bearing HLA-DRB1*0405. Furthermore, HLA-DRB1*0405-bearing patients, but not those with HLA-DRB1*15, recognized an increased breadth of melanocyte epitopes in comparison to HLA-matched control subjects (60% vs. 28%; P < 0.05). These data indicate that patients with VKH are sensitized to melanocyte epitopes and display a peptide-specific Th1 cytokine response. In addition, the data indicate that patients bearing HLA-DRB1*0405 recognize a broader melanocyte-derived peptide repertoire, reinforcing the importance of this allele in susceptibility to the development of VKH disease.

Tagging-via-Substrate Strategy for Probing O-GlcNAc Modified Proteins

Identification of proteins bearing a specific post-translational modification would imply functions of the modification. Proteomic analysis of post-translationally modified proteins is usually challenging due to high complexity and wide dynamic range, as well as unavailability of efficient methods to enrich the proteins of interest. Here, we report a strategy for the detection, isolation, and profiling of O-linked N-acetylglucosamine (O-GlcNAc) modified proteins, which involves three steps: metabolic labeling of cells with an unnatural GlcNAc analogue, peracetylated azido-GlcNAc; chemoselective conjugation of azido-GlcNAc modified proteins via the Staudinger ligation, which is specific between phosphine and azide, using a biotinylated phosphine capture reagent; and detection and affinity purification of the resulting conjugated O-GlcNAc modified proteins. Since the approach relies on a tag (azide) in the substrate, we designated it the tagging-via-substrate (TAS) strategy. A similar strategy was used previously for protein farnesylation, phosphorylation, and sumoylation. Using this approach, we were able to specifically label and subsequently detect azido-GlcNAc modified proteins from the cytosolic lysates of HeLa, 3T3, COS-1, and S2 cell lines, suggesting the azido-substrate could be tolerated by the enzymatic systems among these cells from diverse biological species. We isolated azido-GlcNAc modified proteins from the cytosolic extract of S2 cells and identified 10 previously reported and 41 putative O-GlcNAc modified proteins, by nano-HPLC-MS/MS. Our study demonstrates that the TAS approach is a useful tool for the detection and proteomic analysis of O-GlcNAc modified proteins.

Immunotherapy of Murine Malignant Mesothelioma Using Tumor Lysate–pulsed Dendritic Cells

Exploiting the immunostimulatory capacities of dendritic cells holds great promise for cancer immunotherapy. Currently, dendritic cell-based immunotherapy is evaluated clinically in a number of malignancies, including melanoma and urogenital and lung cancer, showing variable but promising results. To evaluate if pulsed dendritic cells induce protective immunity against malignant mesothelioma in a mouse model. Malignant mesothelioma was induced in mice by intraperitoneal injection of the AB1 mesothelioma cell line, leading to death within 28 days. For immunotherapy, dendritic cells were pulsed overnight either with AB1 tumor cell line lysate, AB1-derived exosomes, or ex vivo AB1 tumor lysate, and injected either before (Days -14 and -7) at the day of (Day 0) or after (Days +1 and +8) tumor implantation. Mice receiving tumor lysate-pulsed dendritic cells before tumor implantation demonstrated protective antitumor immunity with prolonged survival (> 3 months) and even resisted secondary tumor challenge. Tumor protection was associated with strong tumor-specific cytotoxic T-lymphocyte responses. Adoptive transfer of splenocytes or purified CD8+ T lymphocytes transferred tumor protection to unimmunized mice in vivo. When given after tumor implantation in a therapeutic setting, pulsed dendritic cells prevented mesothelioma outgrowth. With higher tumor load and delayed administration after tumor implantation, dendritic cells were no longer effective. We demonstrate in this murine model that immunotherapy using pulsed dendritic cells may emerge as a powerful tool to control mesothelioma outgrowth. In the future, immunotherapy using dendritic cells could be used as adjuvant to control local recurrence after multimodality treatment for malignant mesothelioma.

Role of extracellular cysteine residues in dimerization/oligomerization of the human prostacyclin receptor

Prostacyclin activation of prostanoid IP receptors may result in pain sensation, inflammatory responses, inhibition of platelet aggregation, and vasodilation in vascular tissue. The prostanoid IP receptor is a G-protein-coupled receptor. In the present study, we investigated the determinants responsible, at least in part, for the prostacyclin receptor (IP) dimerization/oligomerization. Using co-immunoprecipitation of differentially tagged IP expressed in COS-7 cells, we demonstrate that IP can form dimers and oligomers. Treatment of IP-expressing cells with the stable agonist carbaprostacyclin failed to alter the ratios of oligomeric/dimeric/monomeric forms of the receptor, suggesting that IP dimerization/oligomerization is an agonist-independent process. The reducing agents dithiothreitol and 2-mercaptoethanol were highly efficient in converting the receptor from its oligomeric form to the monomeric state, indicating the involvement of disulfide bonds in IP oligomerization. Immunoblotting of the osteoblastic MG-63 cell line lysates with an anti-IP specific antibody revealed the presence of endogenous IP oligomers which were converted to dimers and monomers upon treatment with dithiothreitol. Individual substitutions of the four extracellular IP Cys residues (Cys 5, Cys 92, Cys 165 and Cys 170) for Ser resulted in greatly decreased receptor protein expression in COS-7 cells. The C92–170S double mutant showed receptor protein expression level similar to the individual mutants. However, expression of the C92–165S and C165–170S mutants was drastically reduced, suggesting that there was formation of disulfide bonds between Cys 5 and Cys 165, and between Cys 92 and Cys 170. The Cys receptor mutants showed altered oligomer/dimer/monomer ratios. Dimerization/oligomerization likely occurs intracellularly since these Cys receptor mutants could still form dimers/oligomers despite their lack of expression at the cell surface.

Comparison of protein expression patterns between hepatocellular carcinoma cell lines and a hepatoblastoma cell line

Hepatocellular carcinoma (HCC) and hepatoblastoma (HB) are malignancies of the liver with different etiologies, but the HB cell line HepG2 has been frequently used in various studies of HCC. In this study, we compare the protein expression patterns between HepG2 cells and three HCC cell lines, HKCI-2, HKCI-3, and HKCI-4, respectively. The cell lysates of individual cell lines were separated by two-dimensional polyacrylamide gel electrophoresis. The protein spots in the gel images were quantified and compared by image analysis software. The differentially expressing proteins were then identified by tryptic peptide, mass fingerprinting. Compared with the HepG2 cells, the normalized quantities of 49 and 58 protein spots were found to be at least twofold higher and twofold lower, respectively, in all three HCC cell lines. The differentially expressed proteins can be grouped into structural proteins (annexins, transgelin, laminin receptor), stress-induced proteins (HSP27, 60, and 70), enzymes (aldehyde dehydrogenase, pyruvate kinase, α-enolase, ect.), and transcription factors (far upstream element binding protein 2, GTP-binding nuclear protein RAN). Some of these proteins play important roles in regulating homeostasis, drug resistance, apoptosis, cell differentiation, cell growth, and metastasis. In conclusion, our proteomic data indicate that there are considerable differences in the protein expression patterns between HepG2 cells and the HCC cells, suggesting differences in cellular properties. Hence, HepG2 may not be a good cell line model for studying HCC.

Hepatocyte Growth Factor Receptor Tyrosine Kinase Met Is a Substrate of the Receptor Protein-tyrosine Phosphatase DEP-1

The receptor protein-tyrosine phosphatase (PTP) DEP-1 (CD148/PTP-eta) has been implicated in the regulation of cell growth, differentiation, and transformation, and most recently has been identified as a potential tumor suppressor gene mutated in colon, lung, and breast cancers. We have generated constructs comprising the cytoplasmic segment of DEP-1 fused to the maltose-binding protein to identify potential substrates and thereby suggest a physiological function for DEP-1. We have shown that the substrate-trapping mutant form of DEP-1 interacted with a small subset of tyrosine-phosphorylated proteins from lysates of the human breast tumor cell lines MDA-MB-231, T-47D, and T-47D/Met and have identified the hepatocyte growth factor/scatter factor receptor Met, the adapter protein Gab1, and the junctional component p120 catenin as potential substrates. Following ligand stimulation, phosphorylation of specific tyrosyl residues in Met induces mitogenic, motogenic, and morphogenic responses. When co-expressed in 293 cells, the full-length substrate-trapping mutant form of DEP-1 formed a stable complex with the chimeric receptor colony stimulating factor 1 (CSF)-Met and wild type DEP-1 dephosphorylated CSF-Met. Furthermore, we observed that DEP-1 preferentially dephosphorylated a Gab1 binding site (Tyr(1349)) and a COOH-terminal tyrosine implicated in morphogenesis (Tyr(1365)), whereas tyrosine residues in the activation loop of Met (Tyr(1230), Tyr(1234), and Tyr(1235)) were not preferred targets of the PTP. The ability of DEP-1 preferentially to dephosphorylate particular tyrosine residues that are required for Met-induced signaling suggests that DEP-1 may function in controlling the specificity of signals induced by this PTK, rather than as a simple "off-switch" to counteract PTK activity.

Generation of tumor cell lysate-loaded dendritic cells preprogrammed for IL-12 production and augmented T cell response.

Dendritic cells (DC) loaded with tumor associated antigens (TAA) are often used for the vaccination of cancer patients; however methodologies for the vaccine preparation have not yet been standardized. The purpose of this work was to optimize the ex-vivo production of functional TAA-loaded DC that would produce interleukin-2 (IL-12) and enhance the T cell response. We generated ex-vivo DC from human monocytes with granulocyte macrophage-colony stimulating factor (GM-CSF) and IL-4, and whole necrotic tumor cells (cell lysates) of cancer cell lines were used as model TAA. DC were loaded with lysates without or with additional tumor necrosis factor-alpha (TNF-alpha), or cytokine combination treatments and tested for functional ability in vitro. Tumor cell lysates alone did not fully mature DC either phenotypically or functionally. After antigen uptake additional maturation signals were necessary. TNF-alpha matured DC phenotypically, but additional interferon-gamma (IFN-gamma) treatment was necessary to achieve functional maturation, the production of significant amounts of IL-12. Since IL-12 production by DC increased during the first 24 h of maturation and declined by 48 h, proper timing of the ex-vivo DC treatment was crucial for the generation of functionally mature antigen-loaded DC. Our results suggest that after allowing 4 h of tumor lysate uptake by immature DC, further treatment with TNF-alpha and IFN-gamma for 24 h provides the optimal conditions to obtain functional TAA-loaded DC. These TAA-loaded cytokine pretreated DC then prime naïve T cells, and enhance both T helper 1 (Th1), Th2 and cytotoxic T lymphocyte (CTL) responses, that are necessary to achieve an effective, specific anti-tumor response.

Selection of Mimotopes of the Cell Surface Adhesion Molecule Mel-CAM from a Random pVIII-28aa Phage Peptide Library

The cell surface adhesion molecule Mel-CAM is highly expressed in advanced primary and metastatic melanoma. Mel-CAM was first described as an integral membrane glycoprotein of malignant melanoma cells. The murine monoclonal antibody MAd18-5D7 recognizes an epitope of the extracellular domain of Mel-CAM and is able to enhance Mel-CAM mediated adhesion of melanoma cells in aggregation assays. For the characterization of peptides that antigenically mimic surface-exposed areas of Mel-CAM we screened a newly constructed random pVIII-28aa bacteriophage peptide library against MAd18-5D7. After three panning rounds a population of phages binding to MAd18-5D7 was enriched. Peptides expressed on the surface of these phages were then tested for their specificity for the antibody's antigen binding site. DNA sequences coding for two specific peptide ligands were determined. One of the deduced amino acid sequences showed similarity to a portion of the sequence of the third immunoglobulin-like extracellular domain of Mel-CAM. Both peptides blocked the interaction of MAd18-5D7 with Mel-CAM present in a MelJuSo melanoma cell line lysate. Phage displayed as well as synthetic peptides inhibited in a dose-dependent manner the binding of MAd18-5D7 to recombinant Mel-CAM in enzyme-linked immunosorbent assay experiments. No such inhibition was observed using a panel of other anti-Mel-CAM antibodies. Our results clearly indicate that these 28mer peptides are structural equivalents of the MAd18-5D7 epitope of Mel-CAM and that they will be useful tools for further in vitro and in vivo studies of Mel-CAM mediated cell-cell interaction.