RESUMO
Tumor cells initiate platelet activation leading to the secretion of bioactive molecules, which promote metastasis. Platelet receptors on tumors have not been well-characterized, resulting in a critical gap in knowledge concerning platelet-promoted metastasis. We identify a direct interaction between platelets and tumor CD97 that stimulates rapid bidirectional signaling. CD97, an adhesion G protein-coupled receptor (GPCR), is an overexpressed tumor antigen in several cancer types. Purified CD97 extracellular domain or tumor cell-associated CD97 stimulated platelet activation. CD97-initiated platelet activation led to granule secretion, including the release of ATP, a mediator of endothelial junction disruption. Lysophosphatidic acid (LPA) derived from platelets induced tumor invasiveness via proximal CD97-LPAR heterodimer signaling, coupling coincident tumor cell migration and vascular permeability to promote transendothelial migration. Consistent with this, CD97 was necessary for tumor cell-induced vascular permeability in vivo and metastasis formation in preclinical models. These findings support targeted blockade of tumor CD97 as an approach to ameliorate metastatic spread.
Assuntos
Antígenos CD/metabolismo , Plaquetas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Antígenos CD/genética , Plaquetas/citologia , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Dimerização , Fator de Crescimento Epidérmico/farmacologia , Transição Epitelial-Mesenquimal , Humanos , Lisofosfolipídeos/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/genética , Receptores de Ácidos Lisofosfatídicos/antagonistas & inibidores , Receptores de Ácidos Lisofosfatídicos/metabolismo , Junções Íntimas/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Primary cilia arise from the centrosomes of quiescent or post-mitotic cells, and serve as sensory organelles that communicate mechanical and chemical stimuli from the environment to the interior of the cell. Cilium formation may, therefore, become a useful end point signaling exposure to genotoxins or aneugens. Here we have used the aneugen, zidovudine (AZT), an antiretroviral drug that induces DNA replication arrest and centrosomal amplification (>2 centrosomes per quiescent cell), to evaluate cilia formation in retinal epithelial (pigmented) cells. Since cilia are derived from centrosomes, and aneugens can induce centrosomal amplification, the production of multiple cilia arising from multiple centrosomes may reveal the aneugenic nature of the agents. Cells were exposed to AZT to induce centrosomal amplification, cultured without serum to allow the centrioles to develop cilia, and immunostained to visualize cilia and centrosomes. Nuclear DNA was stained with DAPI. Preliminary observations suggest that cells with multiple centrosomes are able to generate extra cilia.
Assuntos
Aneugênicos/toxicidade , Aneuploidia , Cílios/efeitos dos fármacos , Dano ao DNA , Zidovudina/toxicidade , Linhagem Celular , Centrossomo/efeitos dos fármacos , Centrossomo/ultraestrutura , Cílios/genética , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/ultraestruturaRESUMO
The nucleoside reverse transcriptase inhibitor zidovudine (AZT) induces genotoxic damage that includes centrosomal amplification (CA > 2 centrosomes/cell) and micronucleus (MN) formation. Here we explored these end points in mice deficient in DNA repair and tumor suppressor function to evaluate their effect on AZT-induced DNA damage. We used mesenchymal-derived fibroblasts cultured from C57BL/6J mice that were null and wild type (WT) for Xpa, and WT, haploinsufficient and null for p53 (6 different genotypes). Dose-responses for CA formation, in cells exposed to 0, 10, and 100 µM AZT for 24 hr, were observed in all genotypes except the Xpa((+/+)) p53((+/-)) cells, which had very low levels of CA, and the Xpa((-/-)) p53((-/-)) cells, which had very high levels of CA. For CA there was a significant three-way interaction between Xpa, p53, and AZT concentration, and Xpa((-/-)) cells had significantly higher levels of CA than Xpa((+/+)) cells, only for p53((+/-)) cells. In contrast, the MN and MN + chromosomes (MN + C) data showed a lack of AZT dose response. The Xpa((-/-)) cells, with p53((+/+)) or ((+/-)) genotypes, had levels of MN and MN + C higher than the corresponding Xpa((+/+)) cells. The data show that CA is a major event induced by exposure to AZT in these cells, and that there is a complicated relationship between AZT and CA formation with respect to gene dosage of Xpa and p53. The loss of both genes resulted in high levels of damage, and p53 haploinsufficicency strongly protected Xpa((+/+)) cells from AZT-induced CA damage.
Assuntos
Centrossomo/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética , Zidovudina/toxicidade , Animais , Células da Medula Óssea/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Reparo do DNA/genética , Relação Dose-Resposta a Droga , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Testes para Micronúcleos , Proteína de Xeroderma Pigmentoso Grupo A/genéticaRESUMO
The mixed lineage kinase domain-like protein (MLKL) has recently been identified as a key RIP3 (receptor interacting protein 3) downstream component of tumour necrosis factor (TNF)-induced necroptosis. MLKL is phosphorylated by RIP3 and is recruited to the necrosome through its interaction with RIP3. However, it is still unknown how MLKL mediates TNF-induced necroptosis. Here, we report that MLKL forms a homotrimer through its amino-terminal coiled-coil domain and locates to the cell plasma membrane during TNF-induced necroptosis. By generating different MLKL mutants, we demonstrated that the plasma membrane localization of trimerized MLKL is critical for mediating necroptosis. Importantly, we found that the membrane localization of MLKL is essential for Ca(2+) influx, which is an early event of TNF-induced necroptosis. Furthermore, we identified that TRPM7 (transient receptor potential melastatin related 7) is a MLKL downstream target for the mediation of Ca(2+) influx and TNF-induced necroptosis. Hence, our study reveals a crucial mechanism of MLKL-mediated TNF-induced necroptosis.
Assuntos
Apoptose/efeitos dos fármacos , Membrana Celular/metabolismo , Necrose/patologia , Proteínas Quinases/metabolismo , Multimerização Proteica , Fator de Necrose Tumoral alfa/farmacologia , Animais , Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Células HEK293 , Células HT29 , Humanos , Imidazóis/farmacologia , Indóis/farmacologia , Células Jurkat , Camundongos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Proteínas Quinases/química , Multimerização Proteica/efeitos dos fármacos , Proteínas Serina-Treonina Quinases , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Canais de Cátion TRPM/metabolismoRESUMO
Genomic rearrangements commonly occur in many types of cancers and often initiate or alter the progression of disease. Here we describe an in vivo mouse model that recapitulates the most frequent rearrangement in prostate cancer, the fusion of the promoter region of TMPRSS2 with the coding region of the transcription factor, ERG. A recombinant bacterial artificial chromosome including an extended TMPRSS2 promoter driving genomic ERG was constructed and used for transgenesis in mice. TMPRSS2-ERG expression was evaluated in tissue sections and FACS-fractionated prostate cell populations. In addition to the anticipated expression in luminal cells, TMPRSS2-ERG was similarly expressed in the Sca-1(hi)/EpCAM(+) basal/progenitor fraction, where expanded numbers of clonogenic self-renewing progenitors were found, as assayed by in vitro sphere formation. These clonogenic cells increased intrinsic self renewal in subsequent generations. In addition, ERG dependent self-renewal and invasion in vitro was demonstrated in prostate cell lines derived from the model. Clinical studies have suggested that the TMPRSS2-ERG translocation occurs early in prostate cancer development. In the model described here, the presence of the TMPRSS2-ERG fusion alone was not transforming but synergized with heterozygous Pten deletion to promote PIN. Taken together, these data suggest that one function of TMPRSS2-ERG is the expansion of self-renewing cells, which may serve as targets for subsequent mutations. Primary prostate epithelial cells demonstrated increased post transcriptional turnover of ERG compared to the TMPRSS2-ERG positive VCaP cell line, originally isolated from a prostate cancer metastasis. Finally, we determined that TMPRSS2-ERG expression occurred in both castration-sensitive and resistant prostate epithelial subpopulations, suggesting the existence of androgen-independent mechanisms of TMPRSS2 expression in prostate epithelium.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasia Prostática Intraepitelial/metabolismo , Neoplasias da Próstata/metabolismo , Serina Endopeptidases/fisiologia , Transativadores/genética , Androgênios/fisiologia , Animais , Proliferação de Células , Cromossomos Artificiais Bacterianos/genética , Epitélio/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Orquiectomia , Regiões Promotoras Genéticas , Próstata/metabolismo , Próstata/patologia , Neoplasia Prostática Intraepitelial/patologia , Neoplasias da Próstata/patologia , Células-Tronco/metabolismo , Células-Tronco/fisiologia , Transativadores/metabolismo , Regulador Transcricional ERG , Células Tumorais CultivadasRESUMO
Histone deacetylase inhibitors (HDI) have exhibited some efficacy in clinical trials, but it is clear that their most effective applications have yet to be fully determined. In this study, we show that HDIs influence the expression of a common polymorphic variant of the chemotherapy drug efflux transporter ABCG2, which contributes to normal tissue protection. As one of the most frequent variants in human ABCG2, the polymorphism Q141K impairs expression, localization, and function, thereby reducing drug clearance and increasing chemotherapy toxicity. Mechanistic investigations revealed that the ABCG2 Q141K variant was fully processed but retained in the aggresome, a perinuclear structure, where misfolded proteins aggregate. In screening for compounds that could correct its expression, localization, and function, we found that the microtubule-disrupting agent colchicine could induce relocalization of the variant from the aggresome to the cell surface. More strikingly, we found that HDIs could produce a similar effect but also restore protein expression to wild-type levels, yielding a restoration of ABCG2-mediated specific drug efflux activity. Notably, HDIs did not modify aggresome structures but instead rescued newly synthesized protein and prevented aggresome targeting, suggesting that HDIs disturbed trafficking along microtubules by eliciting changes in motor protein expression. Together, these results showed how HDIs are able to restore wild-type functions of the common Q141K polymorphic isoform of ABCG2. More broadly, our findings expand the potential uses of HDIs in the clinic.
Assuntos
Transportadores de Cassetes de Ligação de ATP/efeitos dos fármacos , Transportadores de Cassetes de Ligação de ATP/genética , Inibidores de Histona Desacetilases/farmacologia , Proteínas de Neoplasias/efeitos dos fármacos , Proteínas de Neoplasias/genética , Polimorfismo Genético , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Expressão Gênica/efeitos dos fármacos , Humanos , Mitoxantrona/farmacologia , Proteínas de Neoplasias/metabolismo , RNA Interferente Pequeno/farmacologiaRESUMO
CD97, an adhesion-linked G-protein-coupled receptor (GPCR), is induced in multiple epithelial cancer lineages. We address here the signaling properties and the functional significance of CD97 expression in prostate cancer. Our findings show that CD97 signals through Gα12/13 to increase RHO-GTP levels. CD97 functioned to mediate invasion in prostate cancer cells, at least in part, by associating with lysophosphatidic acid receptor 1 (LPAR1), leading to enhanced LPA-dependent RHO and extracellular signal-regulated kinase activation. Consistent with its role in invasion, depletion of CD97 in PC3 cells resulted in decreased bone metastasis without affecting subcutaneous tumor growth. Furthermore, CD97 heterodimerized and functionally synergized with LPAR1, a GPCR implicated in cancer progression. We also found that CD97 and LPAR expression were significantly correlated in clinical prostate cancer specimens. Taken together, these findings support the investigation of CD97 as a potential therapeutic cancer target.
Assuntos
Antígenos CD/metabolismo , Lisofosfolipídeos/farmacologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Receptores de Ácidos Lisofosfatídicos/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Antígenos CD/genética , Linhagem Celular Tumoral , Progressão da Doença , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Masculino , Invasividade Neoplásica/genética , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Neoplasias da Próstata/genética , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Ácidos Lisofosfatídicos/genética , Transdução de Sinais/genéticaRESUMO
We investigated the PKCdelta-mediated phosphorylation of paxillin within its LIM4 domain and the involvement of this phosphorylation in activation of LFA-1 integrins of the Baf3 pro-B lymphocytic cell line. Using phosphorylated-threonine-specific antibodies, phosphorylated amino acid analysis and paxillin phosphorylation mutants, we demonstrated that TPA, the pharmacological analog of the endogenous second messenger diacyl glycerol, stimulates paxillin phosphorylation at threonine 538 (T538). The TPA-responsive PKC isoform PKCdelta directly binds paxillin in a yeast two-hybrid assay and phosphorylates paxillin at T538 in vitro and also co-immunoprecipitates with paxillin and mediates phosphorylation of this residue in vivo. Recombinant wild-type paxillin, its phospho-inhibitory T538A or phospho-mimetic T538E mutants were expressed in the cells simultaneously with siRNA silencing of the endogenous paxillin. These experiments suggest that phosphorylation of paxillin T538 contributes to dissolution of the actin cytoskeleton, redistribution of LFA-1 integrins and an increase in their affinity. We also show that phosphorylation of T538 is involved in the activation of LFA-1 integrins by TPA.
Assuntos
Antígeno-1 Associado à Função Linfocitária/metabolismo , Linfócitos/citologia , Linfócitos/enzimologia , Paxilina/metabolismo , Fosfotreonina/metabolismo , Proteína Quinase C-delta/metabolismo , Actinas/metabolismo , Animais , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Forma Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Humanos , Imunoprecipitação , Interleucina-3/farmacologia , Linfócitos/efeitos dos fármacos , Camundongos , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Técnicas do Sistema de Duplo-HíbridoRESUMO
ABCG2 is an ATP-binding cassette half-transporter important in normal tissue protection, drug distribution, and excretion. ABCG2 requires homodimerization for function, though the mechanism for dimerization has not been elucidated. We conducted mutational analysis of threonine 402, three residues from the GXXXG motif in TM1, to study its potential role in ABCG2 dimerization (TXXXGXXXG). Single mutations to leucine (T402L) or arginine (T402R) did not have a significant impact on the ABCG2 protein. On the other hand, combining the T402 mutations with the GXXXG glycine to leucine mutations (T402L/G406L/G410L and T402R/G406L/G410L) resulted in a substantially reduced level of expression, altered glycosylation, degradation by a proteosome-independent pathway, and partial retention in the endoplasmic reticulum as suggested by immunostaining, Endo H sensitivity, and MG132 and bafilomycin failed effect. The T402L/G406L/G410L mutant when incubated with the ABCG2 substrate MX showed a shift on immunoblot analysis to the band representing the fully mature glycoprotein. The T402R/G406L/G410L mutant carrying the more drastic substitution was found to primarily localize intracellularly. The same set of mutations also displayed impaired dimerization in the TOXCAT assay for TM1 compared to that of the wild type. Homology modeling of ABCG2 places the TXXXGXXXG motif at the dimer interface. These studies are consistent with a role for the extended TXXXGXXXG motif in ABCG2 folding, processing, and/or dimerization.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Membrana Celular/metabolismo , Mutação , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Multimerização Proteica , Estrutura Quaternária de Proteína , Treonina , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Linhagem Celular , Reagentes de Ligações Cruzadas/farmacologia , Análise Mutacional de DNA , Regulação da Expressão Gênica , Glicosídeo Hidrolases/metabolismo , Humanos , Leupeptinas/farmacologia , Macrolídeos/farmacologia , Mitoxantrona/farmacologia , Dados de Sequência Molecular , Proteínas de Neoplasias/metabolismo , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Transporte Proteico/efeitos dos fármacosRESUMO
BACKGROUND: ABT-751, an orally bioavailable sulfonamide, binds beta-tubulin to inhibit microtubule polymerization. We described response and event-free survival (EFS) in children with neuroblastoma and other solid tumors receiving ABT-751, assessed in vitro cytotoxicity of ABT-751 and evaluated the effect of ABT-751 on tubulin polymerization in peripheral blood mononuclear cells (PBMC) and pediatric tumor cell lines. PROCEDURE: Patients with neuroblastoma (n = 50) or other solid tumors (n = 26) enrolled on the ABT-751 pediatric phase I and pilot trials were reviewed. The sulforhodamine B (SRB) and ACEA Real-Time Cell Electronic Sensing (RT-CES) assays were used to determine the in vitro cytotoxicity. Pharmacodynamic effects on tubulin polymerization/depolymerization were assessed by Western blot and confocal microscopy using antibodies specific for post-translational modifications of polymerized tubulin. RESULTS: Forty-five patients with neuroblastoma were evaluated for anti-tumor response. No complete or partial responses were documented. The median EFS was 9.3 weeks for children with neuroblastoma and 3.3 weeks for children other solid tumors (P < 0.0001). The ABT-751 IC(50) was 0.6-2.6 mcM in neuroblastoma and 0.7-4.6 mcM in other solid tumor cell lines. Following drug exposure, polymerized tubulin decreased in a concentration- and time-dependent manner in cell lines. CONCLUSIONS: In children treated with ABT-751, the EFS is longer in children with neuroblastoma as compared to other diagnoses. In vitro, ABT-751 was cytotoxic at concentrations tolerable in children. Effects of ABT-751 on polymerization and microtubule structure were time- and dose-dependent but not dependent on tumor type.
Assuntos
Proliferação de Células/efeitos dos fármacos , Recidiva Local de Neoplasia/tratamento farmacológico , Neuroblastoma/tratamento farmacológico , Sulfonamidas/uso terapêutico , Moduladores de Tubulina/uso terapêutico , Tubulina (Proteína)/metabolismo , Adolescente , Linhagem Celular Tumoral , Criança , Pré-Escolar , Intervalo Livre de Doença , Feminino , Humanos , Técnicas In Vitro , Masculino , Neuroblastoma/patologia , Projetos Piloto , Prognóstico , Sulfonamidas/farmacologia , Taxa de Sobrevida , Resultado do Tratamento , Moduladores de Tubulina/farmacologiaRESUMO
In cultured cells, exposure to the nucleoside reverse transcriptase inhibitor (NRTI) zidovudine (AZT) induces genomic instability, cell cycle arrest, micronuclei, sister chromatid exchanges, and shortened telomeres. In previous studies, we demonstrated AZT-induced centrosome amplification (>2 centrosomes/cell). Here, we investigate centrosome amplification in cells exposed to other commonly used NRTIs. Experiments were performed using Chinese Hamster ovary (CHO) cells, and two normal human mammary epithelial cell (NHMEC) strains: M99005 and M98040, which are high and low incorporators of AZT into DNA, respectively. Cells were exposed for 24 hr to lamivudine (3TC), stavudine (d4T), didanosine (ddI), and thymidine, and stained with anti-pericentrin antibody. Dose response curves were performed to determine cytotoxicity and a lower concentration at near plasma levels and a 10 fold higher concentration were chosen for the experiments. In CHO cells, there was a concentration-dependent, significant (P < 0.05) increase in centrosome amplification for each of the NRTIs. In NHMEC strain M99005, an NRTI-induced increase (P < 0.05) in centrosome amplification was observed for the high concentrations of each NRTI and the low doses of 3TC and ddI. In NHMEC strain M98040, the high doses of ddI and d4T showed significant increases in centrosome amplification. Functional viability of amplified centrosomes was assessed by arresting microtubule nucleation with nocodazole. In cells with more than two centrosomes, the ability to recover microtubule nucleation was similar to that of unexposed cells. We conclude that centrosome amplification is a consequence of exposure to NRTIs and that cells with centrosome amplification are able to accomplish cell division.
Assuntos
Centrossomo , Amplificação de Genes , Inibidores da Transcriptase Reversa/farmacologia , Animais , Células CHO , Cricetinae , Cricetulus , Didanosina/farmacologia , Relação Dose-Resposta a Droga , Humanos , Lamivudina/farmacologia , Microscopia de Fluorescência , Estavudina/farmacologiaRESUMO
The centrosome directs chromosomal migration by a complex process of tubulin-chromatin binding. In this contribution centrosomal abnormalities, including centrosomal amplification, were explored in Chinese hamster ovary (CHO) and normal human mammary epithelial cells (NHMECs) exposed to the antiretroviral drug zidovudine (3'-azido-3'-deoxythymidine, AZT). Centrosomal amplification/fragmentation was observed in both cell types and kinetochore positive micronuclei were found in AZT-exposed CHO cells in correlation with dose. Normal human mammary epithelial cell (NMHEC) strain M99005, previously identified as a strain that incorporates high levels of AZT into DNA (high incorporator, HI), showed greater centrosomal amplification when compared with a second strain, NHMEC M98040, which did not incorporate AZT into DNA (low incorporator, LI). Additionally, an abnormal tubulin distribution was observed in AZT-exposed HI cells bearing multiple centrosomes. Immunofluorescent staining of human cells with Aurora A, a kinase involved in the maturation of the centrosome, confirmed the induction of centrosomal amplification and revealed multipolar mitotic figures. Flow cytometric studies revealed that cells bearing abnormal numbers of centrosomes and abnormal tubulin distribution had similar S-phase percentages suggesting that cells bearing unbalanced chromosomal segregation could divide. Therefore, AZT induces genomic instability and clastogenicity as well as alterations in proteins involved in centrosomal activation, all of which may contribute to the carcinogenic properties of this compound.
Assuntos
Aneugênicos/toxicidade , Aneuploidia , Centrossomo/efeitos dos fármacos , Zidovudina/toxicidade , Aneugênicos/farmacocinética , Animais , Aurora Quinases , Mama/citologia , Mama/efeitos dos fármacos , Mama/metabolismo , Células CHO , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Centrossomo/metabolismo , Centrossomo/ultraestrutura , Cricetinae , Cricetulus , Adutos de DNA/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Humanos , Micronúcleos com Defeito Cromossômico/induzido quimicamente , Microscopia Eletrônica de Transmissão , Proteínas Serina-Treonina Quinases/metabolismo , Tubulina (Proteína)/metabolismo , Zidovudina/farmacocinéticaRESUMO
ABCG2 is an ATP-binding cassette half-transporter initially identified in multidrug-resistant cancer cell lines and recently suggested to play an important role in pharmacokinetics. Here we report studies of a conserved arginine predicted to localize near the cytoplasmic side of TM1. First, we determined the effect of losing charge and bulk at this position via substitutions with glycine and alanine. The R383G mutant when transfected into HEK cells was not detectable on immunoblot or by functional assay, while the R383A mutant exhibited detectable but significantly decreased levels compared to wild-type, partial retention in the ER and altered glycosylation. Efflux of the ABCG2-substrates mitoxantrone and pheophorbide a was observed. Our experiments suggested rapid degradation of the R383A mutant by the proteasome via a kifunensine-insensitive pathway. Interestingly, overnight treatment of the R383A mutant with mitoxantrone assisted in protein maturation as evidenced by a shift to the N-glycosylated form. The R383A mutant when expressed in insect cells, though detected on the surface, had no measurable ATPase activity. In addition, substitution with the positively charged lysine resulted in significantly decreased protein expression levels in HEK cells, while retaining function. In conclusion, arginine 383 is a crucial residue for ABCG2 biogenesis, where even the most conservative mutations have a large impact.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Arginina/metabolismo , Proteínas de Neoplasias/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Sequência de Aminoácidos , Animais , Arginina/genética , Linhagem Celular , Humanos , Dados de Sequência Molecular , Mutação , Proteínas de Neoplasias/genéticaRESUMO
Activation of hypoxia-inducible factors (HIF), responsible for tumor angiogenesis and glycolytic switch, is regulated by reduced oxygen availability. Normally, HIF-alpha proteins are maintained at low levels, controlled by site-specific hydroxylation carried out by HIF prolyl hydroxylases (PHD) and subsequent proteasomal degradation via the von Hippel-Lindau ubiquitin ligase. Using a yeast two-hybrid screen, we identified an interaction between melanoma antigen-11 (MAGE-11) cancer-testis antigen and the major HIF-alpha hydroxylating enzyme PHD2. The interaction was confirmed by a pull-down assay, coimmunoprecipitation, and colocalization in both normoxic and hypoxic conditions. Furthermore, MAGE-9, the closest homologue of MAGE-11, was also found to interact with PHD2. MAGE-11 inhibited PHD activity without affecting protein levels. This inhibition was accompanied by stabilization of ectopic or endogenous HIF-1alpha protein. Knockdown of MAGE-11 by small interfering RNA results in decreased hypoxic induction of HIF-1alpha and its target genes. Inhibition of PHD by MAGE-11, and following activation of HIFs, is a novel tumor-associated HIF regulatory mechanism. This finding provides new insights into the significance of MAGE expression in tumors and may provide valuable tools for therapeutic intervention because of the restricted expression of the MAGE gene family in cancers, but not in normal tissues.
Assuntos
Antígenos de Neoplasias/metabolismo , Proteínas de Neoplasias/metabolismo , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Sequência de Aminoácidos , Antígenos de Neoplasias/biossíntese , Antígenos de Neoplasias/genética , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Regulação para Baixo , Células HeLa , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Prolina Dioxigenases do Fator Induzível por Hipóxia , Dados de Sequência Molecular , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Interferência de RNA , Transcrição Gênica , Transfecção , Ubiquitina/metabolismoRESUMO
INTRODUCTION: Signaling downstream of Ras is mediated by three major pathways, Raf/ERK, phosphatidylinositol 3 kinase (PI3K), and Ral guanine nucleotide exchange factor (RalGEF). Ras signal transduction pathways play an important role in breast cancer progression, as evidenced by the frequent over-expression of the Ras-activating epidermal growth factor receptors EGFR and ErbB2. Here we investigated which signal transduction pathways downstream of Ras contribute to EGFR-dependent transformation of telomerase-immortalized mammary epithelial cells HME16C. Furthermore, we examined whether a highly transcriptionally regulated ERK pathway target, PHLDA1 (TDAG51), suggested to be a tumor suppressor in breast cancer and melanoma, might modulate the transformation process. METHODS: Cellular transformation of human mammary epithelial cells by downstream Ras signal transduction pathways was examined using anchorage-independent growth assays in the presence and absence of EGFR inhibition. TDAG51 protein expression was down-regulated by interfering small hairpin RNA (shRNA), and the effects on cell proliferation and death were examined in Ras pathway-transformed breast epithelial cells. RESULTS: Activation of both the ERK and PI3K signaling pathways was sufficient to induce cellular transformation, which was accompanied by up-regulation of EGFR ligands, suggesting autocrine EGFR stimulation during the transformation process. Only activation of the ERK pathway was sufficient to transform cells in the presence of EGFR inhibition and was sufficient for tumorigenesis in xenografts. Up-regulation of the PHLDA1 gene product, TDAG51, was found to correlate with persistent ERK activation and anchorage-independent growth in the absence or presence of EGFR inhibition. Knockdown of this putative breast cancer tumor-suppressor gene resulted in increased ERK pathway activation and enhanced matrix-detached cellular proliferation of Ras/Raf transformed cells. CONCLUSION: Our results suggest that multiple Ras signal transduction pathways contribute to mammary epithelial cell transformation, but that the ERK signaling pathway may be a crucial component downstream of EGFR activation during tumorigenesis. Furthermore, persistent activation of ERK signaling up-regulates TDAG51. This event serves as a negative regulator of both Erk activation as well as matrix-detached cellular proliferation and suggests that TDAG51 opposes ERK-mediated transformation in breast epithelial cells.
Assuntos
Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Glândulas Mamárias Humanas/patologia , Proteínas Recombinantes/genética , Transdução de Sinais , Fatores de Transcrição/metabolismo , Anoikis , Linhagem Celular Transformada , Proliferação de Células , Transformação Celular Neoplásica/induzido quimicamente , Transformação Celular Neoplásica/patologia , Doxiciclina/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Feminino , Humanos , Glândulas Mamárias Humanas/metabolismo , Análise em Microsséries , Mutação , Proteína Oncogênica p21(ras)/genética , Proteína Oncogênica p21(ras)/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
Bortezomib (Velcade((R))), a proteasome inhibitor, is approved by the FDA for the treatment of multiple myeloma (MM). While effective, its use has been hampered by peripheral neurotoxicity of unexplained etiology. Since proteasome inhibitors alter protein degradation, we speculated that proteins regulating microtubule (MT) stability may be affected after treatment and examined MT polymerization in cells by comparing the distribution of tubulin between polymerized (P) and soluble (S) fractions. We observed increased MT polymerization following treatment of SY5Y and KCNR [neuroblastoma], HCN2, and 8226 [MM] cells, using five proteasome inhibitors; the baseline proportion of total alpha-tubulin in 'P' fractions ranged from approximately 41-68%, and increased to approximately 55-99% after treatment. Increased acetylated alpha-tubulin, a post-translational marker of stabilized MTs, was observed in the neural cell lines HCN1A and HCN2 and this was sustained up to 144 hours after the proteasome inhibitor was removed. Cell cycle analysis of three cell lines after treatment, showed approximately 50-75% increases in the G(2)M phase. Immunofluorescent localization studies of proteasome inhibitor treated cells did not reveal microtubule bundles in contrast to paclitaxel treated, suggesting MT stabilization via a mechanism other than direct drug binding. We examined the levels of microtubule associated proteins and observed a 1.4-3.7 fold increase in the microtubule associated protein MAP2, in HCN2 cells following treatment with proteasome inhibitors. These data provide a plausible explanation for the neurotoxicity observed clinically and raise the possibility that microtubule stabilization contributes to cytotoxicity.
Assuntos
Ácidos Borônicos/farmacologia , Fase G2/efeitos dos fármacos , Doenças do Sistema Nervoso Periférico/metabolismo , Inibidores de Proteases/farmacologia , Pirazinas/farmacologia , Moduladores de Tubulina/farmacologia , Tubulina (Proteína)/metabolismo , Bortezomib , Linhagem Celular Tumoral , Imunofluorescência , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Doenças do Sistema Nervoso Periférico/etiologiaRESUMO
ASAP3, an Arf GTPase-activating protein previously called DDEFL1 and ACAP4, has been implicated in the pathogenesis of hepatocellular carcinoma. We have examined in vitro and in vivo functions of ASAP3 and compared it to the related Arf GAP ASAP1 that has also been implicated in oncogenesis. ASAP3 was biochemically similar to ASAP1: the pleckstrin homology domain affected function of the catalytic domain by more than 100-fold; catalysis was stimulated by phosphatidylinositol 4,5-bisphosphate; and Arf1, Arf5, and Arf6 were used as substrates in vitro. Like ASAP1, ASAP3 associated with focal adhesions and circular dorsal ruffles. Different than ASAP1, ASAP3 did not localize to invadopodia or podosomes. Cells, derived from a mammary carcinoma and from a glioblastoma, with reduced ASAP3 expression had fewer actin stress fiber, reduced levels of phosphomyosin, and migrated more slowly than control cells. Reducing ASAP3 expression also slowed invasion of mammary carcinoma cells. In contrast, reduction of ASAP1 expression had no effect on migration or invasion. We propose that ASAP3 functions nonredundantly with ASAP1 to control cell movement and may have a role in cancer cell invasion. In comparing ASAP1 and ASAP3, we also found that invadopodia are dispensable for the invasive behavior of cells derived from a mammary carcinoma.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Movimento Celular , Adesões Focais/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Fator 1 de Ribosilação do ADP/genética , Fator 1 de Ribosilação do ADP/metabolismo , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Linhagem Celular Tumoral , Feminino , Adesões Focais/genética , Proteínas Ativadoras de GTPase/genética , Humanos , Camundongos , Células NIH 3T3 , Invasividade Neoplásica , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologia , Fosfatidilinositol 4,5-Difosfato/genética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Estrutura Terciária de Proteína , Homologia de Sequência de AminoácidosRESUMO
CD97 is a member of the adhesion family of G protein-coupled receptors. Alternatively spliced forms of CD97 bind integrins alpha5beta1 and alphavbeta3, decay accelerating factor, or dermatan sulfate. CD97 is expressed on myeloid cells at high levels and a variety of other cell types at lower levels. Little is known about the physiological function of CD97. To begin dissecting the function of CD97, we evaluated the immune response of CD97 null mice to systemic infection by Listeria monocytogenes. CD97 null mice were significantly more resistant to listeriosis than matched wild-type mice. A major determinant of the difference in survival appeared to be the comparatively more robust accumulation of granulocytes in the blood and in infected livers of CD97 null mice within 18 h of inoculation, correlating with a decrease in the number of bacteria. CD97 null mice also displayed a mild granulocytosis in the nonchallenged state. Because there is a strong suggestion that CD97 functions in an adhesive capacity, we examined the migratory properties of granulocytes in CD97 null mice. In chimeric animals, CD97 null and wild-type granulocytes migrated similarly, as determined by inflammation-induced emigration from the bone marrow and accumulation in the peritoneum. Granulocyte development in the bone marrow of CD97 null mice was comparable to that of wild-type mice, and CD97 deficiency did not appear to stimulate granulocytosis secondary to peripheral inflammation and resultant granulocyte colony-stimulating factor induction, unlike various other models of adhesion deficiencies. Our results suggest that CD97 plays a role in peripheral granulocyte homeostasis.
Assuntos
Antígenos CD/genética , Moléculas de Adesão Celular/deficiência , Moléculas de Adesão Celular/genética , Granulócitos/metabolismo , Homeostase/genética , Listeriose/genética , Listeriose/imunologia , Glicoproteínas de Membrana/genética , Animais , Adesão Celular/imunologia , Movimento Celular/imunologia , Granulócitos/imunologia , Granulócitos/patologia , Homeostase/imunologia , Listeriose/metabolismo , Listeriose/patologia , Glicoproteínas de Membrana/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Acoplados a Proteínas GRESUMO
Gem, a member of the Rad,Gem/Kir subfamily of small G-proteins, has unique sequence features. We report here the crystallographic structure determination of the Gem G-domain in complex with nucleotide to 2.4 A resolution. Although the basic Ras protein fold is maintained, the Gem switch regions emphatically differ from the Ras paradigm. Our ensuing biochemical characterization indicates that Gem G-domain markedly prefers GDP over GTP. Two known functions of Gem are distinctly affected by spatially separated clusters of mutations.
Assuntos
Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Cristalografia por Raios X , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Monoméricas de Ligação ao GTP/genética , Mutagênese Sítio-Dirigida , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Eletricidade Estática , Quinases Associadas a rhoRESUMO
We identified a specific interaction between two secreted proteins, thrombospondin-1 and versican, that is induced during a toll-like receptor-3-dependent inflammatory response in vascular smooth muscle cells. Thrombospondin-1 binding to versican is modulated by divalent cations. This interaction is mediated by interaction of the G1 domain of versican with the N-module of thrombospondin-1 but only weakly with the corresponding N-terminal region of thrombospondin-2. The G1 domain of versican contains two Link modules, which are known to mediate TNFalpha-stimulated gene-6 protein binding to thrombospondin-1, and the related G1 domain of aggrecan is also recognized by thrombospondin-1. Therefore, thrombospondin-1 interacts with three members of the Link-containing hyaladherin family. On the surface of poly-I:C-stimulated vascular smooth muscle cells, versican organizes into fibrillar structures that contain elastin but are largely distinct from those formed by hyaluronan. Endogenous and exogenously added thrombospondin-1 incorporates into these structures. Binding of exogenous thrombospondin-1 to these structures, to purified versican and to its G1 domain is potently inhibited by heparin. At higher concentrations, exogenous thrombospondin-1 delays the poly-I:C induced formation of structures containing versican and elastin, suggesting that thrombospondin-1 negatively modulates this component of a vascular smooth muscle inflammatory response.