RESUMO
The ATP-binding cassette (ABC) transporters of class G display a different domain organisation than P-glycoprotein/ABCB1 and bacterial homologues with a nucleotide-binding domain preceding the transmembrane domain. The linker region connecting these domains is unique and its function and structure cannot be predicted. Sequence analysis revealed that the human ABCG2 linker contains a LSGGE sequence, homologous to the canonical C-motif/ABC signature present in all ABC nucleotide-binding domains. Predictions of disorder and of secondary structures indicated that this C2-sequence was highly mobile and located between an α-helix and a loop similarly to the C-motif. Point mutations of the two first residues of the C2-sequence fully abolished the transport-coupled ATPase activity, and led to the complete loss of cell resistance to mitoxantrone. The interaction with potent, selective and non-competitive, ABCG2 inhibitors was also significantly altered upon mutation. These results suggest an important mechanistic role for the C2-sequence of the ABCG2 linker region in ATP binding and/or hydrolysis coupled to drug efflux.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/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/química , Transportadores de Cassetes de Ligação de ATP/genética , Adenosina/análogos & derivados , Adenosina/farmacologia , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dicetopiperazinas , Resistencia a Medicamentos Antineoplásicos , Feminino , Células HEK293 , Compostos Heterocíclicos de 4 ou mais Anéis , Humanos , Mitoxantrona/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Alinhamento de SequênciaRESUMO
PURPOSE: Improved therapies and imaging modalities are needed for the treatment of breast cancer brain metastases (BCBM). ANG1005 is a drug conjugate consisting of paclitaxel covalently linked to Angiopep-2, designed to cross the blood-brain barrier. We conducted a biomarker substudy to evaluate 18F-FLT-PET for response assessment. METHODS: Ten patients with measurable BCBM received ANG1005 at a dose of 550 mg/m2 IV every 21 days. Before and after cycle 1, patients underwent PET imaging with 18F-FLT, a thymidine analog, retention of which reflects cellular proliferation, for comparison with gadolinium-contrast magnetic resonance imaging (Gd-MRI) in brain metastases detection and response assessment. A 20 % change in uptake after one cycle of ANG1005 was deemed significant. RESULTS: Thirty-two target and twenty non-target metastatic brain lesions were analyzed. The median tumor reduction by MRI after cycle 1 was -17.5 % (n = 10 patients, lower, upper quartiles: -25.5, -4.8 %) in target lesion size compared with baseline. Fifteen of twenty-nine target lesions (52 %) and 12/20 nontarget lesions (60 %) showed a ≥20 % decrease post-therapy in FLT-PET SUV change (odds ratio 0.71, 95 % CI: 0.19, 2.61). The median percentage change in SUVmax was -20.9 % (n = 29 lesions; lower, upper quartiles: -42.4, 2.0 %), and the median percentage change in SUV80 was also -20.9 % (n = 29; lower, upper quartiles: -49.0, 0.0 %). Two patients had confirmed partial responses by PET and MRI lasting 6 and 18 cycles, respectively. Seven patients had stable disease, receiving a median of six cycles. CONCLUSIONS: ANG1005 warrants further study in BCBM. Results demonstrated a moderately strong association between MRI and 18F-FLT-PET imaging.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/secundário , Neoplasias da Mama/patologia , Paclitaxel/análogos & derivados , Peptídeos/uso terapêutico , Adulto , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Biomarcadores , Biomarcadores Tumorais , Neoplasias Encefálicas/diagnóstico , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/metabolismo , Terapia Combinada , Feminino , Fluordesoxiglucose F18 , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Imageamento por Ressonância Magnética , Pessoa de Meia-Idade , Paclitaxel/administração & dosagem , Paclitaxel/efeitos adversos , Paclitaxel/uso terapêutico , Peptídeos/administração & dosagem , Peptídeos/efeitos adversos , Tomografia por Emissão de Pósitrons , Resultado do TratamentoRESUMO
Since 1990, the National Cancer Institute (NCI) has screened more than 60,000 compounds against a panel of 60 human cancer cell lines. The 50-percent growth-inhibitory concentration (GI50) for any single cell line is simply an index of cytotoxicity or cytostasis, but the patterns of 60 such GI50 values encode unexpectedly rich, detailed information on mechanisms of drug action and drug resistance. Each compound's pattern is like a fingerprint, essentially unique among the many billions of distinguishable possibilities. These activity patterns are being used in conjunction with molecular structural features of the tested agents to explore the NCI's database of more than 460,000 compounds, and they are providing insight into potential target molecules and modulators of activity in the 60 cell lines. For example, the information is being used to search for candidate anticancer drugs that are not dependent on intact p53 suppressor gene function for their activity. It remains to be seen how effective this information-intensive strategy will be at generating new clinically active agents.
Assuntos
Antineoplásicos/farmacologia , Biologia Computacional , Bases de Dados Factuais , Ensaios de Seleção de Medicamentos Antitumorais , Algoritmos , Antineoplásicos/química , Análise por Conglomerados , Redes de Comunicação de Computadores , Genes p53 , Humanos , Estrutura Molecular , Mutação , Software , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/fisiologiaRESUMO
Obesity is a modern-day epidemic with serious physical, psychological and economic implications for the patients. Tackling obesity is now a priority for most healthcare providers. Managing such patients can be complex, emotional, time consuming and often frustrating. Obesity surgery, in its various forms, has revolutionised this struggle. With appropriate selection of patients, adequate resources and a multidisciplinary team involvement, obesity can now effectively be "cured". It is vital that those who deal with obese patients know how to access these services and understand the processes involved in the journey from initial assessment to postoperative follow-up. Obesity surgery has a major impact in reducing obesity-related comorbidities such as diabetes and hypertension and contributes to society by returning patients to work. Prevention must be at the heart of any strategy to manage obesity, but, for established cases, surgery is taking centre stage and will continue to flourish as new techniques and procedures are developed.
Assuntos
Obesidade Mórbida/terapia , Fármacos Antiobesidade/uso terapêutico , Cirurgia Bariátrica/métodos , Humanos , Seleção de Pacientes , Complicações Pós-Operatórias/etiologia , Encaminhamento e Consulta , Redução de PesoRESUMO
Understanding the mechanisms of multidrug resistance (MDR) could improve clinical drug efficacy. Multidrug resistance is associated with ATP binding cassette (ABC) transporters, but the factors that regulate their expression at clinically relevant drug concentrations are poorly understood. We report that a single-step selection with low doses of anti-cancer agents, similar to concentrations reported in vivo, induces MDR that is mediated exclusively by ABCG2. We selected breast, ovarian and colon cancer cells (MCF-7, IGROV-1 and S-1) after exposure to 14 or 21 nM doxorubicin for only 10 days. We found that these cells overexpress ABCG2 at the mRNA and protein levels. RNA interference analysis confirmed that ABCG2 confers drug resistance. Furthermore, ABCG2 upregulation was facilitated by histone hyperacetylation due to weaker histone deacetylase 1-promoter association, indicating that these epigenetic changes elicit changes in ABCG2 gene expression. These studies indicate that the MDR phenotype arises following low-dose, single-step exposure to doxorubicin, and further suggest that ABCG2 may mediate early stages of MDR development. This is the first report to our knowledge of single-step, low-dose selection leading to overexpression of ABCG2 by epigenetic changes in multiple cancer cell lines.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Antibióticos Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Epigênese Genética , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Acetilação , Antineoplásicos/farmacologia , Western Blotting , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Feminino , Regulação Neoplásica da Expressão Gênica , Histonas/metabolismo , Humanos , Mitoxantrona/farmacologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , RNA Mensageiro/isolamento & purificação , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para CimaRESUMO
The majority of chronic phase chronic myeloid leukemia (CML) patients treated with the tyrosine kinase inhibitor (TKI) imatinib mesylate maintain durable responses to the drug. However, most patients relapse after withdrawal of imatinib and advanced stage patients often develop drug resistance. As CML is considered a hematopoietic stem cell cancer, it has been postulated that inherent protective mechanisms lead to relapse in patients. The ATP binding-cassette transporters ABCB1 (MDR-1; P-glycoprotein) and ABCG2 are highly expressed on primitive hematopoietic stem cells (HSCs) and have been shown to interact with TKIs. Herein we demonstrate a dose-dependent, reversible inhibition of ABCG2-mediated Hoechst 33342 dye efflux in primary human and murine HSC by both imatinib and nilotinib (AMN107), a novel aminopyrimidine inhibitor of BCR-ABL. ABCG2-transduced K562 cells were protected from imatinib and nilotinib-mediated cell death and from downregulation of P-CRKL. Moreover, photoaffinity labeling revealed interaction of both TKIs with ABCG2 at the substrate binding sites as they compete with the binding of [(125)I] IAAP and also stimulate the transporter's ATPase activity. Therefore, our evidence suggests for the role of ABC transporters in resistance to TKI on primitive HSCs and CML stem cells and provides a rationale how TKI resistance can be overcome in vivo.
Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/metabolismo , Resistencia a Medicamentos Antineoplásicos , Células-Tronco Hematopoéticas , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Piperazinas/farmacocinética , Pirimidinas/farmacocinética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Antineoplásicos/farmacocinética , Benzamidas , Sítios de Ligação , Humanos , Mesilato de Imatinib , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Camundongos , Proteínas de Neoplasias/genética , Inibidores de Proteínas Quinases , Recidiva , Transdução GenéticaRESUMO
8-Cl-cAMP, a site-selective analogue of cAMP, decreased mdr-1 expression in multidrug-resistant human breast cancer cells. A sixfold reduction of mdr-1 mRNA expression by 8-Cl-cAMP began within 8 h of treatment and was associated with a decrease in the synthesis of P-glycoprotein and with an increase in vinblastine accumulation. A reduction in mdr-1 expression after 8-Cl-cAMP treatment was also observed in multidrug-resistant human ovarian cancer cell lines. 8-Cl-cAMP is known to change the ratio between the two regulatory subunits, RI and RII, of protein kinase A (PKA). We observed that RI alpha decreased within 24 h of 8-Cl-cAMP treatment, that RII beta increased after as few as 3 h of treatment, and that PKA catalytic activity remained unchanged during 48 h of 8-Cl-cAMP treatment. The results are consistent with the hypothesis that mdr-1 expression is regulated in part by changes in PKA isoenzyme levels. Although 8-Cl-cAMP has been used to differentiate cells in other model systems, the only differentiating effect that could be detected after 8-Cl-cAMP treatment in the MCF-7TH cells was an increase in cytokeratin expression. Evidence that the reduction of mdr-1 mRNA occurred at the level of gene transcription was obtained by measuring chloramphenicol acetyltransferase (CAT) mRNA in MCF-7TH cells transfected with an mdr-1 promoter-CAT construct prior to 8-Cl-cAMP treatment. Thus, 8-Cl-cAMP is able to downregulate mdr-1 expression and suggests a new approach to reversal of drug resistance in human breast cancer.
Assuntos
8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Adenocarcinoma/patologia , Neoplasias da Mama/patologia , AMP Cíclico/fisiologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Neoplasias/biossíntese , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Sequência de Bases , Diferenciação Celular , Subunidade RIIbeta da Proteína Quinase Dependente de AMP Cíclico , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Resistência a Múltiplos Medicamentos/genética , Feminino , Genes Reporter , Humanos , Isoenzimas/fisiologia , Queratinas/biossíntese , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Células Tumorais Cultivadas/efeitos dos fármacos , Vimblastina/metabolismoRESUMO
Expression of a multidrug resistance gene (mdr1) and its protein product, P-glycoprotein (Pgp), has been correlated with the onset of multidrug resistance in vitro in human cell lines selected for resistance to chemotherapeutic agents derived from natural products. Expression of this gene has also been observed in normal tissues and human tumors, including neuroblastoma. We therefore examined total RNA prepared from human neuroblastoma cell lines before and after differentiation with retinoic acid or sodium butyrate. An increase in the level of mdr1 mRNA was observed after retinoic acid treatment of four neuroblastoma cell lines, including the SK-N-SH cell line. Western blot (immunoblot) analysis demonstrated concomitant increases in Pgp. However, studies of 3H-vinblastine uptake failed to show a concomitant Pgp-mediated decrease in cytotoxic drug accumulation. To provide evidence that Pgp was localized on the cell surface, an immunotoxin conjugate directed against Pgp was added to cells before and after treatment with retinoic acid. Incorporation of [3H]leucine was decreased by the immunotoxin in the retinoic acid-treated cells compared with the undifferentiated cells. These results demonstrate that whereas expression of the mdr1 gene can be modulated by differentiating agents, increased levels of expression are not necessarily associated with increased cytotoxic drug accumulation.
Assuntos
ADP Ribose Transferases , Toxinas Bacterianas , Resistência a Medicamentos/genética , Glicoproteínas de Membrana/genética , Neuroblastoma/genética , Tretinoína/farmacologia , Fatores de Virulência , Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Western Blotting , Butiratos/farmacologia , Ácido Butírico , Diferenciação Celular/efeitos dos fármacos , Membrana Celular/análise , Células Clonais , Exotoxinas , Expressão Gênica , Humanos , Imunotoxinas , Glicoproteínas de Membrana/biossíntese , Neuroblastoma/metabolismo , Pseudomonas , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Fatores de Tempo , Células Tumorais Cultivadas , Exotoxina A de Pseudomonas aeruginosaRESUMO
BACKGROUND: Multidrug resistance (MDR) mediated by high levels of mdr-1 (also known as PGY1)/P-glycoprotein (Pgp) has been studied in tissue culture systems; however, most tumor samples which express mdr-1/Pgp have much lower levels. PURPOSE: We wanted to determine if levels seen clinically could be detected by commonly used methods and to determine if these levels conferred MDR reversible by Pgp antagonists. METHODS: We studied multi-drug-resistant cell lines and sublines with levels of mdr-1/Pgp expression comparable to those seen clinically. We evaluated the expression of mdr-1 RNA by Northern blot analysis, slot blot analysis, polymerase chain reaction (PCR) analysis, and in situ hybridization. We evaluated protein expression by immunofluorescence, immunohistochemistry, fluorescence-activated cell sorting, and immunoblotting analyses. Drug resistance and reversibility were determined by cell growth during continuous drug exposure. RESULTS: In most cases, the low level of mdr-1/Pgp present in these cell lines could be detected by each method, but the assays were at the limit of sensitivity for all methods except the PCR method. These low levels of mdr-1/Pgp are capable of conferring MDR, which can be antagonized by verapamil. CONCLUSIONS: Levels of mdr-1/Pgp similar to those found in clinical samples can be detected by each of these methods, but the PCR method was the most sensitive and most reliably quantitative. IMPLICATIONS: In vitro sensitization by the addition of verapamil in cell lines with these low levels of mdr-1/Pgp suggests that clinically detected levels may confer drug resistance in vivo.
Assuntos
Neoplasias do Colo/fisiopatologia , Resistência a Medicamentos , Glicoproteínas de Membrana/análise , Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Citometria de Fluxo , Expressão Gênica , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Glicoproteínas de Membrana/genética , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Neoplásico/genética , Células Tumorais CultivadasRESUMO
BACKGROUND: Growth factor receptor-signaling pathways are potentially important targets for anticancer therapy. The interaction of anticancer agents with specific molecular targets can be identified by correlating target expression patterns with cytotoxicity patterns. We sought to identify new agents that target and inhibit the activity of the epidermal growth factor (EGF) receptor and of c-erbB2 (also called HER2 or neu), by correlating EGF receptor, transforming growth factor (TGF)-alpha (a ligand for EGF receptor), and c-erbB2 messenger RNA (mRNA) expression levels with the results of cytotoxicity assays of the 49000 compounds in the National Cancer Institute (NCI) drug screen database. METHODS: The levels of mRNAs were measured and used to generate a molecular target database for the 60 cell lines of the NCI anticancer drug screen. The computer analysis program, COMPARE, was used to search for cytotoxicity patterns in the NCI drug screen database that were highly correlated with EGF receptor, TGF-alpha, or c-erbB2 mRNA expression patterns. The putative EGF receptor-inhibiting compounds were tested for effects on basal tyrosine phosphorylation, in vitro EGF receptor tyrosine kinase activity, and EGF-dependent growth. Putative ErbB2-inhibiting compounds were tested for effects on antibody-induced ErbB2 tyrosine kinase activity. RESULTS: EGF receptor mRNA and TGF-alpha mRNA levels were highest in cell lines derived from renal cancers, and c-erbB2 mRNA levels were highest in cells derived from breast, ovarian, and colon cancers. Twenty-five compounds with high correlation coefficients (for cytotoxicity and levels of the measured mRNAs) were tested as inhibitors of the EGF receptor or c-erbB2 signaling pathways; 14 compounds were identified as inhibitors of these pathways. The most potent compound, B4, inhibited autophosphorylation (which occurs following activation) of ErbB2 by 50% in whole cells at 7.7 microM. CONCLUSIONS: Novel EGF receptor or c-erbB2 pathway inhibitors can be identified in the NCI drug screen by correlation of cytotoxicity patterns with EGF receptor or c-erbB2 mRNA expression levels.
Assuntos
Antineoplásicos/toxicidade , Receptores ErbB/biossíntese , Receptor ErbB-2/biossíntese , Transcrição Gênica/efeitos dos fármacos , Fator de Crescimento Transformador alfa/biossíntese , Neoplasias da Mama , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Análise por Conglomerados , Neoplasias do Colo , Ensaios de Seleção de Medicamentos Antitumorais , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Renais , Neoplasias Ovarianas , RNA Mensageiro/biossíntese , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
We have characterized the production of transforming growth factor (TGF) activities by five human breast cancer cell lines in culture. The presence of TGF-alpha and -beta activity in medium conditioned by these cells was detected by induction of anchorage-independent colony formation of normal rat kidney cells in soft agar and by epidermal growth factor and TGF-beta receptor competition studies. In MCF-7, an estrogen-receptor positive cell line which requires estrogen for tumorigenesis in vivo, 17 beta-estradiol induced a 2-5-fold increase in a TGF-alpha-like activity (apparent molecular weight, 68,000 and 30,000 by column chromatography). An estrogen induction of lower magnitude (1.5-2.5-fold) was also seen in two other estrogen responsive cell lines, ZR-75-B and T47D. TGF-beta was not induced by 17 beta-estradiol in any of the three cell lines and was decreased by up to 50% of control in the MCF-7 and T47D cell lines. TGF-beta was not detectable by radioreceptor assay in the ZR-75-B cell line. Two hormone-independent and highly tumorigenic cell lines were studied. The MDA-MB-231 cell line produced large amounts of both TGF-alpha-like (Mr 30,000) and TGF-beta activities. In the HS578T cell line, little TGF-alpha was detectable, while large amounts of TGF-beta were produced. No simple correlations between the tumorigenicity of the cell lines in nude mice and production of either TGF activity could be demonstrated.
Assuntos
Neoplasias da Mama/análise , Estrogênios/farmacologia , Peptídeos/análise , Animais , Aprotinina/farmacologia , Neoplasias da Mama/patologia , Linhagem Celular , Cromatografia em Gel , Meios de Cultura/análise , Feminino , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Ensaio Radioligante , Receptores de Estrogênio/análise , Fatores de Crescimento TransformadoresRESUMO
Lack of selectivity in the killing of tumor and normal cells is a major obstacle in cancer therapy. By inhibiting normal but not autonomous cell growth, we exploited the differences in cell cycle regulation to achieve a selective protection of nonautonomous cells against paclitaxel and other microtubule-active drugs. Tubulin polymerization, a primary effect of paclitaxel, can be dissociated from Bcl-2 phosphorylation and cytotoxicity in HL-60 cells. Growth arrest prevented paclitaxel-induced Bcl-2 phosphorylation and apoptosis without affecting paclitaxel-induced tubulin polymerization. We abrogated the effects of paclitaxel on MCF-10A immortalized breast cells, while preserving its effects on MCF-7 cancer cells. Unlike MCF-7 cells, MCF-10A cells were arrested by epidermal growth factor withdrawal, precluding paclitaxel-induced Bcl-2 phosphorylation. Furthermore, the inhibition of the epidermal growth factor receptor kinase with low doses of AG1478 arrested growth of MCF-10A but not MCF-7 cells. Pretreatment with AG1478 did not affect paclitaxel-induced Bcl-2/Raf-1 phosphorylation in MCF-7 but abrogated such phosphorylation in MCF-10A. Exploitation of growth factor dependency may allow the protection of normal cells from microtubule-active drugs.
Assuntos
Ciclo Celular/fisiologia , Microtúbulos/efeitos dos fármacos , Paclitaxel/toxicidade , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Tirfostinas/toxicidade , Apoptose/efeitos dos fármacos , Neoplasias da Mama , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Inibidores Enzimáticos/toxicidade , Fator de Crescimento Epidérmico/farmacologia , Feminino , Células HL-60 , Humanos , Mitose/efeitos dos fármacos , Fosforilação , Quinazolinas , Acetato de Tetradecanoilforbol/farmacologia , Tubulina (Proteína)/efeitos dos fármacos , Tubulina (Proteína)/metabolismo , Células Tumorais CultivadasRESUMO
Reports of multiple distinct mitoxantrone-resistant sublines without overexpression of P-glycoprotein or the multidrug-resistance associated protein have raised the possibility of the existence of another major transporter conferring drug resistance. In the present study, a cDNA library from mitoxantrone-resistant S1-M1-80 human colon carcinoma cells was screened by differential hybridization. Two cDNAs of different lengths were isolated and designated MXR1 and MXR2. Sequencing revealed a high degree of homology for the cDNAs with Expressed Sequence Tag sequences previously identified as belonging to an ATP binding cassette transporter. Homology to the Drosophila white gene and its homologues was found for the predicted amino acid sequence. Using either cDNA as a probe in a Northern analysis demonstrated high levels of expression in the S1-M1-80 cells and in the human breast cancer subline, MCF-7 AdVp3000. Levels were lower in earlier steps of selection, and in partial revertants. The gene is amplified 10-12-fold in the MCF-7 AdVp3000 cells, but not in the S1-M1-80 cells These studies are consistent with the identification of a new ATP binding cassette transporter, which is overexpressed in mitoxantrone-resistant cells.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Antineoplásicos/farmacologia , DNA Complementar/genética , Resistencia a Medicamentos Antineoplásicos/genética , Mitoxantrona/farmacologia , Sequência de Bases , Clonagem Molecular , DNA Complementar/isolamento & purificação , Regulação Neoplásica da Expressão Gênica , Humanos , Dados de Sequência Molecular , Análise de Sequência de DNA , Células Tumorais CultivadasRESUMO
Transforming growth factors-beta (TGF beta) are a family of closely related, ubiquitously expressed growth factors with the common properties of induction of growth inhibition and expression of differentiation-related markers in epithelial cells. We investigated the role of TGF beta 1 in growth regulation of normal human mammary epithelial cells and in benzo(a)pyrene immortalized sublines further transformed by oncogenes in retroviral vectors. The normal cells were markedly growth inhibited by TGF beta 1, produced TGF beta in a latent form, and expressed TGF beta receptors. In the immortalized cells, both TGF beta-induced growth inhibition and TGF beta receptor binding were reduced. With the single oncogenes v-Ha-ras, v-mos, and SV40 T, growth sensitivity to TGF beta 1 increased, but TGF beta production or TGF beta receptor expression was not altered. Transformation to full malignancy by both SV40 T and v-Ha-ras led to escape from growth inhibition by TGF beta under anchorage-independent, but not anchorage-dependent, conditions without affecting TGF beta production or receptor characteristics. Thus, modulation of TGF beta growth responsiveness in these normal and oncogene transformed human mammary epithelial cells apparently occurs at a level distal to TGF beta receptor binding and is not solely correlated to expression of transforming oncogenes. Further, modulation of TGF beta production is not an indicator of malignant transformation in this system.
Assuntos
Transformação Celular Neoplásica , Oncogenes , Fatores de Crescimento Transformadores/biossíntese , Benzo(a)pireno/farmacologia , Mama , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Células Epiteliais , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Feminino , Expressão Gênica , Humanos , Cinética , RNA Mensageiro/análise , RNA Mensageiro/genética , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/metabolismo , Receptores de Fatores de Crescimento Transformadores beta , Transcrição Gênica , Fatores de Crescimento Transformadores/genética , Fatores de Crescimento Transformadores/farmacologiaRESUMO
A disparity was noted in the transport of rhodamine 123 among nine MXR/BCRP/ABCP-overexpressing cells studied; all demonstrated mitoxantrone transport, whereas only two effluxed rhodamine 123. When the MXR/BCRP/ABCP gene was sequenced in the cell lines studied, differences were noted at amino acid 482, predicted to be at the start of the third transmembrane domain. Sequencing genomic DNA revealed wild-type MXR/BCRP/ABCP to have an arginine at position 482. Cells having a threonine or glycine at position 482 were able to efflux rhodamine 123, whereas cells having an arginine were not. A vaccinia virus expression system confirmed that rhodamine as well as doxorubicin efflux is observed with R482T or R482G but not with the wild-type R482; all three MXR/BCRP/ABCP forms transported mitoxantrone. Cross-resistance studies suggest that, compared with wild-type MXR/BCRP/ABCP, cells having an R482T mutation have higher anthracycline resistance, whereas an R482G mutation seems to confer relatively less resistance to SN-38 and topotecan. These results suggest that amino acid 482 has a crucial role in MXR/BCRP/ABCP function and that mutation of a single amino acid residue significantly changes substrate specificity, thus altering the drug resistance phenotype.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Mutação , Proteínas de Neoplasias , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/biossíntese , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Camptotecina/análogos & derivados , Camptotecina/farmacologia , Doxorrubicina/farmacologia , Resistência a Múltiplos Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos , Genes MDR/genética , Vetores Genéticos/genética , Células HeLa , Humanos , Irinotecano , Mitoxantrona/farmacocinética , Mitoxantrona/farmacologia , RNA Mensageiro/genética , Rodamina 123/farmacocinética , Especificidade por Substrato , Topotecan/farmacologia , Transfecção , Células Tumorais Cultivadas , Vaccinia virus/genéticaRESUMO
The mitoxantrone resistance (MXR) gene encodes a recently characterized ATP-binding cassette half-transporter that confers multidrug resistance. We studied resistance to the camptothecins in two sublines expressing high levels of MXR: S1-M1-80 cells derived from parental S1 colon cancer cells and MCF-7 AdVp3,000 isolated from parental MCF-7 breast cancer cells. Both cell lines were 400- to 1,000-fold more resistant to topotecan, 9-amino-20(S)-camptothecin, and the active metabolite of irinotecan, 7-ethyl-10-hydroxycamptothecin (SN-38), than their parental cell lines. The cell lines demonstrated much less resistance to camptothecin and to several camptothecin analogues. Reduced accumulation and energy-dependent efflux of topotecan was demonstrated by confocal microscopy. A significant reduction in cleavable complexes in the resistant cells could be observed after SN-38 treatment but not after camptothecin treatment. In addition to topotecan and SN-38, MXR-overexpressing cells are highly resistant to mitoxantrone and epirubicin. Because these compounds are susceptible to glucuronidation, we examined UDP-glucurono-syltransferase (UGT) activity in parental and resistant cells by TLC. Glucuronides were found at equal levels in both parental and resistant colon cancer cell lines for epirubicin and to a lesser extent for SN-38 and mitoxantrone. Low levels of glucuronidation could also be detected in the resistant breast cancer cells. These results were confirmed by analysis of the UGT1A family mRNAs. We thus conclude that colon and breast cancer cells have a capacity for glucuronidation that could contribute to intrinsic drug resistance in colon cancer cells and may be acquired in breast cancer cells. The lack of selection for higher levels of UGT capacity in the colon cells suggests that high levels of expression of MXR alone are sufficient to confer resistance to the camptothecins.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/toxicidade , Camptotecina/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Resistência a Múltiplos Medicamentos , Glucuronídeos/metabolismo , Glucuronosiltransferase/metabolismo , Mitoxantrona/toxicidade , Antineoplásicos Fitogênicos/toxicidade , Neoplasias da Mama , Camptotecina/análogos & derivados , Dano ao DNA , Epirubicina/toxicidade , Feminino , Humanos , Irinotecano , Microscopia Confocal , Reação em Cadeia da Polimerase , Topotecan/toxicidade , Células Tumorais CultivadasRESUMO
Brain-derived neurotrophic factor (BDNF) and its receptors are necessary for the survival and development of many neuronal cells. Because BDNF and TrkB are expressed in many poor-prognosis neuroblastoma (NB) tumors, we evaluated the role of BDNF in affecting sensitivity to chemotherapeutic agents. We investigated the effects of activation of the BDNF-TrkB signal transduction pathway in two NB cell lines, 15N and SY5Y. 15N cells lack the high-affinity receptor p145TrkB and express BDNF; 15N cells were used along with 15N-TrkB cells, a subline transfected with a TrkB expression vector. In cytotoxicity assays, 15N-TrkB cells were consistently 1.4-2 fold more resistant to vinblastine than 15N cells. Drug accumulation assays showed a 50% reduction in[3H]vinblastine accumulation in 15N-TrkB cells compared with control 15N cells. Addition of 30 ng/ml BDNF resulted in a reduction to 46% of control in 15N cells and a reduction to 28% of control in 15N-TrkB cells. SY5Y cells were chosen as a second model because they lack both endogenous BDNF and TrkB expression. p145TrkB expression is induced by 1 nM retinoic acid. Vinblastine accumulation was not significantly affected by 1 nM retinoic acid in SY5Y cells. Addition of 30 ng/ml BDNF decreased [3H]vinblastine accumulation to 58% of control in SY5Y cells and decreased [3H]vinblastine accumulation to 62% of control in TrkB-expressing SY5Y cells. Although an increase in BDNF expression in seen in multidrug-resistant sublines of SY5Y and BE(2)-C NB cells, the protective effect of BDNF in vinblastine toxicity may be unrelated to mdr-1, because the activity of other agents transported by P-glycoprotein was not affected. There was no increase in mdr-1 expression in 1 nM RA SY5Y cells and 15N-TrkB cells, as assessed by Northern blot analysis. In addition to the effects of BDNF on vinblastine cytotoxicity and accumulation, there was an inhibition in the ability of vinblastine to depolymerize tubulin in BDNF-treated cells. Thus, BDNF and TrkB may partially rescue NB cells from vinblastine toxicity and thereby may contribute to a more chemoresistant phenotype.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Proteínas do Tecido Nervoso/farmacologia , Neuroblastoma/tratamento farmacológico , Vimblastina/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/análise , Fator Neurotrófico Derivado do Encéfalo , Resistência a Medicamentos , Humanos , Proteínas do Tecido Nervoso/genética , RNA Mensageiro/análise , Tretinoína/farmacologia , Tubulina (Proteína)/metabolismo , Células Tumorais Cultivadas , Vimblastina/farmacocinéticaRESUMO
Multidrug resistance (MDR), especially that associated with overexpression of MDR1 and its product, P-glycoprotein (Pgp), is thought to play a role in the outcome of therapy for some human tumors; however, a consensus conclusion has been difficult to reach, owing to the variable results published by different laboratories. Many factors appear to influence the detection of Pgp in clinical specimens, including its low and heterogeneous expression; conflicting definitions of detection end points; differences in methods of sample preparation, fixation, and analysis; use of immunological reagents with variable Pgp specificity and avidity and with different recognition epitopes; use of secondary reagents and chromogens; and differences in clinical end points. Also, mechanisms other than Pgp overexpression may contribute to clinical MDR. The combined effect of these factors is clearly important, especially among tumors with low expression of Pgp. Thus, a workshop was organized in Memphis, Tennessee, to promote the standardization of approaches to MDR1 and Pgp detection in clinical specimens. The 15 North American and European institutions that agreed to participate conducted three preworkshop trials with well-characterized MDR myeloma and carcinoma cell lines that expressed increasing amounts of Pgp. The intent was to establish standard materials and methods for a fourth trial, assays of Pgp and MDR1 in clinical specimens. The general conclusions emerging from these efforts led to a number of recommendations for future studies: (a) although detection of Pgp and MDR1 is at present likely to be more reliable in leukemias and lymphomas than in solid tumors, accurate measurement of low levels of Pgp expression under most conditions remains an elusive goal; (b) tissue-specific controls, antibody controls, and standardized MDR cell lines are essential for calibrating any detection method and for subsequent analyses of clinical samples; (c) use of two or more vendor-standardized anti-Pgp antibody reagents that recognize different epitopes improves the reliability of immunological detection of Pgp; (d) sample fixation and antigen preservation must be carefully controlled; (e) multiparameter analysis is useful in clinical assays of MDR1/Pgp expression; (f) immunostaining data are best reported as staining intensity and the percentage of positive cells; and (g) arbitrary minimal cutoff points for analysis compromise the reliability of conclusions. The recommendations made by workshop participants should enhance the quality of research on the role of Pgp in clinical MDR development and provide a paradigm for investigations of other drug resistance-associated proteins.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/análise , Resistência a Múltiplos Medicamentos , Neoplasias/química , Neoplasias/tratamento farmacológico , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/farmacologia , Estudos de Avaliação como Assunto , Humanos , Imuno-Histoquímica , Células KB , Células Tumorais CultivadasRESUMO
We examined transforming growth factor (TGF) alpha, epidermal growth factor (EGF) and EGF receptor (EGFR) expression and signaling in three drug resistant MCF-7 human breast cancer sublines and asked whether these pathways contribute to the drug resistance phenotype. In the resistant sublines, upregulation of both TGFalpha and EGFR mRNA was observed. In an apparent contrast with upregulated growth factor and receptor gene expression, the drug resistant sublines displayed a reduced growth rate. Defects in the EGFR signaling pathway cascade were found in all examined drug resistant sublines, including altered EGF-induced Shc, Raf-1, or mitogen-activated protein kinase phosphorylation. Induction of c-fos mRNA expression by EGF was impaired in the sublines compared to parental MCF-7 cells. In contrast, the induction of the stress-activated protein kinase activity was similar in both parental and drug resistant cells. Evaluating the link between the reduced growth rate and drug resistance, serum starvation experiments were performed. These studies demonstrated that a reduced proliferative activity resulted in a marked reduction in sensitivity to cytotoxic agents in the parental MCF-7 cells. We propose that the altered EGFR levels frequently observed in drug resistant breast cancer cells are associated with perturbations in the signaling pathway that mediate a reduced proliferative rate and thereby contribute to drug resistance.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Fator de Crescimento Transformador alfa/metabolismo , Anisomicina , Antineoplásicos/farmacologia , Neoplasias da Mama , Divisão Celular , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Doxorrubicina/farmacologia , Resistência a Medicamentos , Receptores ErbB/efeitos dos fármacos , Humanos , Immunoblotting , Proteína Quinase 9 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Paclitaxel/farmacologia , Fenótipo , Testes de Precipitina , Proteínas Proto-Oncogênicas c-fos/genética , RNA Mensageiro/análise , Transdução de Sinais , Células Tumorais CultivadasRESUMO
The fluorescent compounds rhodamine 123, LysoTracker Green DMD-26, mitoxantrone, and BODIPY-prazosin were used with the antagonist fumitremorgin C (FTC) in order to develop functional assays for the half-transporter, MXR/BCRP/ABCP1. A measure of FTC-inhibitable efflux was generated for each compound in a series of MXR-overexpressing drug-selected cell lines and in ten unselected cell lines which were used to determine if the four fluorescent compounds were sensitive enough to detect the low MXR levels found in drug-sensitive cell lines. FTC-inhibitable efflux of mitoxantrone and prazosin was found in four of the ten cell lines, SF295, KM12, NCI-H460, and A549, and low but detectable levels of MXR mRNA were also observed by Northern analysis in these cells. FTC-inhibitable mitoxantrone and prazosin efflux in both selected and unselected cell lines was found to correlate well with MXR levels as determined by Northern blotting, r(2)=0.89 and r(2)=0.70 respectively. In contrast, rhodamine and LysoTracker were not able to reliably detect MXR. Cytotoxicity assays performed on two of the four unselected cell lines confirmed increased sensitivity to mitoxantrone in the presence of FTC. FTC was found to be a specific inhibitor of MXR, with half-maximal inhibition of MXR-associated ATPase activity at 1 microM FTC. Short term selections of the SF295, KM12, NCI-H460 and A549 cell lines in mitoxantrone resulted in a small but measurable increase in MXR by both Northern blot and functional assay. These studies show that flow cytometric measurement of FTC-inhibitable mitoxantrone or prazosin efflux is a sensitive and specific method for measuring the function of the MXR half-transporter in both selected and unselected cell lines.