RESUMEN
BACKGROUND: Local recurrence of rectal cancer after curative resection remains a difficult clinical problem. The aim of this study was to elucidate prognostic risk factors after resection of recurrent cancer. METHODS: Between January 1983 and December 1999, 83 patients with locally recurrent rectal cancer were studied retrospectively for survival benefit by re-resection. Sixty patients underwent resection for recurrent cancer, including total pelvic exenteration in 30 patients and sacrectomy in 23 patients. The extent of locally recurrent tumour was classified by the pattern of pelvic invasion as follows: localized, sacral invasion and lateral invasion. RESULTS: Multivariate analysis showed that the pattern of pelvic invasion was a significant prognostic factor which independently influenced survival after resection of recurrent cancer (P < 0.001). The 5-year survival rates were 38 per cent in the localized type (n = 27), 10 per cent in the sacral invasive type (n = 16) and zero in the lateral invasive type (n = 17). CONCLUSION: Resection for locally recurrent rectal cancer is potentially curative in patients with localized or sacral invasive patterns of recurrence. Alternatives should be explored in patients with recurrence involving the lateral pelvic wall.
Asunto(s)
Recurrencia Local de Neoplasia/cirugía , Neoplasias Pélvicas/patología , Neoplasias del Recto/cirugía , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Análisis Multivariante , Invasividad Neoplásica , Recurrencia Local de Neoplasia/patología , Exenteración Pélvica/métodos , Pronóstico , Modelos de Riesgos Proporcionales , Neoplasias del Recto/patología , Estudios Retrospectivos , Factores de Riesgo , Análisis de Supervivencia , Factores de TiempoRESUMEN
Thyroid carcinoma accounts for the majority of deaths from endocrine cancers. Although effective therapies exist for well differentiated tumors, the treatment options for poorly differentiated and anaplastic tumors are much less effective. In the present study we demonstrate that the thyroglobulin (Tg) promoter can be used to direct specific expression of either luciferase or thymidine kinase in thyroid cancer cells. Furthermore, using a putative enhancer element for the Tg gene, the activity of the Tg promoter in and its specificity for thyroid cells were enhanced. In transient transfectants or in stably transfected thyroid carcinoma cells, treatment with the histone deacetylase inhibitors, depsipeptide (FR9012228) and sodium butyrate, alone or in combination with 8-bromo-cAMP, resulted in further enhancement. In experiments in which the herpes simplex virus thymidine kinase (HSV-TK) gene was driven by the Tg promoter and the putative enhancer, HSV-TK expression and ganciclovir sensitivity were augmented. Similar results were obtained in two cell lines derived from a follicular thyroid carcinoma and in two anaplastic thyroid carcinoma cell lines. In summary, we report the construction of a suicide HSV-TK vector with preferential toxicity for thyroid cells. The results in anaplastic thyroid carcinoma cells suggest that it may be of use in the full spectrum of thyroid malignancies.
Asunto(s)
Antibacterianos/toxicidad , Antibióticos Antineoplásicos/toxicidad , AMP Cíclico/fisiología , Depsipéptidos , Terapia Genética/métodos , Vectores Genéticos , Inhibidores de Histona Desacetilasas , Péptidos Cíclicos , Regiones Promotoras Genéticas , Tiroglobulina/genética , Neoplasias de la Tiroides/tratamiento farmacológico , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Adenocarcinoma Folicular/tratamiento farmacológico , Adenocarcinoma Folicular/patología , Butiratos/farmacología , Carcinoma/tratamiento farmacológico , Carcinoma/patología , Elementos de Facilitación Genéticos , Genes Reporteros , Humanos , Luciferasas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Simplexvirus/genética , Timidina Quinasa/genética , Neoplasias de la Tiroides/patología , Transfección/métodos , Células Tumorales CultivadasRESUMEN
One of the structural characteristics of a neuropeptide nociceptin is the existence of Arg-Lys (RK) residues at positions 8-9 and 12-13; both RKs have been suggested to bind to the acidic amino acid cluster in the second extracellular loop of the seven transmembrane domain receptor ORL1. With a design strategy of attempting to obtain an analog that binds more strongly to the receptor's acidic cluster, we synthesized a series of nociceptin analogs in which the RK dipeptide unit was placed at positions 6-7, 10-11, or 14-15 adjacent to the parent RKs. Among these nociceptin analogs containing the RK triple repeat, [Arg-Lys(6-7)]- and [Arg-Lys(10-11)]nociceptins exhibited weak activities (6-9 and 60-90% of nociceptin, respectively) both in the receptor binding assay and in the [(35)S]GTPgammaS binding functional assay. In contrast, [Arg-Lys(14-15)]nociceptin was found to be very potent in both assays (3-fold in binding and 17-fold in GTPgammaS functional assay). [Arg-Lys(14-15)]nociceptin was the first peptide analog found to be stronger than the parent nociceptin, and structure-activity studies have suggested that the incorporated Arg-Lys(14-15) interacts with either the receptor acidic amino acid cluster or the receptor aromatic amino acid residues.
Asunto(s)
Arginina/química , Lisina/química , Péptidos Opioides/química , Péptidos Opioides/farmacología , Secuencias Repetitivas de Aminoácido , Secuencia de Aminoácidos , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Datos de Secuencia Molecular , Péptidos Opioides/síntesis química , Péptidos Opioides/metabolismo , Unión Proteica , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , NociceptinaRESUMEN
In accordance with detection of a few phospholipase A2 (PLA2) isozyme genes by Southern blot analysis, only two cDNAs, named NnkPLA-I , and NnkPLA-II, encoding group I PLA2s, NnkPLA-I and NnkPLA-II, respectively, were isolated from the venom gland cDNA library of Elapinae Naja naja kaouthia of Malaysia. NnkPLA-I and NnkPLA-II showed four amino acid substitutions, all of which were brought about by single nucleotide substitution. No existence of clones encoding CM-II and CM-III, PLA2 isozymes which had been isolated from the venom of N. naja kaouthia of Thailand, in Malaysian N. naja kaouthia venom gland cDNA library was verified by dot blot hybridization analysis with particular probes. NnkPLA-I and NnkPLA-II differed from CM-II and CM-III with four and two amino acid substitutions, respectively, suggesting that their molecular evolution is regional. The comparison of NnkPLA-I, NnkPLA-II and cDNAs encoding other group I snake venom gland PLA2s indicated that the 5'- and 3'-untranslated regions are more conserved than the mature protein-coding region and that the number of nucleotide substitutions per nonsynonymous site is almost equal to that per synonymous site in the protein-coding region, suggesting that accelerated evolution has occurred in group I venom gland PLA2s possibly to acquire new physiological functions.
Asunto(s)
Evolución Molecular , Glándulas Exocrinas/química , Fosfolipasas A/química , Venenos de Serpiente/química , Secuencia de Aminoácidos , Southern Blotting , Clonación Molecular , Fragmentación del ADN , Biblioteca de Genes , Immunoblotting , Hibridación in Situ , Isoenzimas/química , Isoenzimas/genética , Datos de Secuencia Molecular , Fosfolipasas A/genética , Fosfolipasas A2 , Venenos de Serpiente/genéticaRESUMEN
In preliminary studies we demonstrated that the CYP11B1 (11beta-hydroxylase) promoter could direct specific expression of a suicide gene in adrenocortical cancer cells, providing a potentially specific therapeutic option for adrenocortical cancer. In this present study we describe our attempts to enhance the activity of the CYP11B1 promoter while maintaining its specificity for adrenal cells. Using a putative enhancer element from the cholesterol side-chain cleavage (P450scc) gene, the activity of the CYP11B1 promoter in and its specificity for adrenocortical cells were enhanced. Treatment with 8-bromo-cAMP or forskolin resulted in further enhancement. In stably transfected Y-1 cells, in which the herpes simplex virus thymidine kinase (HSV-TK) gene was driven by the CYP11B1 promoter with the P450scc enhancer element, HSV-TK expression and ganciclovir sensitivity were augmented by treatment with 8-bromo-cAMP, forskolin, and ACTH. In summary, we report the construction of a suicide HSV-TK vector with preferential toxicity to adrenocortical cells. We propose that a similar strategy using differentiating agents may be useful in the gene therapy of tumors with unique differentiated properties, including those arising from other endocrine organs.
Asunto(s)
Corteza Suprarrenal/fisiología , AMP Cíclico/metabolismo , Terapia Genética , Vectores Genéticos , Corteza Suprarrenal/citología , Corteza Suprarrenal/metabolismo , Western Blotting , Supervivencia Celular/genética , Clonación Molecular , Elementos de Facilitación Genéticos , Humanos , Luciferasas/genética , Plásmidos/genética , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Esteroide 11-beta-Hidroxilasa/genética , Esteroide 11-beta-Hidroxilasa/metabolismo , Transfección/genética , Células Tumorales CultivadasRESUMEN
A pair of 35 kDa polypeptides (TAO1/TAO2) are expressed in more than 90% of all primary lung adenocarcinomas but not in other major malignancies. Mass spectrometry of tryptic peptides showed that TAO1/TAO2 is identical to napsin A, a recently described member of the aspartic proteinase family. The site of processing of pronapsin A to the mature form was located. Napsin expression was detected in human lung adenocarcinoma tumors, compatible with the marker nature of TAO1/TAO2 in the diagnosis of primary lung adenocarcinoma. This is important since identification of markers which can distinguish primary lung adenocarcinomas from distant metastases is desirable. Northern blot analysis showed expression of napsin also in normal lung and kidney tissue, and in situ hybridization showed expression in type II alveolar cells of the lung. This protease is concluded to have a specific functional role in the normal alveolar epithelium and is a candidate protease for the proteolytic processing of surfactant precursors.
Asunto(s)
Adenocarcinoma/enzimología , Ácido Aspártico Endopeptidasas/biosíntesis , Riñón/enzimología , Neoplasias Pulmonares/enzimología , Pulmón/enzimología , Proteínas Asociadas a Surfactante Pulmonar , Secuencia de Aminoácidos , Apoproteínas/metabolismo , Ácido Aspártico Endopeptidasas/genética , Clonación Molecular , ADN Complementario/análisis , Humanos , Datos de Secuencia Molecular , Surfactantes Pulmonares/metabolismo , ARN Mensajero/biosíntesis , Distribución TisularRESUMEN
Raf-1 activation and Bcl-2 hyperphosphorylation following treatment with paclitaxel (Taxol) or other microtubule-active drugs is associated with mitotic arrest. Here we show that microtubule-active drugs do not activate the mitogen-activated protein kinase (MAPK) pathway in leukemia cells. PD98059, a MEK inhibitor, and SB202190, a p38 MAP kinase inhibitor, do not abrogate Bcl-2 phosphorylation nor apoptosis. Simultaneously with PARP cleavage, paclitaxel induces cleavage of Bcl-2 protein yielding a potentially pro-apoptotic 22 kDa product. In comparison, the stimulation of Raf-1 by phorbol ester (TPA) activates the MAPK pathway, causes MAPK-dependent p21WAF1/CIP1 induction, Rb dephosphorylation and growth arrest without Bcl-2 phosphorylation or apoptosis. Like TPA, cAMP induces p21WAF1/CIP1 but does not cause Bcl-2 phosphorylation. MEKK1 and Ras, upstream activators of JNK and ERK MAPK, also fail to induce Bcl-2 hyperphosphorylation. Although Lck tyrosine kinase has been recently implicated in Raf-1 activation during mitotic arrest, microtubule-active drugs induce Raf-1/Bcl-2 hyperphosphorylation and apoptosis in a Lck-deficient Jurkat cells. Therefore, microtubule-active drugs induce apoptosis which is associated with Raf-1 and Bcl-2 phosphorylation and Bcl-2 cleavage but is independent of the MAPK pathway. In contrast, TPA-activated MAPK pathway causes p21WAF1/CIP1-dependent growth arrest without apoptosis.
Asunto(s)
Antineoplásicos Fitogénicos/uso terapéutico , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Leucemia Mieloide/tratamiento farmacológico , Quinasa 1 de Quinasa de Quinasa MAP , Microtúbulos/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Apoptosis/efectos de los fármacos , Genes ras , Humanos , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patología , Paclitaxel/uso terapéutico , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Acetato de Tetradecanoilforbol/uso terapéutico , Células Tumorales CultivadasRESUMEN
A ligand containing an SNpys group, i.e. 3-nitro-2-pyridinesulfenyl linked to a mercapto (or thiol) group, can bind covalently to a free mercapto group to form a disulfide bond via the thiol-disulfide exchange reaction. This SNpys chemistry has been successfully applied to the discriminative affinity labeling of mu and delta opioid receptors with SNpys-containing enkephalins [Yasunaga, T. et al. (1996) J. Biochem. 120, 459-465]. In order to explore the mercapto groups conserved at or near the ligand binding sites of three opioid receptor subtypes, we synthesized two Cys(Npys)-containing analogs of dynorphin A, namely, [D-Ala2, Cys(Npys)8]dynorphin A-(1-9) amide (1) and [D-Ala2, Cys(Npys)12]dynorphin A-(1-13) amide (2). When rat (mu and delta) or guinea pig (kappa) brain membranes were incubated with these Cys(Npys)-containing dynorphin A analogs and then assayed for inhibition of the binding of DAGO (mu), deltorphin II (delta), and U-69593 (kappa), the number of receptors decreased sharply, depending upon the concentrations of these Cys(Npys)-containing dynorphin A analogs. It was found that dynorphin A analogs 1 and 2 effectively label mu receptors (EC50 = 27-33 nM), but also label delta receptors fairly well (160-180 nM). However, for kappa receptors they showed drastically different potencies as to affinity labeling; i.e., EC50 = 210 nM for analog 1, but 10,000 nM for analog 2. Analog 2 labeled kappa receptors about 50 times more weakly than analog 1. These results suggested that dynorphin A analog 1 labels the Cys residues conserved in mu, delta, and kappa receptors, whereas analog 2 only labels the Cys residues conserved in mu and delta receptors.
Asunto(s)
Marcadores de Afinidad/química , Dinorfinas/química , Dinorfinas/metabolismo , Receptores Opioides/química , Receptores Opioides/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Encéfalo/citología , Membrana Celular/metabolismo , Cisteína , Disulfuros/química , Cobayas , Datos de Secuencia Molecular , Ratas , Homología de Secuencia de Aminoácido , Compuestos de Sulfhidrilo/químicaRESUMEN
Six cases of carcinosarcoma of the esophagus were studied clinicopathologically, immunohistochemically and with DNA flow cytometry. Transitional areas with morphology intermediate between carcinoma and sarcoma were found microscopically in all cases. Immunohistochemically, the carcinomatous areas contained keratin-positive cell components in all cases while vimentin-positive cells were found in sarcomatous areas in 5 cases. By DNA flow analysis of microdissection, the sarcomatous components of the tumors showed an aneuploid pattern with one exception, in contrast the carcinomatous components were diploid in all cases. In these few cases, PCNA, S-phase fraction and the mitotic rate were extremely high, apparently indicating a correlation with malignant behavior. Accordingly, examination by immunohistochemistry and DNA ploidy is useful for the analysis of biological properties in the so-called carcinosarcoma of the esophagus.
Asunto(s)
Carcinosarcoma/patología , Neoplasias Esofágicas/patología , Anciano , Aneuploidia , Carcinosarcoma/química , Carcinosarcoma/genética , ADN de Neoplasias/análisis , Neoplasias Esofágicas/química , Neoplasias Esofágicas/genética , Citometría de Flujo , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana EdadRESUMEN
Mutations of the p53 gene are the most common abnormalities in human cancer. In contrast to mutant p53, wild-type (wt) p53 protein is present at low levels due to rapid degradation by proteasome. We demonstrated that wt p53 protein stabilization following DNA damage or proteasome inhibition did not abolish the wild-type conformation. DNA damage did not cause accumulation of ubiquitinated forms of wt p53, suggesting abrogation of ubiquitination. Consistent with this, the E6 oncoprotein which targets p53 for ubiquitination abolished stabilization of p53 protein by DNA-damaging drugs but not by proteasome inhibitors. In contrast to the effects on wt p53, inhibitors of proteolysis downregulated mutant p53. Regulation of p53 levels can be explained by a feedback mechanism where wt p53 transcriptionally induces "sensor" proteins (Mdm-2, as an example) and these, in turn, target p53 for degradation. Like p53, Mdm-2 is degraded by proteasome. Therefore, inhibition of proteasome caused accumulation of Mdm-2, leading to degradation of mutant p53 by the remaining proteolytic activity of the cell. We propose that inhibition of transcription should increase wt p53 protein due to inhibition of Mdm-2 synthesis. An inhibitor of transcription, alpha-amanitin, dramatically induced wt p53 protein, whereas Mdm-2 protein was downregulated. Moreover, alpha-amanitin increased p53 protein levels in E6-transfected cells. Although inhibitors of transcription, such as actinomycin D, also damage DNA, reduction of Mdm-2 or other putative "sensor" proteins may contribute to their p53-stabilizing activity. Similarly, antimetabolites augment accumulation of wt p53 due to interference with RNA synthesis.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Daño del ADN/efectos de los fármacos , Complejos Multienzimáticos/metabolismo , Transcripción Genética/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , ADN de Neoplasias/efectos de los fármacos , ADN de Neoplasias/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Retroalimentación/efectos de los fármacos , Retroalimentación/fisiología , Femenino , Humanos , Masculino , Mutación/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal , Conformación Proteica/efectos de los fármacos , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The mechanism of multidrug resistance protein (MRP)-mediated multidrug resistance (MDR) is still unclear. MRP reportedly transports some GSH conjugates. Recently, we demonstrated that a pyridine analog, 2-[4-(diphenylmethyl)-1-piperazinyl]ethyl 5-(trans-4,6-dimethyl-1,3,2-dioxaphosphorinan-2-yl)-2,6-dimethyl-4 -(3-nitrophenyl)-3-pyridinecarboxylate P-oxide (PAK-104P), that reversed P-glycoprotein (P-gp)-mediated MDR directly interacted with MRP and completely reversed the vincristine (VCR) resistance in MRP-mediated MDR C-A120 cells. We investigated the reversing effect of PAK-104P in C-A120 cells in combination with buthionine sulfoximine (BSO), another MDR-reversing agent with a different reversing mechanism. In immunoblots, MRP was overexpressed in C-A120 cells. The level of ATP-dependent [3H]VCR uptake was high in membrane vesicles from KB-C2 cells, but low in those from C-A120 and parental KB-3-1 cells. The sensitivity to VCR of C-A120 cells, but not of KB-C2 cells, was considerably increased by 100 microM BSO. VCR accumulation in C-A120 cells, but not in KB-C2 cells, was also enhanced by BSO. BSO did not inhibit ATP-dependent [3H]LTC4 uptake in C-A120 vesicles. The combination of BSO with PAK-104P at their low concentrations resulted in complete reversal of VCR resistance in C-A120 cells. These findings suggested that BSO might not directly interact with MRP and reversed resistance in MRP-mediated MDR cells by reducing the intracellular glutathione (GSH) level that was needed for the transport of drugs by MRP and suggested a role for the combination of drug resistance-modulating agents with different reversing mechanisms in the reversal of MRP-mediated MDR.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Antineoplásicos Fitogénicos/farmacología , Butionina Sulfoximina/farmacología , Óxidos P-Cíclicos/farmacología , Ácidos Nicotínicos/farmacología , Vincristina/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Antimetabolitos Antineoplásicos/farmacología , Resistencia a Antineoplásicos/fisiología , Sinergismo Farmacológico , Inhibidores Enzimáticos/farmacología , Glutamato-Cisteína Ligasa/antagonistas & inhibidores , Humanos , Células Tumorales CultivadasRESUMEN
A part of the 3'-flanking region of BP-II gene, which is one of Trimeresurus flavoviridis venom gland phospholopase A2 (PLA2) isozyme genes, has a region homologous to avian chicken repeat 1 (CR1)-element. In the present study, ten CR1-like elements were further identified in T. gramineus venom gland PLA2 isozyme genes, T. flavoviridis PLA2 inhibitor (PLI) genes, and T. flavoviridis and T. gramineus TATA-box binding protein (TBP) genes. Southern blot analysis using a probe for CR1 showed that Crotalinae snake genomes contain a number of CR1-like elements.
Asunto(s)
Venenos de Crotálidos/genética , Fosfolipasas A/genética , Secuencias Repetitivas de Ácidos Nucleicos , Trimeresurus/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Venenos de Crotálidos/química , Datos de Secuencia Molecular , Fosfolipasas A/química , Fosfolipasas A2 , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
Four different stereoisomers of cyclopropylphenylalanine (inverted delta Phe) were incorporated into [D-Ala2,Leu5]enkephalin at the position 4. These conformationally restricted enkephalin analogs were evaluated for their binding characteristics to mu and delta opioid receptors in rat brain. A striking finding is that the E-(2R,3S)-isomer binds to a novel class of delta receptors and discriminates this receptor from the ordinary delta receptor. This new type of delta receptor suspected to be a receptor which suppresses the thermal analgesia mediated through mu receptor. The Z-(2R,3R)-isomer was very potent with several times more enhanced affinity to delta receptors than to mu receptors, but could not differentiate the delta receptors. The Z-(2S,3S)-isomer was weak, and E-(2S,3R)-isomer was almost inactive.
Asunto(s)
Encéfalo/metabolismo , Ciclopropanos/química , Ciclopropanos/metabolismo , Fenilalanina/análogos & derivados , Receptores Opioides delta/metabolismo , Animales , Ciclopropanos/farmacología , Relación Dosis-Respuesta a Droga , Encefalinas/química , Encefalinas/metabolismo , Encefalinas/farmacología , Isomerismo , Fenilalanina/química , Fenilalanina/metabolismo , Fenilalanina/farmacología , Ratas , Receptores Opioides delta/efectos de los fármacos , Receptores Opioides mu/efectos de los fármacos , Receptores Opioides mu/metabolismo , Relación Estructura-ActividadRESUMEN
Three agents, verapamil, cepharanthine, and 2-[4-(diphenylmethyl)-1-piperazinyl]ethyl-5-(trans-4,6-dimethyl-1, 3,2-dioxaphosphorinan-2-yl)-2,6-dimethyl-4-(3-nitrophenyl)-3-py ridinecarboxylate P-oxide (PAK-104P), that reverse drug resistance in P-glycoprotein (P-Gp)-mediated multidrug-resistant cells were examined for their activity to reverse drug resistance in multidrug resistance-associated protein (MRP)-mediated multidrug-resistant C-A120 cells. Agents other than PAK-104P could not reverse the resistance to doxorubicin in C-A120 cells. PAK-104P moderately reversed the doxorubicin resistance. In contrast, PAK-104P almost completely reversed the resistance to vincristine (VCR) in C-A120 cells as well as in KB-8-5 cells, and other agents moderately reversed the VCR resistance in C-A120 cells. PAK-104P at 10 microM enhanced the accumulation of VCR in C-A120 cells to the level of that in KB-3-1 cells without the agent. PAK-104P competitively inhibited the ATP-dependent [3H]leukotriene C4 uptake in membrane vesicles isolated from C-A120 cells. These findings demonstrate that PAK-104P can completely reverse the resistance to VCR in both P-Gp- and MRP-mediated multidrug-resistant cells and that PAK-104P directly interacts with MRP and inhibits the transporting activity of MRP.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Óxidos P-Cíclicos/farmacología , Ácidos Nicotínicos/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Alcaloides/farmacología , Bencilisoquinolinas , Óxidos P-Cíclicos/química , ADN-Topoisomerasas de Tipo II/metabolismo , Doxorrubicina/farmacología , Resistencia a Medicamentos , Humanos , Leucotrieno C4/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Ácidos Nicotínicos/química , ARN Mensajero/metabolismo , Células Tumorales Cultivadas , Verapamilo/farmacología , Vincristina/farmacologíaRESUMEN
The intrinsic or acquired resistance of urothelial cancer to chemotherapy is one major obstacle to successful treatment. Generally, the expression level of P-glycoprotein in urothelial cancer is low, so we accordingly investigated the expression of multidrug resistance-associated protein (MRP). We examined the expression of MRP mRNA by means of slot-blotting samples of 11 renal pelvic and/or ureteral tumors, 33 bladder tumors, one lung metastasis from a ureter tumor, 7 non-cancerous urothelia from patients with transitional-cell carcinoma (TCC) and one urothelium from a patient with renal-cell carcinoma (RCC). We also estimated, by Southern blotting, whether or not the MRP gene was amplified in clinical specimens that overexpressed MRP mRNA. MRP was detected immunohistochemically using a polyclonal antibody against MRP. In all, 5 of 11 renal pelvic and/or ureter tumors (45.5%), 17 of 33 bladder tumors (51.5%) and 4 of 7 non-cancerous urothelia of TCC patients (57.1%) expressed more than 2-fold the MRP mRNA levels of drug-sensitive human KB cells. There was no significant difference in the MRP mRNA level between primary and recurrent tumors. Low-grade urothelial carcinomas (G1 and G2 TCCs) expressed significantly higher levels of MRP mRNA than the high-grade G3 TCC. The MRP gene was not amplified in urothelial carcinomas, irrespective of their expression levels of MRP mRNA. Immunohistochemically, MRP was located mainly on the plasma membrane, but also detected on the cytoplasm of cancer cells. MRP may be one mechanism responsible for intrinsic drug resistance in low-grade urothelial cancer.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/biosíntesis , Carcinoma de Células Transicionales/genética , Resistencia a Múltiples Medicamentos/genética , Neoplasias Urológicas/genética , Urotelio/patología , Transportadoras de Casetes de Unión a ATP/análisis , Transportadoras de Casetes de Unión a ATP/genética , Northern Blotting/métodos , Southern Blotting/métodos , Carcinoma de Células Transicionales/patología , Expresión Génica , Humanos , Inmunohistoquímica/métodos , Neoplasias Pulmonares/química , Neoplasias Pulmonares/secundario , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Hibridación de Ácido Nucleico/métodos , ARN Mensajero/análisis , ARN Neoplásico/química , Neoplasias Urológicas/química , Neoplasias Urológicas/patología , Urotelio/químicaRESUMEN
An active efflux pump for cis-diamminedichloroplatinum(II) (cisplatin) has been identified in cisplatin-resistant KCP-4 cells isolated from human epidermoid carcinoma KB-3-1 cells. The adenosine triphosphate(ATP)-dependent transport of leukotriene C4 (LTC4), an endogenous substrate for the glutathione S-conjugate export pump(GS-X pump), has been found in membrane vesicles prepared from KCP-4 cells. Multidrug resistance-associated protein (MRP) has also been identified as an ATP-dependent LTC4 transporter. To examine whether the GS-X pump expressed in KCP-4 cells in MRP, we investigated the expression of MRP in KCP-4 cells and compared the LTC4 transporting activity of GS-X pump expressed in KCP-4 cells with that of MRP. The level of MRP gene expression in KCP-4 cells was low and similar to that in KB-3-1 cells. MRP was not detected in membrane vesicles prepared from KB-3-1 and KCP-4 cells by immunoblot analysis. The ATP-dependent transport of LTC4 in KCP-4 and C-A120 vesicles showed saturable kinetics with an apparent Km of 0.18 microM and 0.25 microM, respectively. [3H]LTC4 transport in KCP-4 vesicles was more inhibited by 2,4-dinitrophenyl-S-glutathione(DNP-SG), bis-(glutathionato)-platinum(II) (GS-platinum) complex and glutathione disulfide(GS-SG) and less by LTD4 compared with that in C-A120 vesicles. The character of the LTC4 transporter expressed in KCP-4 vesicles is similar but not identical to that of MRP. Our results suggest that a glutathione S-conjugate export pump which is different from MRP exists in cisplatin-resistant KCP-4 cells.
Asunto(s)
Adenosina Trifosfato/metabolismo , Cisplatino/farmacología , Leucotrieno C4/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/genética , Secuencia de Aminoácidos , Transporte Biológico , Membrana Celular/metabolismo , Resistencia a Antineoplásicos , Glutatión/metabolismo , Humanos , Datos de Secuencia Molecular , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Concentración Osmolar , ARN Mensajero/genética , Tritio , Células Tumorales Cultivadas , Verapamilo/metabolismoRESUMEN
MRP has been identified as another multidrug-resistance (MDR) gene and may be involved in an alternative MDR mechanism in some solid tumors. We investigated the expression of MRP mRNA in multidrug-resistance KB sublines (KB-8-5, KB-C2, C-A40 and C-A120), human non-small-cell lung carcinomas (NSCLC), gastric and colorectal carcinomas, and compared it with that in drug-sensitive human KB cells. MRP gene expression was elevated in 8 of 9 (89%) squamous-cell carcinomas of the lung. Furthermore, MRP expression in 4 squamous-cell carcinomas (L13, 18, 19 and 20) was more than 3.6 times higher than in KB-3-1 cells, and the average MRP mRNA expression level of all squamous-cell carcinomas was significantly higher than that of adenocarcinoma of the lung and of colorectal and gastric carcinomas. These results suggested that the MRP is responsible, at least in part, for drug resistance in some squamous-cell carcinomas of the lung.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Neoplasias Colorrectales/metabolismo , Resistencia a Múltiples Medicamentos/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Gástricas/metabolismo , Transportadoras de Casetes de Unión a ATP/análisis , Anciano , Anciano de 80 o más Años , Secuencia de Aminoácidos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Colorrectales/tratamiento farmacológico , Femenino , Humanos , Inmunohistoquímica , Neoplasias Pulmonares/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , ARN Mensajero/análisis , Neoplasias Gástricas/tratamiento farmacológico , Células Tumorales CultivadasRESUMEN
Human epidermoid KB cell lines resistant to high levels of adriamycin, C-A90, C-A120, C-A500, and C-A1000, were isolated in selection medium containing increasing concentrations of adriamycin, 1 microgram/ml of cepharanthine, a multidrug-resistance (MDR) reversing agent, and 100 nM of mezerein, a protein kinase C activating agent. One of the adriamycin-resistant KB cell lines, C-A500, was cross-resistant to drugs that typify the classical multidrug resistance phenotype, such as vincristine, actinomycin D, VP-16, and colchicine. The accumulation of adriamycin and vincristine was decreased in C-A500 cells and the efflux of adriamycin from C-A500 was enhanced compared with parental KB-3-1 cells. These adriamycin-resistant KB cells did not contain detectable levels of P-glycoprotein or overexpress MDR1. Multidrug-resistance-associated protein (MRP) and MRP mRNA were expressed in the adriamycin-resistant KB cells, C-A120, C-A500, and C-A1000, but not in parental KB-3-1 and revertant C-AR cells. The MRP gene was amplified in all the MDR cells that overexpressed MRP mRNA. DNA topoisomerase II levels were markedly decreased in C-A500 and C-A1000 cells but only slightly decreased in C-A120 cells. These results indicate that MRP overexpressed in the resistant cells may be responsible for the reduced accumulation of adriamycin and vincristine and that both the increased expression of MRP and decreased levels of topoisomerase II underlie the drug resistance in C-A120, C-A500, and C-A1000 cell lines.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Separación Celular/métodos , Diterpenos , Resistencia a Múltiples Medicamentos , Células Tumorales Cultivadas , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/análisis , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Alcaloides/farmacología , Bencilisoquinolinas , Membrana Celular/química , Medios de Cultivo , ADN-Topoisomerasas de Tipo II/biosíntesis , Doxorrubicina/farmacología , Electroforesis en Gel de Poliacrilamida , Glicoproteínas/análisis , Humanos , Immunoblotting , ARN Mensajero/biosíntesis , Terpenos/farmacología , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
A 55-year-old woman with chronic myelogenous leukemia developed a lymphoid blast crisis (BC) 10 months after diagnosis. By using immunoblotting with a monoclonal antibody against P-glycoprotein (P-gp) C219, her leukemia cells from the first and 3rd crises were shown to be negative for the P-gp, while the cells of the 4th crisis were detected to have a high level of P-gp. This patient did not respond to chemotherapy with several anti-cancer agents in the 4th crisis, although complete remission was achieved in the first, second and third crises after administration of agents including vincristine and prednisolone. Therefore the expression of P-gp in the 4th BC might have been closely related to the resistance to chemotherapy.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Crisis Blástica , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Glicoproteínas de Membrana/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Resistencia a Medicamentos , Femenino , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Persona de Mediana EdadRESUMEN
Serum tumor necrosis factor-beta (TNF-beta) from patients with adult T-cell leukemia (ATL) was studied by a sandwich enzyme-linked immunosorbent assay (ELISA) developed in our laboratory using biotinylated monoclonal anti-TNF-beta and recombinant TNF-beta. Seven of eight patients with hypercalcemia showed elevation of serum TNF-beta. On the other hand, TNF-beta could not be detected by the ELISA in 28 patients without hypercalcemia. The lower detection limit in this assay was 100 pg/mL, corresponding to 500 pg/mL by the conventional method. In two patients serum TNF-beta level decreased after treatment in association with the level of serum calcium. Furthermore, immuno-staining using anti-TNF-beta and avidin-biotin complex showed the presence of cytoplasmic TNF-beta in not only human T-cell leukemia virus type I infected cell lines, but also freshly isolated cells from ATL patients with hypercalcemia. The actual biologic activity of TNF-beta in serum was confirmed by a conventional bioassay in a patient with hypercalcemia, and its cytotoxic activity was inhibited by the addition of anti-TNF-beta antibody in the assay. These results suggested that serum TNF-beta might be one of the factors contributing to the hypercalcemia, at least in patients with ATL.