RESUMEN
Elevated levels of eukaryotic translation initiation factor 4E (eIF4E) enhance translation of many malignancy-related proteins, such as vascular endothelial growth factor (VEGF), c-Myc and osteopontin. In non-small-cell lung cancer (NSCLC), levels of eIF4E are significantly increased compared with normal lung tissue. Here, we used an antisense oligonucleotide (ASO) to inhibit the expression of eIF4E in NSCLC cell lines. eIF4E levels were significantly reduced in a dose-dependent manner in NSCLC cells treated with eIF4E-specific ASO (4EASO) compared with control ASO. Treatment of NSCLC cells with the 4EASO resulted in decreased cap-dependent complex formation, decreased cell proliferation and increased sensitivity to gemcitabine. At the molecular level, repression of eIF4E with ASO resulted in decreased expression of the oncogenic proteins VEGF, c-Myc and osteopontin, whereas expression of ß-actin was unaffected. Based on these findings, we conclude that eIF4E-silencing therapy alone or in conjunction with chemotherapy represents a promising approach deserving of further investigation in future NSCLC clinical trials.
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Carcinoma de Pulmón de Células no Pequeñas/genética , Factor 4E Eucariótico de Iniciación/genética , Neoplasias Pulmonares/genética , Terapia Molecular Dirigida/métodos , Oligonucleótidos Antisentido/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Relación Dosis-Respuesta a Droga , Factor 4E Eucariótico de Iniciación/metabolismo , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Oligonucleótidos Antisentido/genética , Osteopontina/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , GemcitabinaRESUMEN
BACKGROUND: Gemcitabine is used for the treatment of several solid tumours and exhibits high inter-individual pharmacokinetic variability. In this study, we explore possible predictive covariates on drug and metabolite disposition. METHODS: Forty patients were enrolled. Gemcitabine and dFdU concentrations in the plasma and dFdCTP concentrations in peripheral blood mononuclear cell were measured to 72 h post infusion, and pharmacokinetic parameters were estimated by nonlinear mixed-effects modelling. Patient-specific covariates were tested in model development. RESULTS: The pharmacokinetics of gemcitabine was best described by a two-compartment model with body surface area, age and NT5C2 genotype as significant covariates. The pharmacokinetics of dFdU and dFdCTP were adequately described by three-compartment models. Creatinine clearance and cytidine deaminase genotype were significant covariates for dFdU pharmacokinetics. Rate of infusion of <25 mg m(-2) min(-1) and the presence of homozygous major allele for SLC28A3 (CC genotype) were each associated with an almost two-fold increase in the formation clearance of dFdCTP. CONCLUSION: Prolonged dFdCTP systemic exposures (≥72 h) were commonly observed. Infusion rate <25 mg m(-2) min(-1) and carriers for SLC28A3 variant were each associated with about two-fold higher dFdCTP formation clearance. The impacts of these covariates on treatment-related toxicity in more selected patient populations (that is, first-line treatment, single disease state and so on) are not yet clear.
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Antimetabolitos Antineoplásicos/administración & dosificación , Antimetabolitos Antineoplásicos/farmacocinética , Desoxicitidina/análogos & derivados , Proteínas de Transporte de Membrana/genética , Neoplasias/genética , Neoplasias/metabolismo , 5'-Nucleotidasa/genética , 5'-Nucleotidasa/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Antimetabolitos Antineoplásicos/sangre , Citidina Desaminasa/genética , Citidina Desaminasa/metabolismo , Desoxicitidina/administración & dosificación , Desoxicitidina/sangre , Desoxicitidina/farmacocinética , Femenino , Genotipo , Humanos , Infusiones Intravenosas , Leucocitos Mononucleares/metabolismo , Masculino , Proteínas de Transporte de Membrana/metabolismo , Persona de Mediana Edad , Neoplasias/sangre , Neoplasias/tratamiento farmacológico , Adulto Joven , GemcitabinaRESUMEN
Mesothelioma usually leads to death within 8-14 months of diagnosis. To increase the potency of oncolytic measles viruses (MVs) for mesothelioma therapy, we inserted the interferon beta (IFNbeta) gene alone or with the human thyroidal sodium iodide symporter (NIS) gene into attenuated MV of the Edmonston lineage. The corresponding mouse IFNbeta (mIFNbeta) viruses, MV-mIFNbeta and MV-mIFNbeta-NIS, successfully propagated in human mesothelioma cells, leading to intercellular fusion and cell death. High levels of mIFNbeta were detected in the supernatants of the infected cells, and radioiodine uptake was substantial in the cells infected with MV-mIFNbeta-NIS. MV with mIFNbeta expression triggered CD68-positive immune cell infiltration 2-4 times higher than MV-GFP injected into the tumor site. The numbers of CD31-positive vascular endothelial cells within the tumor were decreased at day 7 after intratumoral injection of MV-mIFNbeta or MV-mIFNbeta-NIS, but not after MV-GFP and PBS administration. Immunohistochemical analysis showed that MV-mIFNbeta changed the microenvironment of the mesothelioma by increasing innate immune cell infiltration and inhibiting tumor angiogenesis. Oncolytic MVs coding for IFNbeta effectively retarded growth of human mesotheliomas and prolonged survival time in several mesothelioma tumor models. The results suggest that oncolytic MVs that code for IFNbeta and NIS will be potent and versatile agents for the treatment of human mesothelioma.
Asunto(s)
Interferón beta/genética , Virus del Sarampión/fisiología , Mesotelioma/terapia , Viroterapia Oncolítica/métodos , Simportadores/genética , Animales , Línea Celular Tumoral , Chlorocebus aethiops , Modelos Animales de Enfermedad , Femenino , Humanos , Interferón beta/biosíntesis , Radioisótopos de Yodo , Virus del Sarampión/genética , Mesotelioma/diagnóstico por imagen , Mesotelioma/virología , Ratones , Ratones Desnudos , Ratones SCID , Plásmidos/genética , Simportadores/biosíntesis , Tomografía Computarizada de Emisión de Fotón Único/métodos , Células Vero , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Potency, immunogenicity, and toxicity are three problems that limit the use of targeted toxins in solid tumour therapy. METHODS: To address potency, we used genetic engineering to develop a novel bispecific ligand-directed toxin (BLT) called EGF4KDEL, a novel recombinant anti-mesothelioma agent created by linking human epidermal growth factor (EGF) and interleukin-4 (IL-4) to truncated pseudomonas exotoxin (PE38) on the same single-chain molecule. Immunogenicity was reduced by mutating seven immunodominant B-cell epitopes on the PE38 molecule to create a new agent, EGF4KDEL 7Mut. RESULTS: In vitro, bispecific EGF4KDEL showed superior anti-mesothelioma activity compared with its monospecific counterparts. Toxicity in mice was diminished by having both ligands on the same molecule, allowing administration of a 10-fold greater dose of BLT than a mixture of monomeric IL4KDEL and EGFKDEL. EGF4KDEL 7Mut, retained all of its functional activity and induced about 87% fewer anti-toxin antibodies than mice given the parental, non-mutated form. In vivo, intraperitoneal (IP) injection of the BLT showed significant (P<0.01) and impressive effects against two aggressive, malignant IP mesothelioma models when treatment was begun 14-16 days post tumour innoculation. CONCLUSION: These data show that EGF4KDEL 7Mut is a promising new anti-mesothelioma agent that was developed to specifically address the obstacles facing clinical utility of targeted toxins.
Asunto(s)
ADP Ribosa Transferasas/uso terapéutico , Toxinas Bacterianas/uso terapéutico , Factor de Crecimiento Epidérmico/uso terapéutico , Exotoxinas/uso terapéutico , Inmunotoxinas/uso terapéutico , Interleucina-4/uso terapéutico , Mesotelioma/tratamiento farmacológico , Factores de Virulencia/uso terapéutico , Animales , Línea Celular Tumoral , Femenino , Humanos , Inmunotoxinas/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Neoplasias Peritoneales/tratamiento farmacológico , Proteínas Recombinantes/uso terapéutico , Ensayos Antitumor por Modelo de Xenoinjerto , Exotoxina A de Pseudomonas aeruginosaRESUMEN
BACKGROUND: Insulin-like growth factor (IGF)-I signalling stimulates proliferation, survival, and invasion in malignant mesothelioma and other tumour types. Studies have found that tumourigenesis is linked to dysregulation of cap-dependent protein translation. METHODS: The effect of IGF stimulation on cap-mediated translation activation in mesothelioma cell lines was studied using binding assays to a synthetic 7-methyl GTP-cap analogue. In addition, cap-mediated translation was genetically repressed in these cells with a dominant active motive of 4E-BP1. RESULTS: In most mesothelioma cell lines, IGF-I stimulation resulted in a hyperphosphorylation-mediated inactivation of 4E-BP1 compared with that in normal mesothelial cells. An inhibitor of Akt diminished IGF-I-mediated phosphorylation of 4E-BP1, whereas inhibiting MAPK signalling had no such effect. IGF-I stimulation resulted in the activation of the cap-mediated translation complex as indicated by an increased eIF4G/eIF4E ratio in cap-affinity assays. Akt inhibition reversed the eIF4G/eIF4E ratio. Mesothelioma cells transfected with an activated 4E-BP1 protein (4E-BP1(A37/A46)) were resistant to IGF-I-mediated growth, motility, and colony formation. In a murine xenograft model, mesothelioma cells expressing the dominant active 4E-BP1(A37/A46) repressor protein showed abrogated tumourigenicity compared with control tumours. CONCLUSION: IGF-I signalling in mesothelioma cells drives cell proliferation, motility, and tumourigenesis through its ability to activate cap-mediated protein translation complex through PI3K/Akt/mTOR signalling.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Proteínas Portadoras/fisiología , Factor 4F Eucariótico de Iniciación/fisiología , Factor I del Crecimiento Similar a la Insulina/fisiología , Mesotelioma/etiología , Fosfoproteínas/fisiología , Biosíntesis de Proteínas , Caperuzas de ARN/fisiología , Animales , Proteínas de Ciclo Celular , Línea Celular Tumoral , Cromonas/farmacología , Factores Eucarióticos de Iniciación , Humanos , Mesotelioma/terapia , Ratones , Morfolinas/farmacología , Trasplante de Neoplasias , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Proteínas de Unión a Caperuzas de ARN , Receptor IGF Tipo 1/fisiología , Transducción de Señal , Trasplante HeterólogoRESUMEN
Bortezomib inhibits nuclear factor-kappaB (NF-kappaB). Cetuximab is a chimeric mouse-human antibody targeted against epidermal growth factor receptor (EGFR). We hypothesised that concomitant blockade of NF-kappaB and EGFR signalling would overcome EGFR-mediated resistance to single-agent bortezomib and induce apoptosis through two molecular pathways. The aim of this phase I trial was to establish the maximum tolerated dose (MTD) for bortezomib plus cetuximab in patients with EGFR-expressing epithelial tumours. The 21-day treatment cycle consisted of bortezomib administered on days 1 and 8 through dose escalation (1.3-2 mg m(-2)). Cetuximab was delivered at a dose of 250 mg m(-2) on days 1, 8 and 15 (400 mg m(-2) day 1 cycle 1). A total of 37 patients were enroled and given a total 91 cycles. No grade > or =3 haematological toxicity was noted. Non-hematological grade > or =3 toxicities included fatigue (22% of patients), dyspnoea (16%) and infection (11%). The MTD was not reached at the highest tested bortezomib dose (2.0 mg m(-2)). Efficacy outcomes included disease progression in 21 patients (56.7%) and stable disease (SD) at 6 weeks in 16 patients (43.3%). Five of the six patients with SD at 12 weeks were diagnosed with cancers of the lungs or head and neck. This combination therapy was moderately effective in extensively pretreated patients with non-small cell lung or head and neck cancers and warrants further investigation.
Asunto(s)
Anticuerpos Monoclonales/toxicidad , Protocolos de Quimioterapia Combinada Antineoplásica/toxicidad , Ácidos Borónicos/toxicidad , Receptores ErbB/metabolismo , Neoplasias/tratamiento farmacológico , Pirazinas/toxicidad , Adulto , Anciano , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Antineoplásicos/uso terapéutico , Antineoplásicos/toxicidad , Ácidos Borónicos/uso terapéutico , Bortezomib , Cetuximab , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Femenino , Humanos , Inmunohistoquímica , Masculino , Dosis Máxima Tolerada , Persona de Mediana Edad , FN-kappa B/antagonistas & inhibidores , Neoplasias/patología , Pirazinas/uso terapéutico , Adulto JovenRESUMEN
Malignant mesothelioma causes profound morbidity and nearly universal mortality that is often refractory to conventional treatment modalities of aggressive surgery, radiotherapy, or chemotherapy. Doxycycline, a commonly used antibiotic, has anti-tumor activity against several malignancies, but its anti-tumor effects on malignant mesothelioma have not been evaluated. We report here that concentrations of doxycycline achievable in serum with typical oral doses had cytostatic effects to varying extent on all eight of the mesothelioma cell lines studied but did not affect normal lung fibroblasts. Doxycycline inhibited the production of mitochondrial cytochrome c oxidase, especially in mesothelioma cells more sensitive to its cytostatic effects, and directly inhibited gelatinase A activity; both of these activities are putative mechanisms for the cytostatic activity of doxycycline in other tumor cells. Thus doxycycline may have a role as adjuvant therapy for malignant mesothelioma.
Asunto(s)
Antibacterianos/farmacología , Doxiciclina/farmacología , Mesotelioma , División Celular/efectos de los fármacos , Complejo IV de Transporte de Electrones/metabolismo , Activación Enzimática/efectos de los fármacos , Humanos , Técnicas In Vitro , Metaloproteinasa 2 de la Matriz/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismoRESUMEN
Loss of the G1 checkpoint appears to be extremely common among virtually all neoplasms. A variety of genetic and epigenetic mechanisms have been demonstrated to play significant roles in this process. In a consecutive series of early stage non-small cell lung cancer (NSCLC), we have established the loss of expression of the G1 Cdk inhibitors p15INK4b) and p16INK4a by DNA methylation is very common (37%), and methylation of p16INK4a is tightly correlated with loss of expression of p16INK4a protein (P = 0.0018). Furthermore, methylation of p15INK4b and p16INK4a appear inversely correlated, although methylation of p15INK4b is an infrequent event in this cohort (4%). Methylation was detected in all stages of NSCLC equally, and did not correlate with survival in these patients. Evidence for methylation was more frequent in squamous cell carcinomas in comparison to other tumor histologies (P = 0.0156). In addition, over-expression of cyclin D1 was found to be tightly restricted (P = 0.0032) to those tumors that had retained wild-type expression of pRB, and did not correlate with methylation or expression of p16INK4a gene product. Although loss of p16INK4a function remains tightly correlated with pRB expression, loss of other regulatory elements in NSCLC such as p53 mutation and cyclin D1 over-expression appear independent of loss of the p16INK4a gene product.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Proteínas Portadoras/genética , Proteínas de Ciclo Celular , Ciclo Celular/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Metilación de ADN , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/genética , Proteínas Supresoras de Tumor , Anciano , Secuencia de Aminoácidos , Secuencia de Bases , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Células Escamosas/genética , Proteínas Portadoras/metabolismo , Estudios de Cohortes , Ciclina D1/genética , Ciclina D1/metabolismo , Inhibidor p15 de las Quinasas Dependientes de la Ciclina , Inhibidor p16 de la Quinasa Dependiente de Ciclina/química , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Genes Supresores de Tumor/genética , Genes p53/genética , Genes ras/genética , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Mutación/genética , Estadificación de Neoplasias , Empalme del ARN/genética , Proteína de Retinoblastoma/metabolismoRESUMEN
The absence of expression of the p16INK4a gene product is observed in virtually all mesothelioma tumors and cell lines, whereas wild-type pRB expression is maintained. We have examined the potential therapeutic role of re-expressing the p16INK4a gene product in mice with established human mesothelioma xenografts. Experiments using Adp16 treatments in mesothelioma xenografts demonstrated prolonged survival and potential cure following treatment with p16INK4a-based gene therapy. These results demonstrate that p16INK4a gene transfer may play a therapeutic role in the treatment of mesothelioma.
Asunto(s)
Adenoviridae/genética , Proteínas Portadoras/genética , Terapia Genética/métodos , Mesotelioma/terapia , Animales , Supervivencia Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Vectores Genéticos , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Trasplante de Neoplasias , Fenotipo , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
Poor survival in patients following resection for early stage colorectal cancer is thought to be due in part to the presence of occult micrometastases at the time of surgery. The MUC2 mucin gene is highly expressed in the colon and associated colorectal tumors and may be a candidate marker for colorectal cancer micrometastases. We have used RT-PCR to detect expression of MUC2 mRNA transcripts in order to identify possible lymph node micrometastases in node negative (Stage I and II, or Dukes A and B) colorectal cancer patients. A total of 396 nodes (histologic stage N0) from 34 colon and nine rectal cancers were studied by RT-PCR analysis with nested primers for MUC2 (an average of 7.6 nodes per case). In the primary tumors, 42/43 (98.1%) were positive for MUC2 by RT-PCR. Evidence of the presence of MUC2 was demonstrated in nodes from 0 of 10 (0%) patients with Tis or T1, one of six (16.7%) from T2, 10 of 25 (40.0%) from T3, and one of two (50%) from T4 tumors. MUC2 RT-PCR was negative in six nodes from three patients with non-malignant colon disease and positive in histologically positive lymph nodes from six of six (100%) stage III colon cancers. In this study, using RT-PCR to detect the presence of MUC2 transcripts, we have found preliminary evidence for possible micrometastatic disease in approximately a third of histologically negative N0 colorectal cancer patients. The increased presence of MUC2 expression also correlated with more advanced T stage. We conclude that MUC2 RT-PCR may be a sensitive and specific marker for occult micrometastases. This technique has the potential to identify a group of colorectal cancer patients at risk for early cancer recurrence.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias Colorrectales/genética , ADN de Neoplasias/análisis , Metástasis Linfática/genética , Mucinas/genética , Proteínas de Neoplasias/genética , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/secundario , Cartilla de ADN , Diagnóstico Diferencial , Femenino , Humanos , Ganglios Linfáticos/patología , Metástasis Linfática/diagnóstico , Masculino , Mucina 2 , Estadificación de Neoplasias , Estudios Prospectivos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Células Tumorales CultivadasRESUMEN
Fas ligand (FasL) induces apoptosis in susceptible Fas-bearing cells and is critically involved in regulating T-cell immune responses. It is highly expressed in several human malignancies, and a role in the suppression of antitumor immune responses has been suggested. We evaluated FasL expression in leukemia and normal hematopoietic cells. By Western blotting, all acute leukemic cell lines (n = 9) and primary samples of acute leukemic marrow (n = 4) revealed high levels of FasL. In contrast, much weaker signals were observed in samples of normal marrow (n = 5), and either weak or intermediate expression was seen in chronic myeloid leukemia (CML) in chronic phase (n = 7). Additional leukemic samples were examined by immunohistochemistry. Staining for FasL was negative in 7 of 9 cases of chronic-phase CML, whereas all cases of CML in blast crisis (n = 6), acute lymphoblastic leukemia (n = 6), and acute myeloid leukemia (n = 11) stained strongly in 60 to 100% of nucleated cells. FasL+ leukemic cell lines did not trigger Fas-mediated apoptosis in either Jurkat cells or activated human T lymphocytes, possibly related to the intracellular location of the ligand. Western analysis of normal marrow subpopulations revealed that most FasL in marrow mononuclear cells was expressed by CD7+ lymphocytes. FasL also was strongly expressed in CD34+ hematopoietic progenitor cells from both normal and chronic-phase CML marrow, suggesting a correlation with primitive maturation stage. In summary, high levels of FasL expression were associated with aggressive biologic behavior in leukemia, including transformation of CML to blast crisis. This could potentially represent a response to loss of proapoptotic Fas signaling, which is known to occur in acute leukemic blasts.
Asunto(s)
Crisis Blástica/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Leucemia/metabolismo , Glicoproteínas de Membrana/biosíntesis , Enfermedad Aguda , Apoptosis , Western Blotting , Médula Ósea/metabolismo , Células Cultivadas , Técnicas de Cocultivo , Medios de Cultivo Condicionados/metabolismo , Progresión de la Enfermedad , Ensayo de Inmunoadsorción Enzimática , Proteína Ligando Fas , Citometría de Flujo , Células Madre Hematopoyéticas/metabolismo , Humanos , Inmunohistoquímica , Ganglios Linfáticos/metabolismoRESUMEN
The G1 cell cycle checkpoint regulates entry into S phase for normal cells. Components of the G1 checkpoint, including retinoblastoma (Rb) protein, cyclin D1 and p16INK4a, are commonly altered in human malignancies, abrogating cell cycle control. Using immunohistochemistry, we examined 79 invasive transitional cell carcinomas of the urinary bladder treated by cystectomy, for loss of Rb or p16INK4a protein and for cyclin D1 overexpression. As p53 is also involved in cell cycle control, its expression was studied as well. Rb protein loss occurred in 23/79 cases (29%); it was inversely correlated with loss of p16INK4a, which occurred in 15/79 cases (19%). One biphenotypic case, with Rb+p16- and Rb-p16+ areas, was identified as well. Cyclin D1 was overexpressed in 21/79 carcinomas (27%), all of which retained Rb protein. Fifty of 79 tumours (63%) showed aberrant accumulation of p53 protein; p53 staining did not correlate with Rb, p16INK4a, or cyclin D1 status. Overall, 70% of bladder carcinomas showed abnormalities in one or more of the intrinsic proteins of the G1 checkpoint (Rb, p16INK4a and cyclin D1). Only 15% of all bladder carcinomas (12/79) showed a normal phenotype for all four proteins. In a multivariate survival analysis, cyclin D1 overexpression was linked to less aggressive disease and relatively favourable outcome. In our series, Rb, p16INK4a and p53 status did not reach statistical significance as prognostic factors. In conclusion, G1 restriction point defects can be identified in the majority of bladder carcinomas. Our findings support the hypothesis that cyclin D1 and p16INK4a can cooperate to dysregulate the cell cycle, but that loss of Rb protein abolishes the G1 checkpoint completely, removing any selective advantage for cells that alter additional cell cycle proteins.
Asunto(s)
Carcinoma de Células Transicionales/fisiopatología , Ciclinas/biosíntesis , Fase G1 , Proteína p53 Supresora de Tumor/biosíntesis , Neoplasias de la Vejiga Urinaria/fisiopatología , Anciano , Carcinoma de Células Transicionales/patología , Ciclo Celular , Ciclina G , Ciclina G1 , Ciclinas/farmacología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Pronóstico , Análisis de Supervivencia , Proteína p53 Supresora de Tumor/farmacología , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
Increased apoptosis in the bone marrow (BM) may contribute to the cytopenias that occur in myelodysplastic syndromes (MDS). The Fas receptor, Fas ligand (FasL) pathway is a major mechanism of apoptosis. Since hematopoietic progenitors can express the Fas receptor, they may be susceptible to apoptosis induced by FasL-expressing cells. We examined FasL expression in the BM of patients with MDS (n = 50), de novo acute myeloid leukemia (AML; n = 10), AML following prior MDS (n = 6), and normal controls (n = 6). Compared to controls, FasL expression was increased in MDS, and was highest in AML. In MDS, FasL expression was seen in myeloid blasts, erythroblasts, maturing myeloid cells, megakaryocytes and dysplastic cells, whereas in AML, intense expression was seen in the blasts. FasL expression correlated with the FAB subtype groups of MDS, and also correlated directly with the percentage of abnormal metaphases on cytogenetic analysis. The FasL expressed in MDS BM inhibited the growth of clonogenic hematopoietic progenitors. This inhibition could be blocked by a soluble recombinant FasFc protein. In MDS, FasL expression in the initial diagnostic BM was higher in patients who were more anemic, correlated directly with red cell transfusion requirements over the subsequent course of the disease, and was predictive of survival. These studies indicate that FasL expression in MDS is of prognostic significance, and suggest that pharmacological blockade of the Fas-FasL pathway may be of clinical benefit.
Asunto(s)
Anemia/complicaciones , Leucemia Mieloide/genética , Glicoproteínas de Membrana/genética , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/patología , Enfermedad Aguda , Adulto , Anciano , Anciano de 80 o más Años , Anemia/patología , Crisis Blástica , Médula Ósea/metabolismo , Médula Ósea/patología , Proteína Ligando Fas , Estudios de Seguimiento , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Humanos , Leucemia Mieloide/mortalidad , Leucemia Mieloide/patología , Persona de Mediana Edad , Síndromes Mielodisplásicos/mortalidad , Valor Predictivo de las Pruebas , Pronóstico , Tasa de Supervivencia , Factores de TiempoRESUMEN
Absence of expression of the p16IKN4a gene product is commonly observed in mesothelioma tumors and cell lines, while wild-type pRB expression is maintained. We have examined the biologic and potential therapeutic role of re-expressing p16INK4a gene product in mesothelioma cells and tumors. Following transduction with a p16INK4a expressing adenovirus (Adp16), over-expression of p16INK4a in mesothelioma cells resulted in cell cycle arrest, inhibition of pRB phosphorylation, diminished cell growth, and eventual death of the transduced cells. Expression of p16INK4a protein was accompanied by decreased expression of pRB as detected by immunoblot and immunohistochemistry. Experiments in mesothelioma xenografts demonstrated inhibition of tumor formation, tumor growth arrest and diminished tumor size and spread. p16INK4a gene product expression was also demonstrated in intraperitoneal xenografts of human mesothelioma cells. These results demonstrate that p16INK4a gene transfer may play a therapeutic role in the treatment of mesothelioma.
Asunto(s)
Apoptosis/genética , Ciclo Celular/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Regulación Neoplásica de la Expresión Génica , Mesotelioma/genética , Animales , Terapia Genética , Humanos , Inmunohistoquímica , Mesotelioma/patología , Mesotelioma/terapia , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Proteína de Retinoblastoma/genética , Células Tumorales CultivadasRESUMEN
BACKGROUND: The 5-year survival rate following surgical resection of Stage I or Stage II non-small cell lung carcinoma (NSCLC) is 30% to 50%, probably because of undetected occult micrometastases (OMs) at the time of surgery. Other investigators have detected OMs in bone marrow and histologically negative lymph nodes from patients with NSCLC using immunohistochemical staining to cytokeratins and cell surface glycoproteins. STUDY OBJECTIVE: To develop and evaluate an assay based on the reverse transcriptase-polymerase chain reaction (RT-PCR) for the detection of OMs in NSCLC. PATIENTS: Twenty-eight patients with benign or malignant thoracic pathology. Samples of primary tumors and lymph nodes were collected at the time of surgical resection or mediastinoscopic lymph-node biopsy. RESULTS: Using RT-PCR to detect messenger RNA (mRNA) transcripts for MUC1 (a cell surface glycoprotein present in lung tissue but absent from normal lymph nodes), OMs were identified in 33 of 88 lymph nodes determined to be free of tumor by hematoxylin and eosin staining. Eleven of 11 control mediastinal lymph nodes from patients without malignancy failed to express detectable MUC1 transcripts. Dilutional experiments demonstrate that the assay can detect one MUC1-positive NSCLC cell in 1x10(7) MUC1-negative cells. A comparison of our RT-PCR assay to immunohistochemistry specific for the MUC1 glycoprotein suggests that RT-PCR may be more sensitive than immunohistochemistry for the detection of NSCLC OMs. CONCLUSIONS: This study demonstrates that RT-PCR for MUC1 mRNA can detect the presence of MUC1 mRNA in histologically negative lymph nodes from patients with NSCLC. The prognostic significance of these findings is currently unknown.
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Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/secundario , Neoplasias Pulmonares/patología , Reacción en Cadena de la Polimerasa , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Pulmón de Células no Pequeñas/química , Femenino , Humanos , Inmunohistoquímica , Neoplasias Pulmonares/química , Ganglios Linfáticos/química , Metástasis Linfática , Masculino , Persona de Mediana Edad , Mucina-1/análisis , Mucina-1/genética , ARN Mensajero/análisis , ADN Polimerasa Dirigida por ARN , Sensibilidad y EspecificidadRESUMEN
Patients with non-small-cell lung cancer (NSCLC) survive for variable lengths of time, even when adjustment is made for pathological stage. Numerous reports suggest that biological markers predict survival in patients undergoing surgery for NSCLC with curative intent, but many of these claims are unconfirmed or conflicting. We postulated that the use of multiple putative markers might provide greater power in predicting survival. We studied 101 consecutive patients with NSCLC who underwent exploratory thoracotomy and who were followed for at least 2 yr. We assessed mutations in the p53 tumor suppressor gene (exons 5-8) and the K-ras oncogene (codons 12 and 13) by polymerase chain reaction amplification and single strand conformation polymorphism of the product. We identified 19 K-ras mutations (all adenocarcinomas except for two) and 40 p53 mutations among the 101 cases. We also evaluated p53 protein, bcl-2 protein, c-erbB-1 protein, c-erbB-2 protein, and MIA-15-5 antigen by standard immunocytochemical techniques, and we found that all of these antigens were variably expressed. As expected, we found a strong inverse association between surgical tumor stage and survival. Of the molecular markers studied, only MIA-15-5 antigen expression correlated strongly with survival by univariate analysis (p = 0.001) and it remained a significant predictor by multivariate analysis (p = 0.01). However, in this study, overexpression of MIA-15-5 antigen predicted an improved survival, whereas the original report showed a worse prognosis (N. Engl. J. Med. 1992;327:14). We conclude the multiple cell markers are not clinically useful in predicting survival among patients undergoing surgery for NSCLC. Differences between our results and prior reports may be due to chance, to true population differences, or to other factors.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidad , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales/análisis , Antígenos de Neoplasias/análisis , Biomarcadores de Tumor/análisis , Carcinoma de Pulmón de Células no Pequeñas/cirugía , Receptores ErbB/análisis , Genes p53/genética , Genes ras/genética , Marcadores Genéticos , Humanos , Neoplasias Pulmonares/cirugía , Masculino , Persona de Mediana Edad , Mutación , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-Simple , Pronóstico , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Receptor ErbB-2/análisis , Tasa de Supervivencia , Proteína p53 Supresora de Tumor/análisisRESUMEN
Malignant mesothelioma causes profound morbidity and nearly universal mortality that is refractory to conventional treatment with aggressive surgery, radiotherapy, or chemotherapy. We report that pharmacologic concentrations of lovastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitor, induced apoptosis in human malignant mesothelioma cell lines. Mesothelioma cell viability was decreased in a dose-dependent manner by lovastatin (5 to 30 microM). These effects were not reversed by exogenous growth factors or cholesterol, but were reversed by addition of 100 microM mevalonate, confirming that lovastatin affected mesothelioma viability by inhibiting mevalonate synthesis. Lovastatin appeared to decrease mesothelioma viability by inducing apoptosis, as indicated by morphologic changes, histologic evidence of nuclear condensation and degeneration, and flow-cytometric analysis of DNA content. Lovastatin's effects on cell viability were partially reversed in the presence of farnesol, and treatment of mesothelioma cells with a specific farnesyl-protein transferase (FTP) inhibitor decreased cell viability and induced morphologic changes indistinguishable from those caused by lovastatin. In addition, lovastatin-treated cells showed translocation of ras guanosine triphosphate (GTP)-binding proteins from membrane to cytosolic fractions on Western blots, suggesting that lovastatin's effects on mesothelioma were mediated in part by disrupting acylation of GTP-binding proteins. Thus, lovastatin is a commercially available and clinically well-tolerated agent that reduces viability and induces apoptosis of mesothelioma cells, and may provide the basis for adjunctive treatments of patients with mesothelioma.
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Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Lovastatina/farmacología , Mesotelioma/patología , Transferasas Alquil y Aril/antagonistas & inhibidores , División Celular , Línea Celular , Membrana Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Citosol/metabolismo , ADN de Neoplasias/metabolismo , Relación Dosis-Respuesta a Droga , Farnesol/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Humanos , Mesotelioma/ultraestructura , Ácido Mevalónico/farmacología , Prenilación de Proteína , Células Tumorales Cultivadas/efectos de los fármacos , Proteínas ras/metabolismoRESUMEN
To reach a clinically detectable size, neoplasms must be able to suppress or evade a host immune response. Activated T cells may enter apoptosis in the presence of Fas ligand (FasL) (1), and tissue expression of FasL has been shown to contribute to immune privilege in the eye and testis (2, 3). We have demonstrated that all human lung carcinoma cell lines tested (16 of 16) express a Mr 38,000 protein consistent with FasL by immunoblotting, whereas the majority of resected tumors (23 of 28) show positive staining for FasL by immunohistochemistry. DNA sequencing of reverse transcription-PCR products from lung cancer cells and resected lung tumors confirms the presence of human FasL mRNA in these neoplastic tissues. Furthermore, lung carcinoma cells are capable of killing a Fas-sensitive human T cell line (Jurkat) in coculture experiments; this killing was inhibited by a recombinant form of the soluble portion of the Fas receptor (FasFc). FasL expression by neoplastic cells represents a potential mechanism for peripheral deletion of tumor-reactive T-cell clones.
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Neoplasias Pulmonares/metabolismo , Glicoproteínas de Membrana/biosíntesis , Proteínas de Neoplasias/biosíntesis , ADN de Neoplasias/análisis , Proteína Ligando Fas , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Glicoproteínas de Membrana/genética , Proteínas de Neoplasias/genética , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , ARN Neoplásico/análisis , Proteínas Recombinantes/farmacología , Células Tumorales Cultivadas , Receptor fas/genética , Receptor fas/farmacologíaRESUMEN
We have previously established the efficacy of adenoviral gene delivery vectors for the treatment of bladder carcinoma in vivo. In the present work, we developed a gene therapy strategy for bladder cancer based on the replacement of the tumor suppressor p16, which is known to be mutated or deleted in a variety of human tumors, including those derived from the bladder. Previous reports have demonstrated that reconstitution of p16 has marked effects on the proliferative capacity of tumor cell lines both in vitro and in vivo, and that p16 expression causes resistance to some chemotherapeutic agents. In the present study, we describe the construction of the recombinant adenovirus Adp16, expressing the p16 gene, to evaluate the effects of transient p16 replacement in the context of bladder carcinoma. To identify candidate target cell lines, we screened a panel of bladder cancer cell lines for p16 and retinoblastoma (RB) protein expression. We demonstrate that Adp16 can mediate high-efficiency p16 replacement to the p16-negative cell lines EJ and UMUC-3. In addition, the reconstitution of p16 to the highly transducible p16-negative, RB-positive bladder cancer cell line EJ caused a profound inhibition of cell proliferation mediated by arrest in the G1 phase of the cell cycle. In contrast, the p16-positive, RB-negative cell line J82 was unaffected by this treatment. However, when adenovirally mediated p16 replacement was combined with the chemotherapeutics cisplatin and paclitaxel, a marked chemoresistance was observed in genetically corrected cells. This work has implications for future gene therapy strategies based on p16 replacement.
Asunto(s)
Ciclo Celular/fisiología , Cisplatino/toxicidad , Inhibidor p16 de la Quinasa Dependiente de Ciclina/deficiencia , Resistencia a Antineoplásicos , Genes p16 , Terapia Genética/métodos , Paclitaxel/toxicidad , Transfección/métodos , Adenoviridae , Carcinoma de Pulmón de Células no Pequeñas , Supervivencia Celular/efectos de los fármacos , Inhibidor p16 de la Quinasa Dependiente de Ciclina/biosíntesis , Fase G1 , Genes de Retinoblastoma , Vectores Genéticos , Humanos , Neoplasias Pulmonares , Mesotelioma , Proteínas Recombinantes/biosíntesis , Proteína de Retinoblastoma/biosíntesis , Células Tumorales Cultivadas , Neoplasias de la Vejiga Urinaria/terapiaRESUMEN
Inactivation of the cyclin-dependent kinase inhibitor p16INK4a (CDKN2/MTS1) is documented in a wide variety of cancer cell lines and tumors. We have shown that loss of p16INK4a protein expression is a common event in early stage non-small cell lung cancer (NSCLC), correlates with a significantly worse survival, and is more common in higher stage disease. One hundred NSCLC tumors from patients undergoing definitive thoracotomies at a single institution were examined for p16INK4a and retinoblastoma protein (pRB) expression. Abnormal pRB staining was identified in 15% of the tumors, whereas 51% possessed aberrant p16INK4a protein expression. Tumors with aberrant expression of p16INK4a by immunohistochemistry were associated with a significantly worse survival (P=0.04). Additionally, the inverse correlation of pRB and p16INK4a expression previously noted in lung cancer cell lines and tumors was confirmed in this large cohort of patients, with 65% of the tumors demonstrating inverse expression of pRB and p16INK4a (p=0.00019). A statistically significant increase in aberrant p16INK4a expression, as well as inverse expression of p16INK4a and pRB, was seen with increasing pathological stage of disease. These findings establish the prognostic significance (of the absence of p16INK4, in resected NSCLC and confirm the critical importance of disrupting the pathway of cyclin-dependent kinase-mediated phosphorylation of pRB in the molecular oncogenesis and progression of NSCLC.