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1.
Mol Ther ; 30(11): 3450-3461, 2022 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-35933584

RESUMO

MicroRNA (miRNAs) are pleiotropic post-transcriptional modulators of gene expression. Their inherently pleiotropic nature makes miRNAs strong candidates for the development of cancer therapeutics, yet despite their potential, there remains a challenge to deliver nucleic acid-based therapies into cancer cells. We developed a novel approach to modify miRNAs by replacing the uracil bases with 5-fluorouracil (5-FU) in the guide strand of tumor suppressor miRNAs, thereby combining the therapeutic effect of 5-FU with tumor-suppressive effect of miRNAs to create a potent, multi-targeted therapeutic molecule without altering its native RNAi function. To demonstrate the general applicability of this approach to other tumor-suppressive miRNAs, we screened a panel of 12 novel miRNA mimetics in several cancer types, including leukemia, breast, gastric, lung, and pancreatic cancer. Our results show that 5-FU-modified miRNA mimetics have increased potency (low nanomolar range) in inhibiting cancer cell proliferation and that these mimetics can be delivered into cancer cells without delivery vehicle both in vitro and in vivo, thus representing significant advancements in the development of therapeutic miRNAs for cancer. This work demonstrates the potential of fluoropyrimidine modifications that can be broadly applicable and may serve as a platform technology for future miRNA and nucleic acid-based therapeutics.


Assuntos
MicroRNAs , Neoplasias Pancreáticas , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Genes Supressores de Tumor , Fluoruracila/farmacologia , Neoplasias Pancreáticas/genética , Interferência de RNA , Regulação Neoplásica da Expressão Gênica
2.
Proc Natl Acad Sci U S A ; 118(50)2021 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-34876527

RESUMO

Pancreatic cancer is usually asymptomatic in the early stages; the 5-y survival rate is around 9%; and there is a lack of effective treatment. Here we show that SSEA-4 is more expressed in all pancreatic cancer cell lines examined but not detectable in normal pancreatic cells; and high expression of SSEA-4 or the key enzymes B3GALT5 + ST3GAL2 associated with SSEA-4 biosynthesis significantly lowers the overall survival rate. To evaluate potential new treatments for pancreatic cancer, homogeneous antibodies with a well-defined Fc glycan for optimal effector functions and CAR-T cells with scFv construct designed to target SSEA-4 were shown highly effective against pancreatic cancer in vitro and in vivo. This was further supported by the finding that a subpopulation of natural killer (NK) cells isolated by the homogeneous antibody exhibited enhancement in cancer-cell killing activity compared to the unseparated NK cells. These results indicate that targeting SSEA-4 by homologous antibodies or CAR-T strategies can effectively inhibit cancer growth, suggesting SSEA-4 as a potential immunotherapy target for treating pancreatic disease.


Assuntos
Anticorpos/imunologia , Neoplasias Pancreáticas/tratamento farmacológico , Antígenos Embrionários Estágio-Específicos/imunologia , Animais , Linhagem Celular Tumoral , Terapia Baseada em Transplante de Células e Tecidos , Regulação da Expressão Gênica , Humanos , Imunoterapia , Imunoterapia Adotiva , Camundongos , Camundongos Nus , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Zhonghua Gan Zang Bing Za Zhi ; 21(5): 363-6, 2013 May.
Artigo em Chinês | MEDLINE | ID: mdl-24025138

RESUMO

OBJECTIVE: To observe the dynamic change in expression of protease-activated receptor 2 (PAR2) during onset and progression of liver fibrosis by using a rat model. METHODS: A cholestatic liver fibrosis model was established in Sprague-Dawley rats (aged 8-9 weeks, body weight 350 - 400 g) by bile duct ligation surgery. Rats receiving a sham operation and unoperated rats served as the negative and normal control groups, respectively. At baseline (pre-surgery) and post-surgery weeks 2, 4, 6, and 8, five rats from each group were sacrificed for whole liver resection. The protein and mRNA expressions of PAR2 and collagen I/III were detected by western blotting and RT-PCR, respectively. Between-group differences were assessed by analysis of variance testing. RESULTS: At post-surgery week 2, the liver fibrosis group showed higher expression of PAR2 mRNA and protein than either control group. The expression levels of PAR2 continued to rise over time in the liver fibrosis group (peaking at week 8), and were significantly higher than those detected in the control groups (weeks 4-6: P less than 0.05; week 8, P less than 0.05). A similar trend was observed for the expression of collagen I/III. CONCLUSION: Dynamic expression of PAR2 observed in a cholestatic liver fibrosis rat model may indicate a role for this factor in the formation of liver fibrosis.


Assuntos
Cirrose Hepática Biliar/metabolismo , Fígado/metabolismo , Receptor PAR-2/metabolismo , Animais , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Modelos Animais de Doenças , Fígado/patologia , Cirrose Hepática Biliar/patologia , Masculino , Ratos , Ratos Sprague-Dawley
4.
Pigment Cell Melanoma Res ; 21(4): 451-6, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18710373

RESUMO

Melanoma is a common malignancy which is poorly responsive to chemotherapy and radiation. One of the major reasons melanoma responds poorly to these modalities is constitutive expression of Akt, which protects against apoptosis. The antidepressant sertraline was found to be a potent cytotoxic agent against A375 human melanoma. To determine the mechanism by which sertraline kills melanoma cells, Western blot analysis of signaling molecules, including phosphorylated Akt, caspase 9 and phospho-p70 S6 kinase was performed. Finally, the effects of sertraline on A375 xenografts in mice were assessed. Sertaline potently inhibited the phosphorylation of Akt, and caused cell death through induction of endoplasmic reticulum in vitro. Sertraline monotherapy demonstrated activity against A375 xenografts in vivo. Akt is a major cause of resistance of melanoma to current therapy. Antidepressants are commonly used to prevent interferon-induced depression. Use of antidepressants that decrease Akt may improve the efficacy of interferon and other therapies against melanoma. Further studies are needed to elucidate whether sertraline acts as an Akt inhibitor in melanoma.


Assuntos
Antineoplásicos/farmacologia , Melanoma/patologia , Proteína Oncogênica v-akt/metabolismo , Sertralina/farmacologia , Animais , Antidepressivos/farmacologia , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Humanos , Masculino , Melanoma/genética , Camundongos , Camundongos Nus , Fosforilação/efeitos dos fármacos , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
5.
J Clin Invest ; 115(6): 1492-502, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15902309

RESUMO

Overcoming resistance to chemotherapy and radiation therapy has been a difficult but important goal in the effort to cure cancer. We used gene-expression microarrays to identify differentially expressed genes involved in colorectal cancer resistance to chemotherapy and identified secreted protein, acidic and rich in cysteine (osteonectin) (SPARC) as a putative resistance-reversal gene by demonstrating low SPARC expression in refractory human MIP101 colon cancer cells. We were able to achieve restoration of their radiosensitivity and sensitivity to 5-fluorouracil and irinotecan by reexpression of SPARC in tumor xenografts. Moreover, treatment of mice with SPARC conferred increased sensitivity to chemotherapy and led to significant regression of xenografted tumors. The results show that modulation of SPARC expression affects colorectal cancer sensitivity to radiation and chemotherapy. SPARC-based gene or protein therapy may ameliorate the emergence of resistant clones and eradicate existing refractory clones and offers a novel approach to treating cancer.


Assuntos
Antineoplásicos/administração & dosagem , Camptotecina/análogos & derivados , Neoplasias do Colo/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Experimentais/tratamento farmacológico , Osteonectina/administração & dosagem , Animais , Camptotecina/administração & dosagem , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Fluoruracila/administração & dosagem , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Irinotecano , Camundongos , Camundongos Nus , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Osteonectina/metabolismo , Transplante Heterólogo
6.
Carcinogenesis ; 26(5): 908-15, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15731169

RESUMO

Several factors have been shown to promote the growth of colorectal cancers. Here, we provide evidence that periostin, a protein with structural and sequence homology with a TGF-beta-inducible gene, beta ig-h3, is upregulated in colorectal cancers and their liver metastasis, and it may play a role in promoting growth in these tumors. In vitro studies reveal that periostin promotes growth and cell proliferation in colorectal cancers and that this effect can be abrogated with antibodies to periostin. Furthermore, exposure of colorectal cancer cells to anti-periostin antibodies activates apoptosis and potentiates the effects of 5-fluorouracil chemotherapy. The results demonstrate the growth-promoting properties of periostin, and a possible role of targeting this protein as a therapeutic option in colorectal cancers.


Assuntos
Anticorpos/imunologia , Moléculas de Adesão Celular/metabolismo , Proliferação de Células , Neoplasias Colorretais/metabolismo , Apoptose/imunologia , Moléculas de Adesão Celular/imunologia , Colo/metabolismo , Colo/patologia , Colo/ultraestrutura , Neoplasias Colorretais/imunologia , Humanos , Intestino Delgado/metabolismo , Intestino Delgado/ultraestrutura , Fígado/patologia , Fígado/ultraestrutura , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/secundário , Regulação para Cima
7.
Proc Natl Acad Sci U S A ; 101(29): 10501-4, 2004 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-15249663

RESUMO

Although a reliable method for detection of cancer cells in blood would be an important tool for diagnosis and monitoring of solid tumors in early stages, current technologies cannot reliably detect the extremely low concentrations of these rare cells. The preferred method of detection, automated digital microscopy (ADM), is too slow to scan the large substrate areas. Here we report an approach that uses fiber-optic array scanning technology (FAST), which applies laser-printing techniques to the rare-cell detection problem. With FAST cytometry, laser-printing optics are used to excite 300,000 cells per sec, and emission is collected in an extremely wide field of view, enabling a 500-fold speed-up over ADM with comparable sensitivity and superior specificity. The combination of FAST enrichment and ADM imaging has the performance required for reliable detection of early-stage cancer in blood.


Assuntos
Tecnologia de Fibra Óptica , Neoplasias/sangue , Neoplasias/diagnóstico , Células Neoplásicas Circulantes/metabolismo , Biomarcadores Tumorais , Citofotometria/instrumentação , Citofotometria/métodos , Tecnologia de Fibra Óptica/instrumentação , Tecnologia de Fibra Óptica/métodos , Células HT29/metabolismo , Humanos , Programas de Rastreamento/métodos , Fibras Ópticas , Sensibilidade e Especificidade
8.
Clin Cancer Res ; 10(9): 3020-8, 2004 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-15131038

RESUMO

PURPOSE: The purpose of this study was to assess the feasibility of using rare event imaging system (REIS)-assisted analysis to detect occult tumor cells (OTCs) in peripheral blood (PB). The study also sought to determine whether REIS-assisted OTC detection presents a clinically viable alternative to manual microscopic detection to establish the true significance of OTC from solid epithelial tumors. EXPERIMENTAL DESIGN: We recently demonstrated proof of concept using a fluorescence-based automated microscope system, REIS, for OTC detection from the PB. For this study, the prototype of the system was adopted for high-throughput and high-content cellular analysis. RESULTS: The performance of the improved REIS was examined using normal blood (n = 10), normal blood added to cancer cells (n = 20), and blood samples obtained from cancer patients (n = 80). Data from the screening of 80 clinical slides from breast and lung cancer patients, by manual microscopy and by the REIS, revealed that as many as 14 of 35 positive slides (40%) were missed by manual screening but positively identified by REIS. In addition, REIS-assisted scanning reliably and reproducibly quantified the total number of cells analyzed in the assay and categorized positive cells based on their marker expression profile. CONCLUSIONS: REIS-assisted analysis provides excellent sensitivity and reproducibility for OTC detection. This approach may enable an improved method for screening of PB samples and for obtaining novel information about disease staging and about risk evaluation in cancer patients.


Assuntos
Neoplasias da Mama/diagnóstico , Carcinoma de Células Pequenas/diagnóstico , Neoplasias Pulmonares/diagnóstico , Microscopia de Fluorescência/métodos , Neoplasias da Mama/sangue , Carcinoma de Células Pequenas/sangue , Contagem de Células , Linhagem Celular Tumoral , Humanos , Processamento de Imagem Assistida por Computador/métodos , Imuno-Histoquímica , Queratinas/análise , Neoplasias Pulmonares/sangue , Células Neoplásicas Circulantes/química , Células Neoplásicas Circulantes/patologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
9.
Anticancer Res ; 23(1A): 49-62, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-12683352

RESUMO

Lung cancer accounts for approximately 30% of all cancer mortalities in the United States. Small cell lung cancer (SCLC), which is an aggressive malignancy with frequent and early metastases, accounts for about 15% of all of the lung cancer cases with a dismal 5-year survival rate of < 5% with current standard therapies. Early detection of SCLC is challenging, in part due to the lack of adequate serum tumor markers. The goal of this review is to summarize the current knowledge of circulating tumor cells and serum biomarkers in small cell lung cancer. The role of circulating tumor cells in prognostication is controversial, but may be better defined with advancing technologies of detection of such cells with higher precision, and improved clinico-pathological correlations. The current knowledge on the known serum cytokines and tumor biomarkers of SCLC, such as CEA, chromogranin-A and neuron-specific enolase will be presented. Serum cytokines, such as vascular endothelial growth factor (VEGF), stem cell factor (SCF) and hepatocyte growth factor/scatter factor (HGF/SF) are also discussed. New findings in the search for novel diagnostic and therapeutic molecular markers using the emerging genomics and proteomics technologies are emphasized. It is our hope that validation of these new research platforms and technologies will result in improved early detection, prognostication and finally treatment of SCLC with potential novel molecularly-targeted therapeutics.


Assuntos
Biomarcadores Tumorais/sangue , Carcinoma de Células Pequenas/sangue , Neoplasias Pulmonares/sangue , Células Neoplásicas Circulantes/patologia , Carcinoma de Células Pequenas/patologia , Humanos , Neoplasias Pulmonares/patologia
10.
Blood ; 102(1): 289-96, 2003 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-12637335

RESUMO

The effect of 2-methoxyestradiol, 2ME2, an endogenous metabolite of 17beta-estradiol (E2), on cell growth and cytoskeletal functions in a BCR-ABL-transformed cell line model was investigated. We determined the interaction of 2ME2 with STI571 (Gleevec, imatinib mesylate) in STI571 drug-sensitive and -resistant cell lines. In cells expressing BCR-ABL, STI571 cooperated with 2ME2 in reducing cell growth, and STI571-resistant cells were sensitive to 2ME2 treatment. 2ME2 also inhibited growth of several cancer cell lines by a mechanism independent of BCR-ABL. BCR-ABL transformation leads to altered motility, increased adhesion, and spontaneous migration in different in vitro model systems. 2ME2 was found to specifically inhibit the spontaneous motility of BCRABL-transformed Ba/F3 cells and to change the morphology and volume of treated cells. Cells attached to fibronectin-coated surfaces showed a reduced number of filipodia and lamellipodia. In addition, 2ME2 significantly reduced BCRABL-mediated adhesion to fibronectin. The spontaneous migration of BCR-ABL-transformed cells through a transwell membrane also was found to be significantly decreased by 2ME2. Cytoskeletal changes were accompanied by alteration of tubulin formation, distinct from paclitaxel treatment. These results demonstrate that 2ME2 treatment of transformed cells strongly reduces cytoskeletal functions and may also be useful for the treatment of cancers with high metastatic potential. Combination of 2ME2 with other anticancer drugs may be beneficial to treatment of drug-resistant cancers.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Fenômenos Fisiológicos Celulares/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Estradiol/farmacologia , 2-Metoxiestradiol , Animais , Benzamidas , Adesão Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Transformada , Movimento Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Citoesqueleto/fisiologia , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Estradiol/análogos & derivados , Humanos , Mesilato de Imatinib , Camundongos , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas , Pirimidinas/farmacologia , Superóxidos/análise , Tubulina (Proteína)/efeitos dos fármacos , Tubulina (Proteína)/metabolismo , Células Tumorais Cultivadas
11.
Breast Cancer Res Treat ; 77(3): 245-52, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12602924

RESUMO

Periostin is a recently identified gene that is preferentially expressed in periosteum, indicating a potential role in bone formation and maintenance of structure. We independently identified and isolated periostin from cancer tissue, using the palindromic PCR-driven cDNA Differential Display technique. For the present work, we developed a novel sandwich chemiluminescence assay to detect serum periostin level using newly developed monoclonal and polyclonal antibodies. We investigated serum periostin levels in breast cancer and small cell lung cancer patients, especially in patients with bone metastasis. The study included 58 breast cancer and 44 small cell lung cancer patients. Serum periostin levels were elevated in breast cancer patients presenting with bone metastases (92.0 +/- 28.6 ng/ml) compared to similar breast cancer patients without evidence of bone metastasis (55.0 +/- 16.6 ng/ml, p = 0.04). No correlation was found between the serum periostin level and any other prognostic factors, such as clinical stage and lymph node metastasis in breast cancer. Serum periostin levels thus appear to serve as a marker of bone metastasis from breast cancer. In contrast, serum periostin levels were similar in samples from patients with small cell lung cancer who did or did not have bone metastasis. However, increasing T-stage and N-stage of patients with small cell lung cancer were correlated with higher periostin levels (T4, 126.5 +/- 29.7 ng/ml v.s. T2, 64.9 +/- 16.1 ng/ml, p = 0.03; and T4 v.s. T1, 36.3+/- 7.5 ng/ml, p = 0.01; N3, 108.7 +/- 17.3 ng/ml v.s. N2, 49.7+/- 10.9 ng/ml, p = 0.01). Periostin has a substantial homology with the insect cell adhesion molecule, fasciclin I. Thus, expression of periostin may facilitate tumor cell adhesion to the bone surface. In fact, we found by in situ RNA hybridization, that the periostin gene was highly expressed in the stromal cells immediately surrounding the tumor, but not within the breast cancer cells themselves.


Assuntos
Biomarcadores Tumorais/sangue , Neoplasias Ósseas/secundário , Neoplasias da Mama/patologia , Carcinoma de Células Pequenas/secundário , Moléculas de Adesão Celular/sangue , Neoplasias Pulmonares/patologia , Adulto , Neoplasias Ósseas/sangue , Neoplasias da Mama/sangue , Carcinoma de Células Pequenas/sangue , Feminino , Humanos , Hibridização In Situ , Medições Luminescentes , Neoplasias Pulmonares/sangue , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Estadiamento de Neoplasias , Reação em Cadeia da Polimerase , Valor Preditivo dos Testes
13.
Blood ; 101(9): 3606-14, 2003 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-12480690

RESUMO

Our previous study demonstrated that 2-methoxyestradiol (2ME2), an estrogen derivative, induces apoptosis in multiple myeloma (MM) cells; however, the related transcriptional events are unclear. In the present study, we used oligonucleotide microarrays to identify genes altered during 2ME2-induced apoptosis in MM cells. 2ME2 triggers an early transient induction of genes known to trigger cell death and repression of growth/survival-related genes. Many genes regulating cell defense/repair machinery also were transiently induced. Since 2ME2 also induces apoptosis in MM cells resistant to conventional therapies such as dexamethasone (Dex), we compared the gene profiles of 2ME2-treated and Dex-resistant MM cells. Our results suggest that 2ME2 overcomes Dex resistance by modulating genes that confer chemoresistance in MM cells. Microarray results were confirmed by Northern and Western blot analyses. A comparative analysis of selected genes from freshly isolated MM patient cells and 2ME2-treated MM.1S MM cells further provides an in vivo relevance of our in vitro studies. Collectively, these findings suggest genetic events mediating anti-MM activity of 2ME2, as well as mechanisms whereby 2ME2 overcomes Dex resistance in MM cells. These studies may therefore allow improved therapeutic use of 2ME2, based upon targeting genes that regulate MM cell growth and survival.


Assuntos
Estradiol/farmacologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Mieloma Múltiplo/patologia , Proteínas de Neoplasias/biossíntese , 2-Metoxiestradiol , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Cisteína Endopeptidases/metabolismo , Proteínas do Citoesqueleto/biossíntese , Proteínas do Citoesqueleto/genética , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Dexametasona/farmacologia , Resistencia a Medicamentos Antineoplásicos , Retículo Endoplasmático/metabolismo , Estradiol/análogos & derivados , Genes Reporter , Proteínas de Choque Térmico/biossíntese , Proteínas de Choque Térmico/genética , Interleucina-6/farmacologia , Complexos Multienzimáticos/metabolismo , Proteínas de Neoplasias/genética , Análise de Sequência com Séries de Oligonucleotídeos , Complexo de Endopeptidases do Proteassoma , Dobramento de Proteína , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Fatores de Transcrição de Fator Regulador X , Técnica de Subtração , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Transfecção , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Ubiquitina/metabolismo
14.
Exp Hematol ; 30(3): 212-20, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11882358

RESUMO

OBJECTIVE: Since the central hallmarks of human multiple myeloma (MM) are abnormalities in immunoglobulin (Ig) gene rearrangement, IgH class switching, and DNA damage repair, and since Ku86 and Ku70 proteins are central to these processes, aberrant Ku function may play a role in MM pathogenesis. Our prior studies demonstrated a 69-kDa Ku86 variant in freshly isolated patient MM cells that confers sensitivity to DNA damage. We also showed that Ku86 on the cell surface of CD40-activated MM cells mediates homotypic tumor cell adhesion, as well as heterotypic adhesion to bone marrow stromal cells. We here define the mechanism and functional significance of CD40-induced Ku translocation from the cytoplasm to the cell membrane in MM cells vs normal B cells. MATERIALS AND METHODS: We examined Ku86 and Ku70 translocation following CD40 activation in human MM cells vs normal tonsillar B lymphocytes. We then identified the functional sequelae of membrane Ku86 and Ku70 expression on CD40-activated human MM cells. RESULTS: CD40 activation induces translocation of both Ku86 and Ku70 to the cell surface of MM cells, but not normal tonsillar B cells. Moreover, CD40 activation triggers Ku association with CD40 only in CD40-activated MM cells. Finally, CD40-activated MM cells adhere to fibronectin and are protected against apoptosis triggered by irradiation or doxorubicin; conversely, antibodies to Ku both inhibit tumor cell binding and restore sensitivity to these agents. CONCLUSION: These results demonstrate functional significance of Ku translocation to the cell membrane of CD40-activated human MM cells. Therefore, targeting Ku86 and Ku70, with blocking peptides for example, might serve as a novel treatment strategy in human MM.


Assuntos
Antígenos Nucleares , Membrana Celular/imunologia , DNA Helicases , Proteínas de Ligação a DNA/fisiologia , Mieloma Múltiplo/imunologia , Proteínas Nucleares/fisiologia , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Linfócitos B/imunologia , Transporte Biológico , Antígenos CD40/imunologia , Adesão Celular , Fracionamento Celular , Citoplasma/metabolismo , Doxorrubicina/farmacologia , Fibronectinas/metabolismo , Citometria de Fluxo , Imunofluorescência , Humanos , Autoantígeno Ku , Mieloma Múltiplo/ultraestrutura , Tonsila Palatina/citologia
15.
Mol Biol Cell ; 13(3): 767-81, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11907260

RESUMO

Here we demonstrate that multiple tetraspanin (transmembrane 4 superfamily) proteins are palmitoylated, in either the Golgi or a post-Golgi compartment. Using CD151 as a model tetraspanin, we identified and mutated intracellular N-terminal and C-terminal cysteine palmitoylation sites. Simultaneous mutations of C11, C15, C242, and C243 (each to serine) eliminated >90% of CD151 palmitoylation. Notably, palmitoylation had minimal influence on the density of tetraspanin protein complexes, did not promote tetraspanin localization into detergent-resistant microdomains, and was not required for CD151-alpha 3 beta 1 integrin association. However, the CD151 tetra mutant showed markedly diminished associations with other cell surface proteins, including other transmembrane 4 superfamily proteins (CD9, CD63). Thus, palmitoylation may be critical for assembly of the large network of cell surface tetraspanin-protein interactions, sometimes called the "tetraspanin web." Also, compared with wild-type CD151, the tetra mutant was much more diffusely distributed and showed markedly diminished stability during biosynthesis. Finally, expression of the tetra-CD151 mutant profoundly altered alpha 3 integrin-deficient kidney epithelial cells, such that they converted from a dispersed, elongated morphology to an epithelium-like cobblestone clustering. These results point to novel biochemical and biological functions for tetraspanin palmitoylation.


Assuntos
Antígenos CD/metabolismo , Tamanho Celular , Integrina alfa3beta1/metabolismo , Proteínas de Membrana/metabolismo , Palmitatos/metabolismo , Sequência de Aminoácidos , Antígenos CD/genética , Transporte Biológico/fisiologia , Brefeldina A/farmacologia , Linhagem Celular , Proteínas de Fluorescência Verde , Humanos , Indicadores e Reagentes/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Tetraspanina 24
16.
Oncogene ; 21(9): 1346-58, 2002 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-11857078

RESUMO

Our previous studies have characterized Dexamethasone (Dex)-induced apoptotic signaling pathways in multiple myeloma (MM) cells; however, related transcriptional events are not fully defined. In the present study, gene expression profiles of Dex-treated MM cells were determined using oligonucleotide arrays. Dex triggers early transient induction of many genes involved in cell defense/repair-machinery. This is followed by induction of genes known to mediate cell death and repression of growth/survival-related genes. The molecular and genetic alterations associated with Dex resistance in MM cells are also unknown. We compared the gene expression profiles of Dex-sensitive and Dex-resistant MM cells and identified a number of genes which may confer Dex-resistance. Finally, gene profiling of freshly isolated MM patient cells validates our in vitro MM cell line data, confirming an in vivo relevance of these studies. Collectively, these findings provide insights into the basic mechanisms of Dex activity against MM, as well as mechanisms of Dex-resistance in MM cells. These studies may therefore allow improved therapeutic uses of Dex, based upon targeting genes that regulate MM cell growth and survival.


Assuntos
Dexametasona/farmacologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Mieloma Múltiplo/genética , Análise de Sequência com Séries de Oligonucleotídeos , Apoptose/efeitos dos fármacos , Apoptose/genética , Western Blotting , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Cisteína Endopeptidases/metabolismo , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Citometria de Fluxo , Proteínas de Choque Térmico/genética , Humanos , Complexos Multienzimáticos/metabolismo , NF-kappa B/metabolismo , Complexo de Endopeptidases do Proteassoma , RNA Mensageiro/metabolismo , Receptores de Glucocorticoides/genética , Receptores de Interleucina-6/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Transdução de Sinais , Células Tumorais Cultivadas , Ubiquitina/metabolismo , Regulação para Cima/efeitos dos fármacos
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