RESUMEN
These studies explore the molecular effect of arsenicals on MM cells. Freshly isolated cells derived from patients with advanced, chemo-refractory myeloma as well as human myeloma cell lines, ARP-1, RPMI-8226 and H929 were exposed to the organic arsenical melarsoprol and to the inorganic compound AT. Both agents potently induced apoptosis in myeloma cells. Exposure to 1-5 microM AT or melarsoprol for 6 hours suppressed NF-kappa B DNA binding and enhanced of c-Jun kinase (JNK) activity. Arsenic also activated caspase-3 resulting in the cleavage of poly (ADP-ribose) polymerase (PARP) and Fas/TNF alpha related receptor interacting protein (RIP). In contrast to reported observations in acute promyelocytic leukemia, myeloma cell apoptosis was not associated with either the downregulation of Bcl-2 protein or with alterations in the expression of other Bcl-2 family members, Bax, Bak, Bag, and Bcl-xl. This study first shows that arsenic induces apoptotic signaling in MM through the cleavage of TNF alpha related receptor interacting protein (RIP). RIP is a key downstream protein in FasL/ TNF alpha /TRAIL induced apoptosis and a major antiapoptotic adaptor of pathways through NF-kappa B and JNK. RIP has not been previously characterized in myeloma. This study supports the hypothesis that arsenicals share common mediators (RIP, NF-kappa B, PARP, caspase-3) with death receptor induced apoptosis. These studies provide an important insight into the molecular mechanism of AT induced apoptosis and can be used in the development of adjuvant therapy for MM, presently an incurable disease.
Asunto(s)
Apoptosis/fisiología , Arsénico/farmacología , Mieloma Múltiple/patología , Apoptosis/efectos de los fármacos , Caspasa 3 , Caspasas/metabolismo , División Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Humanos , Melarsoprol/farmacología , FN-kappa B/metabolismoRESUMEN
The purpose of this study was to investigate the anti-proliferative and pro-apoptotic effects of the butyrate analogues, tributyrin (TB) and phenylbutyrate (PB), in a colon cancer model. We demonstrate that HT-29 colon cancer cells exposed to PB and TB result in growth inhibition associated with an induction of apoptosis mediated through the activation of caspase-3 activity. A block in the G1/S cell cycle traverse associated with a decrease in CDK2 (cyclin dependent kinase) protein levels and retinoblastoma protein hypophosphorylation was also noted after PB and TB exposure. Importantly, TB proved to be the most potent agent in its ability to induce these phenotypic changes, and potentially may represent a novel therapy for patients with advanced colorectal cancer.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Quinasas CDC2-CDC28 , Caspasas/metabolismo , Proteínas de Ciclo Celular , Proteínas de Neoplasias/metabolismo , Triglicéridos/farmacología , Adenocarcinoma/enzimología , Adenocarcinoma/patología , Butiratos/farmacología , Caspasa 3 , Neoplasias del Colon/enzimología , Neoplasias del Colon/patología , Quinasa 2 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción E2F , Factor de Transcripción E2F4 , Activación Enzimática/efectos de los fármacos , Fase G1/efectos de los fármacos , Proteínas Nucleares/metabolismo , Fenotipo , Fenilbutiratos/farmacología , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína de Retinoblastoma/metabolismo , Factores de Transcripción/metabolismo , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/enzimologíaRESUMEN
The inhibition of blood platelet aggregation and secretion was studied using covalent thiol reagents, maleimides, or mercuribenzoates, or using inhibitors of protein disulfide isomerase (PDI), bacitracin or antibodies to PDI. As expected, both types of inhibitors were effective against stimulation by normal physiologic stimuli. On the other hand, when stimulation was initiated with the peptide LSARLAF, that specifically activates the integrin alphaIIbbeta3 (the fibrinogen receptor), the PDI inhibitors were without effect. LSARLAF-induced aggregation was, however, inhibited by the sulfhydryl reagents. To further investigate the role of sulfhydryl-containing proteins and alphaIIbbeta3, platelets were labeled with membrane-impermeant sulfhydryl reagents. Nine bands were found labeled on gel electrophoresis. Two of the labeled bands were identified as alphaIIb and beta3. The conclusions are that while PDI is required for platelet aggregation and secretion, an additional sulfhydryl-dependent step or protein is also required. This latter reaction occurs at the level of alphaIIbbeta3. In distinction to most literature reports, at least a subpopulation of alphaIIbbeta3 contains free sulfhydryl groups, consistent with the possibility that it is a substrate for PDI or part of the sulfhydryl-dependent response.
Asunto(s)
Plaquetas/enzimología , Activación Plaquetaria , Proteína Disulfuro Isomerasas/fisiología , Transducción de Señal , Compuestos de Sulfhidrilo/fisiología , Plaquetas/efectos de los fármacos , Plaquetas/fisiología , Colágeno/farmacología , Ácido Ditionitrobenzoico/metabolismo , Ácido Ditionitrobenzoico/farmacología , Inhibidores Enzimáticos/farmacología , Humanos , Lisina/análogos & derivados , Lisina/metabolismo , Lisina/farmacología , Maleimidas/metabolismo , Maleimidas/farmacología , Modelos Químicos , Oligopéptidos/farmacología , Activación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Proteína Disulfuro Isomerasas/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Compuestos de Sulfhidrilo/metabolismo , Reactivos de Sulfhidrilo/farmacología , Ácido p-Cloromercuribenzoico/metabolismo , Ácido p-Cloromercuribenzoico/farmacologíaRESUMEN
Differentiation agents use existing cellular systems to induce neoplastic cells to regain a normal phenotype and/or to cause growth arrest and therefore may offer novel chemotherapeutic approaches to treating solid tumors. In this study, we demonstrate in Caco-2 colon cancer cells that the differentiation agent phenylbutyrate (PB) causes a decrease in viable cells, an increase in cell differentiation, and a G1-S-phase block. The mechanism of this last effect is related to a PB-induced increase in p27Kip1, leading to a decrease in the activity of cyclin-dependent kinase 2 (CDK2), a positive regulator of the G1-S-phase cell cycle transition. Consistent with the decreased CDK2 kinase activity, we also observed a decrease in the phosphorylation state of the retinoblastoma protein after PB treatment. This was associated with increased binding and consequent inactivation of E2F, a transactivator of genes that regulate the G1 to S phase cell cycle transition. These data suggest that the differentiation agent PB inhibits tumor growth by limiting the availability of active E2F, with a subsequent G1-S-phase block. Additional studies should show whether PB is a clinically effective therapeutic agent against colorectal cancer.
Asunto(s)
Quinasas CDC2-CDC28 , Proteínas Portadoras , Proteínas de Ciclo Celular , Ciclo Celular/fisiología , Diferenciación Celular/fisiología , Proteínas de Unión al ADN , Fenilbutiratos/farmacología , Proteínas Proto-Oncogénicas , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor , Fosfatasa Alcalina/metabolismo , Células CACO-2 , Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Quinasa 2 Dependiente de la Ciclina , Quinasa 4 Dependiente de la Ciclina , Quinasa 6 Dependiente de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Factores de Transcripción E2F , Fase G1 , Humanos , Cinética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína 1 de Unión a Retinoblastoma , Fase S , Factores de Tiempo , Factor de Transcripción DP1RESUMEN
In this study, purified preparations of platelet protein disulfide isomerase (PDI), vitronectin, alpha-thrombin, and antithrombin (AT) were used to demonstrate that PDI catalyzes formation of vitronectin-thrombin-AT complexes. Complex formation requires reduced glutathione (GSH) and can be prevented by N-ethymaleimide, and the formed complex is dissociated by reducing agents such as mercaptoethanol. No vitronectin-thrombin complex formed in the absence of AT, indicating that the thrombin-AT complex is an obligate intermediate in the reaction. Under optimal conditions, the majority of the thrombin-AT is incorporated into the complex in 60 min. Thrombospondin-1, known to form disulfide-linked complexes with thrombin-AT [Milev, Y., and Essex, D. W. (1999) Arch. Biochem. Biophys. 361, 120-126], competes with vitronectin for thrombin-AT in the low-Ca(2+) environment that favors the active form of thrombospondin. The results presented here may also explain previous studies showing that vitronectin-thrombin-AT complexes form better in plasma (which contains PDI) than with purified proteins (where PDI was not used). We were able to purify a PDI from plasma that was immunologically identical to the platelet enzyme. We used the scrambled RNase assay to show that added purified PDI can function in a plasma environment. Complex formation in plasma was inhibited by inhibitors of PDI. PDI was released from the platelet surface in a soluble form at high pH (around the physiologic range), suggesting a source of the plasma PDI. In summary, these studies indicate that PDI functions to form disulfide-linked complexes of vitronectin with thrombin-AT.
Asunto(s)
Antitrombina III/metabolismo , Disulfuros/sangre , Péptido Hidrolasas/metabolismo , Proteína Disulfuro Isomerasas/sangre , Trombina/metabolismo , Vitronectina/sangre , Animales , Plaquetas/metabolismo , Western Blotting , Catálisis , Relación Dosis-Respuesta a Droga , Glutatión/sangre , Humanos , Sustancias Macromoleculares , Proteínas de la Membrana/sangre , Propiedades de Superficie , Porcinos , Factores de TiempoRESUMEN
The molecular mechanisms by which multiple myeloma (MM) cells evade glucocorticoid-induced apoptosis have not been delineated. Using a human IgAkappa MM cell line (ARP-1), we found that dexamethasone (Dex)-induced apoptosis is associated with decreased NF-kappaB DNA binding and kappaB-dependent transcription. Both nuclear p50:p50 and p50:p65 NF-kappaB complexes are detected in ARP-1 cells by supershift electrophoretic mobility shift assay (EMSA). Dex-mediated inhibition of NF-kappaB DNA binding precedes a notable increase in annexin V binding, thereby indicating that diminished NF-kappaB activity is an early event in Dex-induced apoptosis. Overexpression of bcl-2 in ARP-1 cells prevents Dex-mediated repression of NF-kappaB activity and apoptosis. Sustained NF-kappaB DNA binding is also observed in two previously characterized Dex-resistant MM cell lines (RPMI8226 and ARH-77) that express moderate levels of endogenous bcl-2 and IkappaBalpha proteins. In addition, enforced bcl-2 expression in ARP-1 cells did not prevent the augmentation of IkappaBalpha protein by Dex. We also noted a possible association between Dex-mediated downregulation of NF-kappaB in freshly obtained primary myeloma cells and the patients' responsiveness to glucocorticoid-based chemotherapy. Collectively, our data suggest that the protective effects of bcl-2 in MM cells act upstream in the NF-kappaB activation-signaling pathway and the potential use of NF-kappaB as a biomarker in progressive MM.
Asunto(s)
Apoptosis/fisiología , Dexametasona/farmacología , Genes bcl-2 , Glucocorticoides/farmacología , Mieloma Múltiple/genética , Mieloma Múltiple/patología , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Apoptosis/efectos de los fármacos , Médula Ósea/patología , ADN de Neoplasias/metabolismo , Humanos , Luciferasas/genética , Proteínas Recombinantes de Fusión/metabolismo , Transcripción Genética , Transfección , Células Tumorales CultivadasRESUMEN
Multiple myeloma (MM) is a B cell malignancy characterized by the expansion of monoclonal Ig-secreting plasma cells with low proliferative activity. It is postulated that inhibition of physiologic cell death is an underlying factor in the pathophysiology of MM. The development of chemoresistance is a common feature in patients with MM. In the present studies, hexamethylene bisacetamide (HMBA), a hybrid polar compound that is a potent inducer of terminal differentiation of various transformed cells, is shown to inhibit the growth of several human myeloma cell lines (ARP-1, U266, and RPMI 8226), including doxorubicin-resistant RPMI 8226 variants that overexpress the multidrug-resistance gene, MDR-1, and its product, p-glycoprotein. In addition to growth arrest and suppression of clonogenicity, HMBA induces apoptosis both in freshly isolated human myeloma cells and in cell lines, as determined by morphologic alterations, cell cycle distribution and endonucleosomal DNA fragmentation. Further, HMBA decreases BCL-2 protein expression in myeloma cells within 12-48 hr. Overexpression of BCL-2 protein in ARP-1 cells confers resistance to HMBA-induced apoptosis. Taken together, these data suggest that HMBA is a potent inducer of apoptosis in human myeloma cells, which may act through suppressing the anti-apoptotic function of the bcl-2 gene. HMBA, and related hybrid polar compounds, may prove useful in the management of this presently incurable disease.
Asunto(s)
Acetamidas/farmacología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Regulación hacia Abajo , Mieloma Múltiple/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Humanos , Técnicas para Inmunoenzimas , Proteínas Proto-Oncogénicas c-bcl-2/genética , Transfección , Células Tumorales CultivadasRESUMEN
A tripartite domain of the immunoglobulin mu heavy-chain gene enhancer that activates transcription in B cells contains binding sites for PU.1, Ets-1, and a leucine zipper-containing basic helix-loop-helix factor. Because PU.1 is expressed only in B cells and macrophages, we tested the activity of a minimal mu enhancer fragment in macrophages by transient transfections. The minimal mu enhancer activated transcription in macrophages, and the activity was dependent on all three sites. Analysis of mutated enhancers, in which spacing and orientation of the ETS protein binding sites had been changed, suggested that the mechanisms of enhancer activation were different in B cells and macrophages. Thus, ETS protein binding sites may be combined in different ways to generate tissue-specific transcription activators. Despite the activity of the minimal enhancer in macrophages, a larger mu enhancer fragment was inactive in these cells. We propose that formation of the nucleoprotein complex that is formed on the minimal enhancer in macrophages cannot be helped by the neighboring muE elements that are essential for activity of the monomeric enhancer.
Asunto(s)
Linfocitos B/fisiología , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Genes de Inmunoglobulinas , Cadenas mu de Inmunoglobulina/genética , Macrófagos/fisiología , Transcripción Genética , Animales , Células Cultivadas , Huella de ADN , Análisis Mutacional de ADN , Proteínas de Unión al ADN/metabolismo , Ratones , Proteínas Nucleares/metabolismo , Distribución TisularRESUMEN
Specific cytogenetic abnormalities have been identified in multiple myeloma that confer a poor prognosis, even with intensive chemotherapy and autotransplants. The identification and characterization of potential genes involved in these different chromosomal changes and their interplay with oncogenes and tumor suppressor genes controlling cellular growth and apoptosis is the major focus of this review.
Asunto(s)
Mieloma Múltiple/genética , Aberraciones Cromosómicas , HumanosRESUMEN
Since the introduction of melphalan-prednisone for the treatment of multiple myeloma (MM) three decades ago, the prognosis of patients has not been improved by the addition of other agents, probably due to marked resistance of tumor cells, even at diagnosis, to commonly employed cytotoxic drugs. The biological basis for drug resistance is reviewed and current methods of diagnosis and staging delineated. The overview on treatment focuses on recurrent advances with myeloablative therapy demonstrating, in randomized and historically controlled trials, that high-dose therapy increases the incidence of true complete remission (CR) from 5% to approximately 40%, with an extension of median event-free (EFS) and overall (OS) survival durations to 3.5 and > or = 5 years, respectively. It is concluded that high-dose therapy should be offered to all patients with symptomatic meyloma, and current therapeutic research explores posttransplant immunotherapy.
Asunto(s)
Mieloma Múltiple/fisiopatología , Mieloma Múltiple/terapia , Antineoplásicos/uso terapéutico , Trasplante de Médula Ósea , Terapia Combinada , Humanos , Cariotipificación , Mieloma Múltiple/patología , Estadificación de Neoplasias , Pronóstico , Ensayos Clínicos Controlados Aleatorios como AsuntoRESUMEN
New developments are constantly introduced in the search for the optimal treatment modality to restore a single anterior tooth. The patient attention has shifted to aesthetics of the restoration, biocompatibility of the dental materials utilized, conservative preparation of the teeth to be restored, and the retention of intact adjacent dentition. The learning objective of this article is to review the methods currently utilized and to present a recently introduced treatment modality--the two-component bridge, which combines the strength and resiliency of composite resin with the aesthetic advantages of porcelain. The technology of the material is reviewed, the predominantly lingual tooth preparation procedures are outlined, and the bridge try-in is described. The advantages of the two-component bridge are presented along with the contraindications and suggestions of careful case selection. Three cases with congenitally missing maxillary lateral incisors in youthful patients are presented to supplement the theoretical outline and to describe and illustrate the clinical procedure.
Asunto(s)
Resinas Compuestas , Diseño de Dentadura , Dentadura Parcial Fija con Resina Consolidada , Adolescente , Adulto , Femenino , Humanos , Incisivo , Masculino , Maxilar , Selección de Paciente , Preparación Protodóncica del DienteAsunto(s)
Proteínas de Unión al ADN/metabolismo , Mieloma Múltiple/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Factores de Transcripción/metabolismo , Acetamidas/farmacología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Dexametasona/farmacología , Humanos , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Células Tumorales CultivadasRESUMEN
In the search for an optimal treatment to restore the aesthetically prominent maxillary anterior dentition, new materials are continually introduced. The attention of the patients has shifted from function to aesthetics, biocompatibility of the materials utilized, and conservative preparation of teeth to be restored. To fulfill patient expectations, an advanced treatment modality has recently been developed. It combines porcelain with composite resin, thereby integrating the strength and resilience of composite resin with the aesthetic advantages of porcelain. To achieve this combination, bundles of fibers are impregnated with resin in the fabrication phase of the material. The learning objective of this article is to briefly review the past methods of fabricating a 3-unit prosthesis in the maxillary anterior region and describe the recently introduced treatment alternative.
Asunto(s)
Resinas Compuestas/uso terapéutico , Porcelana Dental/uso terapéutico , Coronas con Frente Estético , Dentadura Parcial Fija con Resina Consolidada , Adolescente , Terapia Combinada , Coronas , Prótesis Dental de Soporte Implantado/métodos , Estética Dental , Femenino , Humanos , Incisivo , MaxilarRESUMEN
Various technologies within the dental armamentarium are applied to achieve the precise tooth preparations required for the different types of restorations. This article reintroduces the air microabrasion technique, which was first presented in 1945. The popular use of this technique had been postponed, pending the decrease in cost and development of compatible restorative materials to repair the tooth structure. With increased patient demand for less invasive preparation techniques and the decrease of the equipment cost, the use of air microbrasion for tooth preparation has been recognized. The history of the technology, its function, indications, advantages, and limitations are discussed, and a step-by-step clinical procedure is presented. The learning objective of this article is to familiarize the readers with this preparation procedure, enhancing the knowledge of preparation options.