RESUMO
The ATP-binding cassette (ABC) transporter superfamily includes several membrane-bound proteins that are critical to drug pharmacokinetics and disposition. Pharmacologic evaluation of these proteins in vitro remains a challenge. In this study, human ABC transporters were expressed in polarized epithelial cell monolayers transduced using the BacMam baculovirus gene transfer system. The purpose of the study was to evaluate the efficacy of BacMam baculovirus to transduce cells grown in monolayers. In a porcine kidney cell line, LLC-PK1 cells, baculoviral transduction is successful only via the apical side of a polarized monolayer. We observed that recombinant ABC transporters were expressed on the cell surface with post-translational modification. Furthermore, sodium butyrate played a critical role in recombinant protein expression, and preincubation in the presence of tunicamycin or thapsigargin enhanced protein expression. Cells overexpressing human P-glycoprotein (P-gp) showed vectorial basolateral-to-apical transport of [(3)H]-paclitaxel, which could be reversed by the inhibitor tariquidar. Similarly, coexpression of human P-gp and ABCG2 in LLC-PK1 cells resulted in higher transport of mitoxantrone, which is a substrate for both transporters, than in either P-gp- or ABCG2-expressing cells alone. Taken together, our results indicate that a high level of expression of efflux transporters in a polarized cell monolayer is technically feasible with the BacMam baculovirus system.
Assuntos
Baculoviridae/metabolismo , Polaridade Celular/fisiologia , Células Epiteliais/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Preparações Farmacêuticas/metabolismo , Proteínas Recombinantes/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Transporte Biológico/fisiologia , Células CACO-2 , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Cães , Humanos , Rim/metabolismo , Células LLC-PK1 , Células Madin Darby de Rim Canino , Mitoxantrona/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , SuínosRESUMO
Since its development, tariquidar (TQR; XR9576; N-[2-[[4-[2-(6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)ethyl]phenyl]carbamoyl]-4,5-dimethoxyphenyl]quinoline-3-carboxamide) has been widely regarded as one of the more potent inhibitors of P-glycoprotein (P-gp), an efflux transporter of the ATP-binding cassette (ABC) transporter family. A third-generation inhibitor, TQR exhibits high affinity for P-gp, although it is also a substrate of another ABC transporter, breast cancer resistance protein (BCRP). Recently, several studies have questioned the mechanism by which TQR interfaces with P-gp, suggesting that TQR is a substrate for P-gp instead of a noncompetitive inhibitor. We investigated TQR and its interaction with human and mouse P-gp to determine if TQR is a substrate of P-gp in vitro. To address these questions, we used multiple in vitro transporter assays, including cytotoxicity, flow cytometry, accumulation, ATPase, and transwell assays. A newly generated BCRP cell line was used as a positive control that demonstrates TQR-mediated transport. Based on our results, we conclude that TQR is a potent inhibitor of both human and mouse P-gp and shows no signs of being a substrate at the concentrations tested. These in vitro data further support our position that the in vivo uptake of [(11)C]TQR into the brain can be explained by its high-affinity binding to P-gp and by it being a substrate of BCRP, followed by amplification of the brain signal by ionic trapping in acidic lysosomes.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Quinolinas/metabolismo , Quinolinas/farmacologia , Células 3T3 , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Encéfalo/metabolismo , Linhagem Celular , Linhagem Celular Transformada , Humanos , Células KB , Células MCF-7 , CamundongosRESUMO
BACKGROUND: Intrinsic and acquired resistance to drug therapies remains a challenge for malignant melanoma patients. Intratumoral heterogeneities within the tumor microenvironment contribute additional complexity to the determinants of drug efficacy and acquired resistance. METHODS: We use 3D biomimetic platforms to understand dynamics in extracellular matrix (ECM) biogenesis following pharmaceutical intervention against mitogen-activated protein kinases (MAPK) signaling. We further determined temporal evolution of secreted ECM components by isogenic melanoma cell clones. RESULTS: We found that the cell clones differentially secrete and assemble a myriad of ECM molecules into dense fibrillar and globular networks. We show that cells can modulate their ECM biosynthesis in response to external insults. Fibronectin (FN) is one of the key architectural components, modulating the efficacy of a broad spectrum of drug therapies. Stable cell lines engineered to secrete minimal levels of FN showed a concomitant increase in secretion of Tenascin-C and became sensitive to BRAF(V600E) and ERK inhibition as clonally- derived 3D tumor aggregates. These cells failed to assemble exogenous FN despite maintaining the integrin machinery to facilitate cell- ECM cross-talk. We determined that only clones that increased FN production via p38 MAPK and ß1 integrin survived drug treatment. CONCLUSIONS: These data suggest that tumor cells engineer drug resistance by altering their ECM biosynthesis. Therefore, drug treatment may induce ECM biosynthesis, contributing to de novo resistance.
Assuntos
Matriz Extracelular/metabolismo , Melanoma/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Transdução de Sinais , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Movimento Celular , Sobrevivência Celular , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos , Proteínas da Matriz Extracelular/metabolismo , Feminino , Fibronectinas/metabolismo , Xenoenxertos , Humanos , Melanoma/tratamento farmacológico , Melanoma/patologia , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Metástase Neoplásica , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Tenascina/metabolismo , Microambiente TumoralRESUMO
Multidrug resistance (MDR) has been associated with expression of ABC transporter genes including P-glycoprotein (Pgp, MDR1, ABCB1). However, deregulation of apoptotic pathways also renders cells resistant to chemotherapy. To discover apoptosis-related genes affected by Pgp expression, we used the HeLa MDR-off system. We found that using doxycycline to control Pgp expression has a significant advantage over tetracycline, in that doxycycline caused less endogenous gene expression modification/perturbation, and was more potent than tetracycline in suppressing Pgp expression. Cells overexpressing Pgp have lower TNFSF10 (TRAIL) expression than their parental cells. Controlled downregulation of Pgp increased endogenous TRAIL protein expression. Also, ectopic overexpression of TRAIL in Pgp-positive cells was associated with a reduction in Pgp levels. However, cells expressing a functionally defective mutant Pgp showed an increase in TRAIL expression, suggesting that Pgp function is required for TRAIL suppression. Cells in which Pgp is knocked down by upregulation of TRAIL expression are less susceptible to TRAIL ligand (sTRAIL)-induced apoptosis. Our findings reveal an inverse correlation between functional Pgp and endogenous TRAIL expression. Pgp function plays an important role in the TRAIL-mediated apoptosis pathway by regulating endogenous TRAIL expression and the TRAIL-mediated apoptosis pathway in MDR cancer cells.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Apoptose/genética , Resistencia a Medicamentos Antineoplásicos/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Linhagem Celular Tumoral , Regulação para Baixo , Doxiciclina/farmacologia , Resistência a Múltiplos Medicamentos/genética , Células HeLa , Humanos , Interferência de RNA , RNA Interferente Pequeno , Ligante Indutor de Apoptose Relacionado a TNF/biossíntese , Tetraciclina/farmacologiaRESUMO
Intratumoral heterogeneity has been found to be a major cause of drug resistance. Cell-to-cell variation increases as a result of cancer-related alterations, which are acquired by stochastic events and further induced by environmental signals. However, most cellular mechanisms include natural fluctuations that are closely regulated, and thus lead to asynchronization of the cells, which causes intrinsic heterogeneity in a given population. Here, we derive two novel mathematical models, a stochastic agent-based model and an integro-differential equation model, each of which describes the growth of cancer cells as a dynamic transition between proliferative and quiescent states. These models are designed to predict variations in growth as a function of the intrinsic heterogeneity emerging from the durations of the cell-cycle and apoptosis, and also include cellular density dependencies. By examining the role all parameters play in the evolution of intrinsic tumor heterogeneity, and the sensitivity of the population growth to parameter values, we show that the cell-cycle length has the most significant effect on the growth dynamics. In addition, we demonstrate that the agent-based model can be approximated well by the more computationally efficient integro-differential equations when the number of cells is large. This essential step in cancer growth modeling will allow us to revisit the mechanisms of multidrug resistance by examining spatiotemporal differences of cell growth while administering a drug among the different sub-populations in a single tumor, as well as the evolution of those mechanisms as a function of the resistance level.
Assuntos
Modelos Biológicos , Neoplasias/patologia , Apoptose , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Simulação por Computador , Humanos , Fatores de TempoRESUMO
Acquired drug resistance in cancer continues to be a challenge in cancer therapy, in part due to overexpression of the drug efflux transporter P-glycoprotein (P-gp, MDR1, ABCB1). NSC73306 is a thiosemicarbazone compound that displays greater toxicity against cells expressing functional P-gp than against other cells. Here, we investigate the cellular uptake of NSC73306, and examine its interaction with P-gp and copper transporter 1 (CTR1, SLC31A1). Overexpression of P-gp sensitizes LLC-PK1 cells to NSC73306. Cisplatin (IC50 = 77 µM), cyclosporin A (IC50 = 500 µM), and verapamil (IC50 = 700 µM) inhibited cellular accumulation of [(3)H]NSC73306. Cellular hypertoxicity of NSC73306 to P-gp-expressing cells was inhibited by cisplatin in a dose-dependent manner. Cells transiently expressing the cisplatin uptake transporter CTR1 (SLC31A1) showed increased [(3)H]NSC73306 accumulation. In contrast, CTR1 knockdown decreased [(3)H]NSC73306 accumulation. The presence of NSC73306 reduced CTR1 levels, similar to the negative feedback of CTR1 levels by copper or cisplatin. Surprisingly, although cisplatin is a substrate of CTR1, we found that CTR1 protein was overexpressed in high-level cisplatin-resistant KB-CP20 and BEL7404-CP20 cell lines. We confirmed that the CTR1 protein was functional, as uptake of NSC73306 was increased in KB-CP20 cells compared to their drug-sensitive parental cells, and downregulation of CTR1 in KB-CP20 cells reduced [(3)H]NSC73306 accumulation. These results suggest that NSC73306 is a transport substrate of CTR1.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Indóis/farmacocinética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Células COS , Chlorocebus aethiops , Cisplatino/administração & dosagem , Cisplatino/química , Cobre/química , Transportador de Cobre 1 , Ciclosporina/química , Células HEK293 , Humanos , Concentração Inibidora 50 , Células LLC-PK1 , RNA Interferente Pequeno/metabolismo , Reprodutibilidade dos Testes , Suínos , Tiossemicarbazonas/química , Verapamil/administração & dosagemRESUMO
Tasigna (Nilotinib) is a BCR-ABL kinase inhibitor recently approved by the Food and Drug Administration, which is indicated for the treatment of drug-resistant chronic myelogenous leukemia (CML). The efflux of tyrosine kinase inhibitors by ATP-binding cassette (ABC) drug transporters, which actively pump these drugs out of cells utilizing ATP as an energy source, has been linked to the development of drug resistance in CML patients. We report here the synthesis and characterization of a fluorescent derivative of Tasigna to study its interaction with two major ABC transporters, P-glycoprotein (Pgp) and ABCG2, in in vitro and ex vivo assays. A fluorescent derivative of Tasigna, BODIPY FL Tasigna, inhibited the BCR-ABL kinase activity in K562 cells and was also effluxed by Pgp- and ABCG2-expressing cells in both cultured cells and rat brain capillaries expressing Pgp and ABCG2. In addition, [(3)H]-Tasigna was found to be transported by Pgp-expressing polarized LLC-PK1 cells in a transepithelial transport assay. Consistent with these results, both Tasigna and BODIPY FL Tasigna were less effective at inhibiting the phosphorylation of Crkl (a substrate of BCR-ABL kinase) in Pgp- and ABCG2-expressing K562 cells due to their reduced intracellular concentration. Taken together, these data provide evidence that BODIPY FL Tasigna is transported by Pgp and ABCG2, and Tasigna is transported by Pgp. Further, we propose that BODIPY FL Tasigna can potentially be used as a probe for functional analysis of Pgp and ABCG2 in cancer cells and in other preclinical studies.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Compostos de Boro/química , Pirimidinas/química , Pirimidinas/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Linhagem Celular Tumoral , Citometria de Fluxo , Humanos , Immunoblotting , Técnicas In Vitro , Células LLC-PK1 , Masculino , Microscopia Confocal , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Ratos , Ratos Sprague-Dawley , SuínosRESUMO
The MDR1 (ABCB1) gene encodes a membrane-bound transporter that actively effluxes a wide range of compounds from cells. The overexpression of MDR1 by multidrug-resistant cancer cells is a serious impediment to chemotherapy. MDR1 is expressed in various tissues to protect them from the adverse effect of toxins. The pharmacokinetics of drugs that are also MDR1 substrates also influence disease outcome and treatment efficacy. Although MDR1 is a well-conserved gene, there is increasing evidence that its polymorphisms affect substrate specificity. Three single nucleotide polymorphisms (SNPs) occur frequently and have strong linkage, creating a common haplotype at positions 1236C>T (G412G), 2677G>T (A893S) and 3435C>T (I1145I). The frequency of the synonymous 3435C>T polymorphism has been shown to vary significantly according to ethnicity. Existing literature suggests that the haplotype plays a role in response to drugs and disease susceptibility. This review summarizes recent findings on the 3435C>T polymorphism of MDR1 and the haplotype to which it belongs. A possible molecular mechanism of action by ribosome stalling that can change protein structure and function by altering protein folding is discussed.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Etnicidade/genética , Haplótipos , Humanos , Camundongos , Farmacocinética , Polimorfismo de Nucleotídeo Único , Ribossomos/efeitos dos fármacos , Ribossomos/fisiologia , Especificidade por SubstratoRESUMO
The deleted in liver cancer 2 (DLC2) is a tumor suppressor gene, frequently found to be underexpressed in hepatocellular carcinoma. DLC2 is a multidomain protein containing a sterile alpha-motif (SAM) domain, a GTPase-activating protein (GAP) domain, and a lipid-binding StAR-related lipid-transfer (START) domain. The SAM domain of DLC2, DLC2-SAM, exhibits a low level of sequence homology (15-30%) with other SAM domains, and appears to be the prototype of a new subfamily of SAM domains found in DLC2-related proteins. In the present study, we have determined the three-dimensional solution structure of DLC2-SAM using NMR methods together with molecular dynamics simulated annealing. In addition, we performed a backbone dynamics study. The DLC2-SAM packed as a unique four alpha-helical bundle stabilized by interhelix hydrophobic interactions. The arrangement of the four helices is distinct from all other known SAM domains. In contrast to some members of the SAM domain family which form either dimers or oligomers, both biochemical analyses and rotational correlation time (tau(c)) measured by backbone 15N relaxation experiments indicated that DLC2-SAM exists as a monomer in solution. The interaction of DLC2-SAM domain with sodium dodecyl sulfate (SDS) micelles and 1,2-dimyristoyl-sn-glycerol-3-phosphatidylglycerol (DMPG) phospholipids was examined by CD and NMR spectroscopic techniques. The DLC2-SAM exhibits membrane binding properties accompanied by minor loss of the secondary structure of the protein. Deletion studies showed that the self-association of DLC2 in vivo does not require SAM domain, instead, a protein domain consisting of residues 120-672 mediates the self-association of DLC2.
Assuntos
Ressonância Magnética Nuclear Biomolecular , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Dicroísmo Circular , Micelas , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Dodecilsulfato de Sódio , Soluções/química , Proteínas Supressoras de Tumor/genéticaRESUMO
One of the hallmarks of the malignant transformation of epithelial tissue is the modulation of stromal components of the microenvironment. In particular, aberrant extracellular matrix (ECM) remodeling and stiffening enhances tumor growth and survival and promotes metastasis. Type I collagen is one of the major ECM components. It serves as a scaffold protein in the stroma contributing to the tissue's mechanical properties, imparting tensile strength and rigidity to tissues such as those of the skin, tendons, and lungs. Here we investigate the effects of intrinsic spatial heterogeneities due to fibrillar architecture, pore size and ligand density on the microscale and bulk mechanical properties of the ECM. Type I collagen hydrogels with topologies tuned by polymerization temperature and concentration to mimic physico-chemical properties of a normal tissue and tumor microenvironment were measured by in situ-calibrated Active Microrheology by Optical Trapping revealing significantly different microscale complex shear moduli at Hz-kHz frequencies and two orders of magnitude of strain amplitude that we compared to data from bulk rheology measurements. Access to higher frequencies enabled observation of transitions from elastic to viscous behavior that occur at ~200Hz to 2750Hz, which largely was dependent on tissue architecture well outside the dynamic range of instrument acquisition possible with SAOS bulk rheology. We determined that mouse melanoma tumors and human breast tumors displayed complex moduli ~5-1000 Pa, increasing with frequency and displaying a nonlinear stress-strain response. Thus, we show the feasibility of a mechanical biopsy in efforts to provide a diagnostic tool to aid in the design of therapeutics complementary to those based on standard histopathology.
RESUMO
The drug efflux function of P-glycoprotein (P-gp) encoded by MDR1 can be influenced by genetic polymorphisms, including two synonymous changes in the coding region of MDR1. Here we report that the conformation of P-gp and its drug efflux activity can be altered by synonymous polymorphisms in stable epithelial monolayers expressing P-gp. Several cell lines with similar MDR1 DNA copy number were developed and termed LLC-MDR1-WT (expresses wild-type P-gp), LLC-MDR1-3H (expresses common haplotype P-gp), and LLC-MDR1-3HA (a mutant that carries a different valine codon in position 3435). These cell lines express similar levels of recombinant mRNA and protein. P-gp in each case is localized on the apical surface of polarized cells. However, the haplotype and its mutant P-gps fold differently from the wild-type, as determined by UIC2 antibody shift assays and limited proteolysis assays. Surface biotinylation experiments suggest that the non-wild-type P-gps have longer recycling times. Drug transport assays show that wild-type and haplotype P-gp respond differently to P-gp inhibitors that block efflux of rhodamine 123 or mitoxantrone. In addition, cytotoxicity assays show that the LLC-MDR1-3H cells are more resistant to mitoxantrone than the LLC-MDR1-WT cells after being treated with a P-gp inhibitor. Expression of polymorphic P-gp, however, does not affect the host cell's morphology, growth rate, or monolayer formation. Also, ATPase activity assays indicate that neither basal nor drug-stimulated ATPase activities are affected in the variant P-gps. Taken together, our findings indicate that "silent" polymorphisms significantly change P-gp function, which would be expected to affect interindividual drug disposition and response.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Regulação Neoplásica da Expressão Gênica , Polimorfismo Genético , Subfamília B de Transportador de Cassetes de Ligação de ATP , Adenosina Trifosfatases/metabolismo , Animais , Transporte Biológico , Biotinilação , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos , Haplótipos , Humanos , Mitoxantrona/química , Mutação , Estabilidade Proteica , Proteínas Recombinantes/química , Rodamina 123/química , SuínosRESUMO
Cancer cells often become resistant to chemotherapy through a phenomenon known as multidrug resistance (MDR). Several factors are responsible for the development of MDR, preeminent among them being the accelerated drug efflux mediated by overexpression of ATP binding cassette (ABC) transporters. Some small molecule tyrosine kinase inhibitors (TKIs) were recently reported to modulate the activity of ABC transporters. Therefore, the purpose of this study was to determine if motesanib, a multikinase inhibitor, could reverse ABCB1-mediated MDR. The results showed that motesanib significantly sensitized both ABCB1-transfected and drug-selected cell lines overexpressing this transporter to its substrate anticancer drugs. Motesanib significantly increased the accumulation of [(3)H]-paclitaxel in ABCB1 overexpressing cells by blocking the efflux function of ABCB1 transporter. In contrast, no significant change in the expression levels and localization pattern of ABCB1 was observed when ABCB1 overexpressing cells were exposed to 3µM motesanib for 72h. Moreover, motesanib stimulated the ATPase activity of ABCB1 in a concentration-dependent manner, indicating a direct interaction with the transporter. Consistent with these findings, the docking studies indicated favorable binding of motesanib within the transmembrane region of homology modeled human ABCB1. Here, we report for the first time, motesanib, at clinically achievable plasma concentrations, antagonizes MDR by inhibiting the efflux activity of the ABCB1 transporter. These findings may be useful for cancer combination therapy with TKIs in the clinic.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Indóis/farmacologia , Niacinamida/análogos & derivados , Inibidores de Proteínas Quinases/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos Fitogênicos/farmacocinética , Western Blotting , Linhagem Celular Tumoral , Imunofluorescência , Humanos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Niacinamida/farmacologia , Oligonucleotídeos , Paclitaxel/farmacocinéticaRESUMO
The efflux pump P-glycoprotein (ATP-binding cassette B1, multidrug resistance [MDR] 1, P-gp) has long been known to contribute to MDR against cancer chemotherapeutics. We describe the development of a dual-fluorescent cell line system to allow multiplexing of drug-sensitive and P-gp-mediated MDR cell lines. The parental OVCAR-8 human ovarian carcinoma cell line and the isogenic MDR NCI/ADR-RES subline, which stably expresses high levels of endogenous P-gp, were transfected to express the fluorescent proteins Discosoma sp. red fluorescent protein DsRed2 and enhanced green fluorescent protein, respectively. Co-culture conditions were defined, and fluorescent barcoding of each cell line allowed for the direct, simultaneous comparison of resistance to cytotoxic compounds in sensitive and MDR cell lines. We show that this assay system retains the phenotypes of the original lines and is suitable for multiplexing using confocal microscopy, flow cytometry, or laser scanning microplate cytometry in 1,536-well plates, enabling the high-throughput screening of large chemical libraries.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Resistência a Múltiplos Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos/genética , Antineoplásicos/metabolismo , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células , Corantes , Avaliação Pré-Clínica de Medicamentos , Feminino , Fluorescência , Genótipo , Humanos , Citometria de Varredura a Laser , Microscopia Confocal , Mitoxantrona/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sais de Tetrazólio , Tiazóis , TransfecçãoRESUMO
Brassica juncea BjCHI1 is a unique chitinase with two chitin-binding domains. Here, we show that, unlike other chitinases, potato-expressed BjCHI1 shows hemagglutination ability. BjCHI1 expression in B. juncea seedlings is induced by Rhizoctonia solani infection, suggesting its protective role against this fungus. To verify this, transgenic potato (Solanum tuberosum L. cv. Desiree) plants expressing BjCHI1 generated by Agrobacterium-mediated transformation were challenged with R. solani. We also transformed potato with a cDNA encoding Hevea brasiliensis beta-1,3-glucanase, designated HbGLU, and a pBI121-derivative that contains cDNAs encoding both BjCHI1 and HbGLU. In vitro fungal bioassays using Trichoderma viride showed that extracts from transgenic potato lines co-expressing BjCHI1 and HbGLU inhibited fungal growth better than extracts from transgenic potato expressing either BjCHI1 or HbGLU, suggesting a synergistic effect. Consistently, in vivo fungal bioassays with soil-borne R. solani on young transgenic potato plants indicated that the co-expressing plants showed healthier root development than untransformed plants or those that expressed either BjCHI1 or HbGLU. Light microscopy and transmission electron microscopy revealed abundant intact R. solani hyphae and monilioid cells in untransformed roots and disintegrated fungus in the BjCHI1-expressing and the BjCHI1 and HbGLU co-expressing plants. Observations of collapsed epidermal cells in the co-expressing potato roots suggest that these proteins effectively degrade the fungal cell wall, producing elicitors that initiate other defense responses causing epidermal cell collapse that ultimately restricts further fungal penetration.
Assuntos
Quitinases/metabolismo , Mostardeira/enzimologia , Doenças das Plantas/microbiologia , Rhizoctonia , Solanum tuberosum/genética , Sequência de Aminoácidos , Quitinases/genética , Quitinases/fisiologia , Regulação da Expressão Gênica de Plantas , Glucana 1,3-beta-Glucosidase/genética , Glucana 1,3-beta-Glucosidase/metabolismo , Hevea/enzimologia , Doenças das Plantas/genética , Raízes de Plantas/microbiologia , Raízes de Plantas/ultraestrutura , Plantas Geneticamente Modificadas , RNA Mensageiro/análiseRESUMO
We have previously isolated a Brassica juncea cDNA encoding BjCHI1, a novel chitinase with two chitin-binding domains, and have shown that its mRNA is induced by wounding and methyl jasmonate treatment (K.-J. Zhao and M.-L. Chye, Plant Mol. Biol. 40 (1999) 1009-1018). By the presence of two chitin-binding domains, BjCHI1 resembles the precursor of UDA (Urtica dioica agglutinin) but, unlike UDA, BjCHI1 retains its chitinase catalytic domain after post-translational processing. Here, we indicate the role of BjCHI1 in plant defense by demonstrating its mRNA induction upon Aspergillus niger infection or caterpillar Pieris rapae (L.) feeding. To further investigate the biological properties of BjCHI1, we transformed tobacco with a construct expressing the BjCHI1 cDNA from the CaMV 35S promoter. Subsequently, we purified BjCHI1 from the resultant transgenic Ro plants using a regenerated chitin column followed by fast protein liquid chromatography (FPLC). Also, the significance of the second chitin-binding domain in BjCHI1 was investigated by raising transgenic tobacco plants expressing BjCHI2, a deletion derivative of BjCHI1 lacking one chitin-binding domain. Colorimetric chitinase assays at 25 degrees C, pH 5, showed no significant differences between the activities of BjCHI1 and BjCHI2, suggesting that chitinase activity, due to the catalytic domain, is not enhanced by the presence of a second chitin-binding domain. Both BjCHI1 and BjCHI2 show in vitro anti-fungal activity toward Trichoderma viride, causing reductions in hyphal diameter, hyphal branching and conidia size.