RESUMO
Calmodulin is an essential regulator of intracellular processes in response to extracellular stimuli mediated by a rise in Ca(2+) ion concentration. To profile protein-protein interactions of calmodulin in human brain, we probed a high content human protein array with fluorophore-labeled calmodulin in the presence of Ca(2+). This protein array contains 37,200 redundant proteins, incorporating over 10,000 unique human neural proteins from a human brain cDNA library. We designed a screen to find high affinity (K(D) < or = 1 microm) binding partners of calmodulin and identified 76 human proteins from all intracellular compartments of which 72 are novel. We measured the binding kinetics of 74 targets with calmodulin using a high throughput surface plasmon resonance assay. Most of the novel calmodulin-target complexes identified have low dissociation rates (k(off) < or = 10(-3) s(-1)) and high affinity (K(D) = 1 mum), consistent with the design of the screen. Many of the identified proteins are known to assemble in neural tissue, forming assemblies such as the spectrin scaffold and the postsynaptic density. We developed a microarray of the identified target proteins with which we can characterize the biochemistry of calmodulin for all targets in parallel. Four novel targets were verified in neural cells by co-immunoprecipitation, and four were selected for exploration of the calmodulin-binding regions. Using synthetic peptides and isothermal titration calorimetry, calmodulin binding motifs were identified in the potassium voltage-gated channel Kv6.1 (residues 474-493), calmodulin kinase-like vesicle-associated protein (residues 302-316), EF-hand domain family member A2 (residues 202-216), and phosphatidylinositol-4-phosphate 5-kinase, type I, gamma (residues 400-415).
Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Neurônios/metabolismo , Análise Serial de Proteínas/métodos , Motivos de Aminoácidos , Animais , Western Blotting , Proteínas de Ligação a Calmodulina/química , Calorimetria , Humanos , Imunoprecipitação , Camundongos , Peptídeos/metabolismo , Ligação Proteica , Reprodutibilidade dos Testes , Software , Ressonância de Plasmônio de SuperfícieRESUMO
Doxorubicin is considered to be the most effective agent in the treatment of breast cancer patients. Unfortunately, resistance to this agent is common, representing a major obstacle to successful treatment. The identification of novel biomarkers that are able to predict treatment response may allow therapy to be tailored to individual patients. Antibody microarrays provide a powerful new technique, enabling the global comparative analysis of many proteins simultaneously. This technology may identify a panel of proteins to discriminate between drug-resistant and drug-sensitive samples. The Panorama Cell Signaling Antibody Microarray was exploited to analyze the MDA-MB-231 breast cancer cell line and a novel derivative, which displays significant resistance to doxorubicin at clinically relevant concentrations. The microarray comprised 224 antibodies selected from a variety of pathways, including apoptotic and cell signaling pathways. A standard >/=2.0-fold cutoff value was used to determine differentially expressed proteins. A decrease in the expression of mitogen-activated protein kinase-activated monophosphotyrosine (phosphorylated extracellular signal-regulated kinase; 2.8-fold decrease), cyclin D2 (2.5-fold decrease), cytokeratin 18 (2.5-fold decrease), cyclin B1 (2.4-fold decrease), and heterogeneous nuclear ribonucleoprotein m3-m4 (2.0-fold decrease) was associated with doxorubicin resistance. Western blotting was exploited to confirm results from the antibody microarray experiment. These results suggest that antibody microarrays can be used to identify novel biomarkers and further validation may reveal mechanisms of chemotherapy resistance and identify potential therapeutic targets. [Mol Cancer Ther 2006;5(8):2115-20].
Assuntos
Antibióticos Antineoplásicos/farmacologia , Anticorpos , Neoplasias da Mama/tratamento farmacológico , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Análise Serial de Proteínas/métodos , Western Blotting , Feminino , Humanos , Células Tumorais CultivadasRESUMO
Apoptosis proteins may play a role in prognosis and therapy response; however, they have not been fully investigated in gastric cancer. We aimed to assess the expression of proteins in the Bcl-2 family. Immunohistochemistry was employed to examine the expression of the antiapoptotic proteins Bcl-2 and Bcl-XL and the proapoptotic proteins Bad, Bak, Bax, Bid, Bim, and p53 in 21 cases of gastric cancer. Immunopositivity was observed in 12/21 (57%) cases for p53, 16/21 (76%) cases for Bcl-XL, and 5/21 (23%) cases for Bcl-2. For the proapoptotic members of the Bcl family, loss of protein expression was observed: Bid (14/21 cases; 66%), Bad (13/21 cases; 61%), Bax (12/21 cases; 57%), Bak (9/21 cases; 42%), and Bim (4/21 cases; 19%). This study identified apoptosis proteins that exhibit heterogeneous expression between primary gastric carcinomas.
Assuntos
Apoptose , Biomarcadores Tumorais/análise , Imuno-Histoquímica , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Idoso , Idoso de 80 Anos ou mais , Proteínas Reguladoras de Apoptose/análise , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/análise , Proteína 11 Semelhante a Bcl-2 , Feminino , Humanos , Masculino , Proteínas de Membrana/análise , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas c-bcl-2/análise , Neoplasias Gástricas/imunologia , Proteína Supressora de Tumor p53/análise , Proteína Killer-Antagonista Homóloga a bcl-2/análise , Proteína X Associada a bcl-2/análise , Proteína de Morte Celular Associada a bcl/análise , Proteína bcl-X/análiseRESUMO
One of the most valuable objectives for oncologists is the ability to predict patient response to chemotherapy before drugs are administered in order to maximise the therapeutic benefit of treatment whilst limiting the toxicity. This is particularly relevant in non-small cell lung cancer as the initial treatment decision is important due to the inherent drug resistance of many tumours and short survival times of patients. We established a homogeneous series of pre-treatment archival biopsy samples from patients receiving first-line single-agent vinorelbine for non-small cell lung cancer. Cases were selected following strict inclusion criteria and patient response was assessed using the Response Evaluation Criteria in Solid Tumours guideline. The expression of 7 proteins was investigated and correlated with response data. Chi-square analysis revealed no association between expression of Bcl-2, Bcl-XL, Bad, Bak, Bid or p53 proteins and response to vinorelbine therapy. There was a trend for Hsp27-positive tumours to show progression but this did not reach significance (p=0.068). The results suggest that Hsp27 expression may be useful as a predictor of response to single-agent vinorelbine chemotherapy in non-small cell lung cancer patients but a larger study is required to confirm this.
Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Proteínas de Choque Térmico/análise , Neoplasias Pulmonares/tratamento farmacológico , Proteínas de Neoplasias/análise , Vimblastina/análogos & derivados , Idoso , Proteínas Reguladoras de Apoptose/análise , Feminino , Proteínas de Choque Térmico HSP27 , Humanos , Masculino , Pessoa de Meia-Idade , Chaperonas Moleculares , Prognóstico , Vimblastina/uso terapêutico , VinorelbinaRESUMO
Autoantibodies represent an attractive biomarker for diagnostic assays principally due to the stability of immunoglobulin in patient serum facilitating measurement with conventional assays. Immune responses to tumorigenesis may facilitate detection of ovarian cancer in the early stages of the disease with identification of a panel of tumour specific autoantibodies. Despite the reporting of many tumour associated autoantibodies using arrays of tumour antigens, this has not led to the advance in diagnostic capability as rapidly as was initially expected. Here we examine the potential diagnostic utility of candidate autoantibody biomarkers identified via screening of serum samples on a high content human protein array from a unique cohort of early stage and late stage ovarian cancer patients. We analyse the performance of autoantibodies to the tumour suppressor protein p53 and the novel autoantigens alpha adducin and endosulfine alpha identified in our array screen. Each antigen has different performance characteristics using conventional ELISA format and Western blot immunoassay. The high attrition rate of promising autoantigens identified by array screening can in part be explained by the presentation of the epitope of the antigen in the subsequent method of validation and this study provides directions on maximising the potential of candidate biomarkers. This article is part of a Special Issue entitled: Translational Proteomics.
Assuntos
Anticorpos Antineoplásicos/sangue , Antígenos de Neoplasias/química , Autoanticorpos/sangue , Epitopos/química , Proteínas de Neoplasias/química , Neoplasias Ovarianas , Análise Serial de Proteínas/métodos , Feminino , Humanos , Neoplasias Ovarianas/sangue , Neoplasias Ovarianas/diagnósticoRESUMO
Profiling the autoantibody (AAb) repertoire in serum has been routinely used for many years for the diagnosis of autoimmune diseases, including rheumatoid arthritis, scleroderma, and lupus. In recent years, AAb profiling of cancers has become a prominent field in oncology research. Protein arrays enable high-throughput screening of clinical samples, characterising the serum profile using low volumes of samples. This chapter describes the use of a protein array comprising 37,200 redundant proteins (containing over 10,000 non-redundant human recombinant proteins) for identification of the proteins bound by the antibodies in human sera using a test set of serum samples. The proteins identified have the potential to be candidate biomarkers. These recombinant proteins are expressed, purified, and robotically spotted on microarrays or chips to facilitate the screening of additional serum samples with the aim of identifying a candidate biomarker or panel of potential biomarkers for applications in disease diagnosis, stage, progression, or response to therapy.
Assuntos
Autoanticorpos/sangue , Biomarcadores/sangue , Análise Serial de Proteínas/métodos , Proteômica/métodos , Eletroforese em Gel de Poliacrilamida , HumanosRESUMO
BACKGROUND: Although malignant pleural mesothelioma is a rare tumour, its incidence is increasing. The prognosis remains very poor with an average survival of 10 months from diagnosis. The choice of chemotherapy regimens for mesothelioma patients is limited and new approaches are required. COX-2 inhibition induces apoptosis in a variety of tumour cell lines. The cytotoxic effect of conventional drugs may be enhanced by the addition of a COX-2 inhibitor. In order to identify possible new therapeutic approaches we aimed to determine whether the addition of COX-2 inhibitors would enhance the cytotoxic effect of chemotherapeutic agents in mesothelioma cell lines. MATERIALS AND METHODS: Three mesothelioma cell lines MSTO-211H, NCI-H2052 and NCI-H2452 were utilised. Using the COX-2 positive A549 lung cancer cell line as control, all cell lines were assayed using an MTT assay with non-specific COX-2 inhibitors (sulindac and flurbiprofen), specific COX-2 inhibitors (DuP-697 and NS-398), and chemotherapeutic agents (cisplatin, vinorelbine and pemetrexed). RESULTS: All cell lines exhibited COX-2 expression by western blotting using two antibodies. The addition of either DuP-697 or NS-398 increased the sensitivity to pemetrexed in all cell lines. CONCLUSION: These findings suggest that the design of novel pemetrexed-containing combination regimens with increased cytotoxicity may be feasible.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase 2/administração & dosagem , Glutamatos/administração & dosagem , Guanina/análogos & derivados , Mesotelioma , Neoplasias Pleurais , Western Blotting , Linhagem Celular Tumoral , Cisplatino/administração & dosagem , Sinergismo Farmacológico , Guanina/administração & dosagem , Humanos , Mesotelioma/tratamento farmacológico , Pemetrexede , Neoplasias Pleurais/tratamento farmacológico , Vimblastina/administração & dosagem , Vimblastina/análogos & derivados , VinorelbinaRESUMO
Little is known about the Bcl-2 family members in mesothelioma. These proteins are involved in the control of apoptosis, carrying out both pro- and anti- apoptotic functions. Immunohistochemistry was used to examine the expression of p53 and Bcl-2 family members in 54 archival mesothelioma samples (39 epithelial, 15 sarcomatoid tumours). Overexpression of p53 was observed in 81% (44/54). For anti-apoptotic proteins, overexpression was recorded as follows: Bcl-2 40% (22/54), Bcl-XL 24% (13/54), Mcl-1 92% (50/54). For pro-apoptotic proteins, loss of expression was recorded as follows: Bad 25% (14/54), Bak 24% (13/54), Bax 42% (23/54), Bid 37% (20/54), Bim 18% (10/54). Statistically significant differences between epithelial and sarcomatoid tumours were observed for Bid (p < 0.001), Bad (p = 0.012) and Bcl-XL (p = 0.03). Significant differences in abnormal expression of apoptosis proteins were found between epithelial and sarcomatoid subtypes but histological subtype was the only factor with significant association to patient prognosis.
Assuntos
Mesotelioma/metabolismo , Neoplasias Pleurais/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Adulto , Idoso , Apoptose , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Feminino , Humanos , Masculino , Mesotelioma/patologia , Pessoa de Meia-Idade , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Pleurais/patologia , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/metabolismoRESUMO
BACKGROUND: Although BRCA genes have been implicated in certain tumors, particularly breast tumors, their role in colon tumorigenesis has not been fully explored. We aimed to investigate the association of the BRCA2 and putative "BRCA3" genes in a homogeneous series of right-sided colon cancer specimens. METHODS: Twenty-three Dukes' stage C, replication error-negative carcinomas were selected from patients with right-sided colon cancer. After histological examination and microdissection, DNA was extracted from normal colon and carcinoma from each patient. Five microsatellite markers spanning the region of BRCA2 and BRCA3 on chromosome 13 (D13S218, D13S219, D13S165, D13S156, and D13S160) and two markers intragenic to BRCA2 and BRCA3 (D13S171 and D13S1308, respectively) were used. Polymerase chain reaction products were analyzed by using a fluorescent allele imbalance assay. RESULTS: Markers demonstrating the highest allelic imbalance were D13S1308 (53%), D13S171 (33%), and D13S160 (37%). CONCLUSIONS: The intragenic markers D13S1308 (BRCA3) and D13S171 (BRCA2) on chromosome 13 demonstrated a high frequency of allelic imbalance in primary colon carcinoma. This suggests an involvement of BRCA2 and putative BRCA3 in colon tumorigenesis in right-sided, replication error-negative, Dukes' stage C cancers. Further studies are needed to confirm the precise role of these genes, and any prognostic significance, in colon cancer.
Assuntos
Neoplasias do Colo/genética , Genes BRCA2 , Alelos , Cromossomos Humanos Par 13 , Neoplasias do Colo/patologia , Dano ao DNA , DNA de Neoplasias/genética , Genes Supressores de Tumor , Humanos , Perda de Heterozigosidade , Repetições de Microssatélites , Estadiamento de Neoplasias , Reação em Cadeia da PolimeraseRESUMO
BACKGROUND: Proteins of the Bcl-2 family play a key role in the control of apoptosis and carry out both proapoptotic and antiapoptotic functions. However, with the exception of Bcl-2 itself, little is known about the expression of these potentially critical proteins in nonsmall cell lung carcinoma. METHODS: Immunohistochemistry was used to study the expression of Bcl-2 and 6 other Bcl-2 family proteins in a pilot series of 41 archival nonsmall cell lung carcinoma specimens (19 adenocarcinomas and 22 squamous cell carcinomas). RESULTS: Overexpression of the apoptosis inhibitors Bcl-2 and Bcl-X(L) was observed in 10 of 41 samples (24%) and in 11 of 41 samples (27%), respectively. Loss of expression of proapoptotic proteins was observed as follows: Bak, 24 of 41 samples (59%); Bad, 21 of 41 samples (51%); Bid, 20 of 41 samples (49%); Bax, 14 of 41 samples (34%); and Bim/Bod, 2 of 41 samples (5%). Statistically significant differences in expression between adenocarcinoma samples and squamous cell carcinoma samples were observed for Bcl-X(L) (overexpression in 11 of 19 adenocarcinomas [58%] vs. 0 of 22 squamous cell carcinomas [0%]; P < 0.001) and for Bad (loss of expression in 5 of 19 adenocarcinomas [26%] vs. 16 of 22 squamous cell carcinomas [73%]; P = 0.004). CONCLUSIONS: Although this was only a pilot study, the results revealed significant differences in the expression of apoptosis-related proteins both between individual samples of nonsmall cell lung carcinoma and between the two main histologic subtypes. Such differences may play a role in the development of lung tumors; and, if it is found that these differences are of clinical importance, then it may be required to regard nonsmall cell lung carcinoma subtypes as separate entities rather than as one disease.