RESUMEN
The prevalence of non-tuberculous mycobacterium (NTM) appears to be increasing. Much of the experience in the literature about this emerging organism comes from specialised units or populations such as cystic fibrosis patients. We, therefore, aim to evaluate the experience in a general respiratory population of dealing with patients with positive culture of NTM. We did a retrospective review of medical notes of general respiratory patients from whom NTM were isolated from January 2007 to July 2012. Cystic fibrosis patients were excluded. We reviewed 37 patients' (19 males, 18 females) medical records. A total of 73 positive cultures were reviewed. 28 isolates were from sputum samples alone, 34 isolates were from bronchoalveolar lavage alone and 11 isolates were from a combination of sputum and bronchoalveor lavage (11 isolates), We found that Mycobacterium avium was the most frequently isolated Mycobacterium in our laboratory with 22 (60%) patients had Mycobacterium avium in their pulmonary cultures. Interestingly, Mycobacterium gordonae and mycobacterium intracellulare were the second commonest mycobacterium (4, 11%) cultured. We noted 2 (5%), cases of Mycobacterium szulgai, 2 (5%) cases of Mycobacterium chelonae and 2 (5%) cases of Mycobacterium abscessus. There was 1(3%) case of Mycobacterium malmoense. There is prevalence of NTM in male COPD patients (7, 89%) and femal bronchiectasis (10, 77%) patients. Of our 8 COPD patients, 6 (75%) were on inhaled corticosteroids while 2 (25%) were not. 9 (24%) patients were smokers, 11 (30%) were ex-smokers, 14 (38%) were non-smokers and the smoking status of the remaining 3 (8%) was unknown. Of the 37 patients, only 6 (16%) received treatment. However, 2 patients stopped their treatment due to treatment toxicity. We concluded that the isolation of NTM is not uncommon. Defining NTM disease is difficult and deciding which patient to be treated needs careful evaluation as treatment can potentially be very toxic.
Asunto(s)
Infecciones por Mycobacterium no Tuberculosas/epidemiología , Infecciones por Mycobacterium no Tuberculosas/microbiología , Micobacterias no Tuberculosas/aislamiento & purificación , Enfermedad Pulmonar Obstructiva Crónica/microbiología , Anciano , Bronquiectasia/epidemiología , Bronquiectasia/microbiología , Líquido del Lavado Bronquioalveolar/microbiología , Femenino , Humanos , Irlanda/epidemiología , Masculino , Persona de Mediana Edad , Enfermedad Pulmonar Obstructiva Crónica/epidemiología , Estudios Retrospectivos , Esputo/microbiologíaAsunto(s)
Antirreumáticos/efectos adversos , Infecciones Oportunistas/inducido químicamente , Tuberculosis/inducido químicamente , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales/efectos adversos , Anticuerpos Monoclonales/uso terapéutico , Antirreumáticos/uso terapéutico , Artritis Psoriásica/tratamiento farmacológico , Femenino , Humanos , Huésped Inmunocomprometido , Infliximab , Masculino , Persona de Mediana Edad , Infecciones Oportunistas/inmunología , Tuberculosis/inmunologíaRESUMEN
Deregulation of the hedgehog (HH) pathway results in overexpression of the GLI target BCL2 and is an initiating event in specific tumor types including basal cell carcinoma of the skin. Regulation of the HH pathway during keratinocyte differentiation is not well understood. We measured HH pathway activity in response to differentiation stimuli in keratinocytes. An upregulation of suppressor of fused (SUFU), a negative regulator of the HH pathway, lowered HH pathway activity and was accompanied by loss of BCL2 expression associated with keratinocyte differentiation. We used in vitro and in vivo models to demonstrate that ΔNp63α, a crucial regulator of epidermal development, activates SUFU transcription in keratinocytes. Increasing SUFU protein levels inhibited GLI-mediated gene activation in suprabasal keratinocytes and promoted differentiation. Loss of SUFU expression caused deregulation of keratinocyte differentiation and BCL2 overexpression. Using in vivo murine models, we also provide evidence of GLI-mediated regulation of the TP63 pathway. p63 expression appears essential to establish an optimally functioning HH pathway. These observations present a regulatory mechanism by which SUFU acts as an interacting node between the HH and TP63 pathways to mediate differentiation and maintain epidermal homeostasis. Disruption of this regulatory node can be an important contributor to multistep carcinogenesis.
Asunto(s)
Células Epidérmicas , Proteínas Hedgehog/fisiología , Homeostasis/fisiología , Queratinocitos/citología , Fosfoproteínas/fisiología , Transducción de Señal/fisiología , Transactivadores/fisiología , Animales , Diferenciación Celular/fisiología , Línea Celular , Proliferación Celular , Supervivencia Celular/fisiología , Células Cultivadas , Epidermis/fisiología , Femenino , Técnicas In Vitro , Queratinocitos/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Noqueados , Modelos Animales , Fosfoproteínas/deficiencia , Fosfoproteínas/genética , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Proteínas Represoras/fisiología , Transactivadores/deficiencia , Transactivadores/genéticaRESUMEN
The European Respiratory Society COPD audit was a cross-sectional, multicentre study that analysed outcomes for COPD patients admitted to hospital with an exacerbation across Europe. We present the data on patients admitted to 11 Irish hospitals that participated in the audit. Among 237 patients (123 Male), the median age was 71 years and 79 (33%) patients were current smokers. 82 (35%) patients received high-flow oxygen before admission and 43 (18%) were cared for in a dedicated respiratory ward. 54 (23%) patients required ventilatory support. Median length of stay was 7 days, 98 (41%) patients were readmitted and 211 (89%) patients were alive at the 90 day follow up point. Irish patients were more likely to receive high-flow oxygen before admission, less likely to be managed in a dedicated respiratory ward and had a higher likelihood of readmission or death within 90 days than the European average.
Asunto(s)
Enfermedad Pulmonar Obstructiva Crónica/terapia , Anciano , Anciano de 80 o más Años , Estudios Transversales , Europa (Continente)/epidemiología , Femenino , Hospitalización/estadística & datos numéricos , Humanos , Irlanda/epidemiología , Tiempo de Internación/estadística & datos numéricos , Masculino , Persona de Mediana Edad , Enfermedad Pulmonar Obstructiva Crónica/epidemiología , Fumar/epidemiología , Resultado del TratamientoRESUMEN
PURPOSE: The National Lung Screening Trial (NLST) has renewed interest in low-dose computed tomography (LDCT) screening for lung cancer. Smokers may be less receptive toward LDCT screening, however, compared with never smokers. The views of patients with COPD, a particularly high-risk group, toward LDCT screening for lung cancer are currently unknown. We therefore evaluated attitudes of patients with COPD toward LDCT screening for lung cancer. METHODS: Interviews with Irish patients with COPD who satisfied NLST eligibility criteria were conducted in clinical settings using a questionnaire based on that of a comparable study of U.S. current/former smokers of unspecified disease status. RESULTS: A total of 142 subjects had a mean age of 65.09 ± 6.07 years (46.4 % were male, mean pack years 54.5 ± 33.3, mean FEV1 59.16 ± 23 %); 97.8 % had an identifiable usual source of healthcare. Compared with data from a U.S. cohort of current/former smokers, a higher proportion of Irish COPD smokers: believed that they were at risk for lung cancer (63.6 vs. 15.7 %); believed that early detection improved chances of survival (90 vs. 51.2 ); were willing to consider LDCT screening (97.9 vs. 78.6 %); were willing to pay for a LDCT scan (68.6 vs. 36.2 %); and were willing to accept treatment recommendations arising (95.7 vs. 56.2 %; p < 0.0001 for all comparisons). CONCLUSIONS: Urban Irish smokers with COPD who would be eligible for LDCT screening are almost universally in favor of being screened and treated for screening-detected lung cancers. This readily accessible high-risk population should be actively targeted in future screening programs.
Asunto(s)
Actitud Frente a la Salud , Detección Precoz del Cáncer/psicología , Neoplasias Pulmonares/diagnóstico , Aceptación de la Atención de Salud , Enfermedad Pulmonar Obstructiva Crónica/psicología , Tomografía Computarizada por Rayos X , Población Urbana , Anciano , Femenino , Humanos , Irlanda , Masculino , Persona de Mediana Edad , Dosis de Radiación , FumarAsunto(s)
Calidad de Vida , Síndrome de Dificultad Respiratoria/fisiopatología , Síndrome de Dificultad Respiratoria/terapia , Adulto , Femenino , Humanos , Masculino , Modalidades de Fisioterapia , Recuperación de la Función , Síndrome de Dificultad Respiratoria/epidemiología , Síndrome de Dificultad Respiratoria/etiología , Tasa de SupervivenciaRESUMEN
The potential for pharmaceuticals to produce side effects and drug interactions is well known to medical practitioners and the lay public alike. However, the potential for alternative medicines to produce such effects is less widely known. We describe a potentially dangerous interaction between a herbal medicine and concomitant selective serotonin re-uptake inhibitor (SSRI) ingestion.
Asunto(s)
Citalopram/efectos adversos , Interacciones de Hierba-Droga , Fitoterapia , Rhodiola/efectos adversos , Inhibidores Selectivos de la Recaptación de Serotonina/efectos adversos , Adulto , Atención/efectos de los fármacos , Femenino , HumanosRESUMEN
Trisomy 11 in myelodysplastic syndromes (MDS) is rare, with undefined clinical significance and is currently assigned to the International Prognostic Scoring System (IPSS) intermediate-risk group. Over a 15-year period, we identified 17 MDS patients with trisomy 11 either as a sole abnormality (n=10) or associated with one or two additional alterations (n=7), comprising 0.3% of all MDS cases reviewed. Of 16 patients with Bone Marrow material available for review, 14 (88%) patients presented with excess blasts, 69% patients evolved to acute myeloid leukemia (AML) in a 5-month median interval and the median survival was 14 months. For comparison, we studied 19 AML patients with trisomy 11 in a noncomplex karyotype, of which, a substantial subset of patients had morphologic dysplasia, and/or preexisting cytopenia(s)/MDS. Genomic DNA PCR showed MLL partial tandem duplication in 5 of 10 MDS and 7 of 11 AML patients. A review of literature identified 17 additional cases of MDS with trisomy 11, showing similar clinicopathologic features to our patients. Compared with our historical data comprising 1165 MDS patients, MDS patients with trisomy 11 had a significantly inferior survival to patients in the IPSS intermediate-risk cytogenetic group (P=0.0002), but comparable to the poor-risk group (P=0.97). We conclude that trisomy 11 in MDS correlates with clinical aggressiveness, may suggest an early/evolving AML with myelodysplasia-related changes and is best considered a high-risk cytogenetic abnormality in MDS prognostication.
Asunto(s)
Trastornos de los Cromosomas/genética , Cromosomas Humanos Par 11/genética , Leucemia Mieloide Aguda/genética , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/patología , Trisomía/genética , Adulto , Anciano , Anciano de 80 o más Años , Southern Blotting , ADN de Neoplasias/genética , Femenino , Duplicación de Gen , Genes ras/genética , N-Metiltransferasa de Histona-Lisina , Humanos , Hibridación Fluorescente in Situ , Cariotipificación , Masculino , Persona de Mediana Edad , Mutación/genética , Síndromes Mielodisplásicos/clasificación , Proteína de la Leucemia Mieloide-Linfoide/genética , Pronóstico , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas p21(ras) , Estudios Retrospectivos , Tasa de Supervivencia , Tirosina Quinasa 3 Similar a fms/genética , Proteínas ras/genéticaRESUMEN
BACKGROUND: The Bacille-Calmette-Guérin (BCG) vaccine is used in Mycobacterium tuberculosis prophylaxis in at risk tuberculin-negative healthcare workers. Its use is contraindicated however in individuals with HIV infection. AIMS: We herein highlight the case of a healthcare worker who developed a localised reaction at a BCG vaccination site and who was subsequently found to be HIV positive. CONCLUSION: This case emphasises the importance of eliciting risk factors for immunocompromise in individuals for whom BCG vaccination is being considered.
Asunto(s)
Vacuna BCG/efectos adversos , Seropositividad para VIH , Úlcera Cutánea/etiología , Adulto , Técnicos Medios en Salud , Antibióticos Antituberculosos/uso terapéutico , Vacuna BCG/inmunología , Contraindicaciones , Seropositividad para VIH/inmunología , Humanos , Masculino , Mycobacterium bovis/aislamiento & purificación , Rifampin/uso terapéutico , Tuberculosis/prevención & controlAsunto(s)
Antiinflamatorios no Esteroideos/efectos adversos , Fumaratos/efectos adversos , Inmunoglobulina G/efectos adversos , Tuberculosis Ganglionar/inducido químicamente , Adulto , Susceptibilidad a Enfermedades , Combinación de Medicamentos , Etanercept , Humanos , Masculino , Psoriasis/tratamiento farmacológico , Receptores del Factor de Necrosis Tumoral , Resultado del Tratamiento , Prueba de TuberculinaRESUMEN
A consistent, if not invariant, feature of cancer cells is the acquired ability to evade apoptosis. The pioneering work of Dr. Stan Korsmeyer was invaluable in characterizing the molecular foundations of cell death signaling mechanisms during normal development and during multistep carcinogenesis. This foundation now forms the basis for the rational design of therapeutic strategies to selectively activate cell death in cancer cell populations. These strategies are currently being evaluated in an increasing number of clinical trials targeting diverse tumor types.
Asunto(s)
Carcinoma/metabolismo , Carcinoma/patología , Animales , Carcinoma/genética , Carcinoma/terapia , Muerte Celular , Transformación Celular Neoplásica/patología , Progresión de la Enfermedad , Humanos , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Transducción de SeñalRESUMEN
Many diseases, especially solid tumors, involve the disruption or deregulation of cellular processes. Most current work using gene expression and other high-throughput data, simply list a set of differentially expressed genes. We propose a new method, PAPES (predicting altered pathways using extendable scaffolds), to computationally reverse-engineer models of biological systems. We use sets of genes that occur in a known biological pathway to construct component process models. We then compose these models to build larger scale networks that capture interactions among pathways. We show that we can learn process modifications in two coupled metabolic pathways in prostate cancer cells.
Asunto(s)
Biología Computacional/métodos , Perfilación de la Expresión Génica , Neoplasias de la Próstata/diagnóstico , Neoplasias de la Próstata/patología , Teorema de Bayes , Glutatión/metabolismo , Humanos , Masculino , Modelos Genéticos , Análisis de Secuencia por Matrices de Oligonucleótidos , Estrés Oxidativo , Oxígeno/metabolismo , Ingeniería de Proteínas , Sensibilidad y Especificidad , Programas Informáticos , Urea/metabolismoRESUMEN
Acute exacerbations of chronic obstructive pulmonary disease (AECOPD) are a major cause of hospital admissions. Because of the consequent morbidity, mortality and burden on hospital resources, COPD management guidelines have been formulated. We reviewed 62 consecutive patients with AECOPD admitted from September 1st to December 18th 2000 in St. Vincents University Hospital, Ireland, including 3 months follow-up data, to evaluate the quality of care and in particular to assess the care of such patients by respiratory and non-respiratory physicians. There was a frequent failure to objectively confirm the diagnosis of COPD by spirometry (completed in 39 of the 51 patients who, at admission, had been previously labelled with COPD (76%), and in 53 out of 62 patients (85%) at the end of the study period), or to estimate severity by quantifying the FEV1 as a percentage of the normal predicted range (estimated in only 21 of the 39 patients who had spirometry previously performed (53%)). Those patients managed with input from respiratory physicians were more likely to have their diagnosis of COPD confirmed with spirometry (p < 0.05). They were also more likely to have out-patient follow-up arranged at discharge (p < 0.05). There was a trend towards the more frequent prescribing of oxygen to hypoxic patients in "respiratory" than in "non-respiratory" managed cases (p = 0.182) and a shorter hospital stay (0.1 < p < 0.5). 4 out of 11 severely hypoxaemic patients at admission (PO2 < 7.3kPa) were not screened at discharge for possible long term oxygen therapy (36%). 20 patients received combination antibiotic therapy with no infiltrate on CXR (32%). Pulmonary rehabilitation was offered to 12 patients (19%). 5 out of 18 current smokers had documented smoking cessation advice (28%) and none received smoking cessation pharmacotherapy. Finally we noted that the Hospital In-Patient Enquiry (HIPE) data and casualty department admission books were frequently misleading or medical records unlocatable (in 30 out of 92 cases (33%)). We conclude that the management of AECOPD at St. Vincent's University Hospital is frequently suboptimal, and may be managed better with respiratory physician involvement. In particular, there could be more frequent spirometric confirmation of the diagnosis of COPD, better screening for long term oxygen therapy and more conservative use of antibiotics. Audit is complicated by difficulty accessing relevant data.
Asunto(s)
Pautas de la Práctica en Medicina , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Irlanda , Masculino , Auditoría Médica , Persona de Mediana Edad , Estudios RetrospectivosRESUMEN
PURPOSE: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor (TNF) family that preferentially kills tumor cells. In this study, we sought to determine whether chemotherapeutic agents augment TRAIL-induced cytotoxicity in human prostate cancer cells, and whether this sensitivity can be blocked by overexpression of bcl-2. METHODS: Prostate cancer cells, PC3 and LNCaP, were treated with TRAIL alone, drug alone or a combination of both for 24 h. Cytotoxicity was determined by DNA fragmentation and clonogenic survival assay. RESULTS: Treatment with the conventional chemotherapeutic agents cisplatin (2 and 5 microg/ml), etoposide (10 microM and 20 microM) and doxorubicin (30 and 60 n M) dramatically augmented TRAIL-induced apoptosis in LNCaP and PC3 cells. TRAIL-induced apoptosis was partially abrogated by overexpression of bcl-2 in these two cell lines when it was used in combination with the above agents. Similar results were obtained using clonogenic survival assays where bcl-2 overexpression was also found to marginally protect against TRAIL- and chemotherapy-induced cell killing. CONCLUSIONS: This study demonstrates that combination treatment of prostate cancer cells with TRAIL and chemotherapeutic agents overcomes their resistance by triggering caspase activation. This greater than additive effect of cotreatment with TRAIL and chemotherapy may provide the basis for a new therapeutic approach to induce apoptosis in otherwise resistant cancer cells.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Glicoproteínas de Membrana/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Factor de Necrosis Tumoral alfa/farmacología , Proteínas Reguladoras de la Apoptosis , Western Blotting , Caspasas/metabolismo , Supervivencia Celular/efectos de los fármacos , Cisplatino/farmacología , Terapia Combinada , Doxorrubicina/farmacología , Sinergismo Farmacológico , Etopósido/farmacología , Humanos , Masculino , Neoplasias de la Próstata/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Recombinantes/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF , Células Tumorales Cultivadas/efectos de los fármacos , Ensayo de Tumor de Célula MadreRESUMEN
Mantle cell lymphoma (MCL) is a distinct type of B-cell non-Hodgkin's lymphoma characterized by cyclin D1 overexpression and the cytogenetic abnormality, the t(11;14)(q13;q32). MCL cell lines have been difficult to establish and in vitro studies of these neoplasms are scarce. We describe the establishment and characteristics of a new MCL cell line, Mino. The cells are large, growing singly and in small clumps in vitro. By flow cytometry, the immunophenotype was compatible with MCL (i.e. CD5+CD20+CD23-FMC7+). Conventional cytogenetics showed hyperdiploidy with multiple complex karyotypic abnormalities, but no evidence of the t(11;14), proven to be present only by fluorescence in situ hybridization and polymerase chain reaction (PCR) methods. Western blots showed expression of cyclin D1 but no detectable cyclin D2 and cyclin D3; the retinoblastoma protein was predominantly phosphorylated. There was expression of tumor suppressor gene products including p53, p16(INK4a), and p21(WAF1). Sequencing of the TP53 gene revealed a mutation (codon 147(valine-->glycine)) in exon 5. Epstein Barr virus was absent. In summary, Mino is a new MCL cell line that may be useful to study the pathogenesis of MCL.
Asunto(s)
Linfoma de Células del Manto/patología , Células Tumorales Cultivadas , Sustitución de Aminoácidos , Aneuploidia , Antígenos CD/análisis , Western Blotting , Proteínas de Ciclo Celular/análisis , Tamaño de la Célula , Aberraciones Cromosómicas , Codón/genética , Ciclinas/análisis , Exones/genética , Resultado Fatal , Femenino , Genes p53 , Herpesvirus Humano 4 , Humanos , Inmunofenotipificación , Hibridación Fluorescente in Situ , Cariotipificación , Linfoma de Células del Manto/química , Linfoma de Células del Manto/genética , Persona de Mediana Edad , Mutación Missense , Proteínas de Neoplasias/análisis , Mutación Puntual , Reacción en Cadena de la Polimerasa , Células Tumorales Cultivadas/química , Células Tumorales Cultivadas/patologíaRESUMEN
Strong evidence exists to support the tenet that activation of E2F transcription factors, via alterations in the p16-cyclin D-Rb pathway, is a key event in the malignant progression of most human malignant gliomas. The oncogenic ability of E2F has been related to the E2F-mediated up-regulation of several proteins that positively regulate cell proliferation. However, E2F may indirectly enhance proliferation by activating antiapoptotic molecules. In this work, we sought to ascertain whether E2F-1-mediated events involve the up-regulation of the antiapoptotic molecule Bcl-2. Western blot analyses showed up-regulation of Bcl-2 but not of Bcl-x(L) by 24 h after the transfer of E2F-1. Northern blot studies showed that transfer of E2F-1 also up-regulated Bcl-2 RNA. In support of these findings and the concept that E2F-1 has a direct effect in the induction of Bcl-2, we found a putative E2F binding site within the Bcl-2 sequence. Subsequent gel-mobility shift and supershift experiments involving the CTCCGCGC site in the bcl-2 promoter showed that E2F-1 bound Bcl-2. Transactivation experiments consistently showed that ectopic E2F-1 activated responsive elements located in the -1448/-1441 region in the P1 promoter region of the bcl-2 gene. As expected, other members of the E2F family of transcription factors such as E2F-2 and E2F-4 also transactivated the bcl-2 promoter. Our results demonstrate that E2F-1 modulates the expression of the antiapoptotic molecule Bcl-2 and suggest that up-regulation of Bcl-2 may favor the oncogenic role of E2F-1 and other members of the E2F family of transcription factors.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Glioma/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Factores de Transcripción/genética , Activación Transcripcional , Sitios de Unión , Ciclo Celular/fisiología , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Factor de Transcripción E2F2 , Factor de Transcripción E2F4 , Regulación Neoplásica de la Expresión Génica , Técnicas de Transferencia de Gen , Genes bcl-2/genética , Glioma/metabolismo , Humanos , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Factores de Transcripción/biosíntesis , Regulación hacia ArribaRESUMEN
The bcl-2 proto-oncogene is frequently expressed in human cancer. Although bcl-2 was first cloned as the t(14;18) translocation breakpoint from human follicular B-cell lymphoma, it has become apparent that many cell types express bcl-2 because of transcriptional regulation. As such, several transcription factors have been demonstrated to activate expression of bcl-2, including NF-kappaB. We investigated the role of NF-kappaB1 (p50) homodimers in the expression of Bcl-2 in two murine B-cell lymphoma cell lines: LY-as, an apoptosis-proficient line with low Bcl-2 protein expression and no nuclear NF-kappaB activity, and LY-ar, a nonapoptotic line with constitutive p50 homodimer activity and 30 times more Bcl-2 protein expression than LY-as. We found that nuclear p50 homodimer activity correlated with Bcl-2 expression in these cell types and identified several sites within the bcl-2 5'-flanking region that p50 was capable of binding. In vitro transcription revealed that recombinant p50 enhanced the production of run-off transcripts from the bcl-2 P1 promoter. Additional in vitro transcription experiments suggested the sites by which p50 afforded this effect. We conclude that the p50 homodimer is capable of transcriptional activation of the bcl-2 gene and suggest that its nuclear activity contributes to the expression of bcl-2 in LY-ar cells.
Asunto(s)
FN-kappa B/química , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transcripción Genética , Animales , Apoptosis , Sitios de Unión , Western Blotting , Calpaína/farmacología , Línea Celular , Núcleo Celular/metabolismo , Dimerización , Inhibidores Enzimáticos/farmacología , Ratones , Subunidad p50 de NF-kappa B , Regiones Promotoras Genéticas , Unión Proteica , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-bcl-2/genética , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
In prostate carcinoma, overexpression of the anti-apoptotic gene Bcl-2 has been found to be associated with resistance to therapies including radiation and androgen ablation. Restoring the balance of Bcl-2 family members may result in the induction of apoptosis in prostate cancer cells previously resistant to treatment. To accomplish this, a strategy involving overexpression of the pro-apoptotic gene Bax was executed. The use of cytotoxic genes such as Bax require selective expression of the gene. In this study, we examined the ability of selective expression of Bax protein directed by a prostate-specific promoter to induce apoptosis in human prostate carcinoma. A second-generation adenoviral vector was constructed with the modified prostate-specific probasin promoter, ARR2PB, directing expression of an HA-tagged Bax gene and a green fluorescent protein reporter translated from an internal ribosome entry site (ARR2PB.Bax.GFP). ARR2PB promoter activity is tightly regulated and highly prostate specific and is responsive to androgens and glucocorticoids. The prostate-specific promoter-Bax-GFP transgene cassette was inserted into a cloning site near the right inverted terminal repeat of the adenoviral vector to retain specificity of the promoter. LNCaP cells infected with Ad/ARR(2)PB.Bax.GFP showed high levels of Bax expression 48 h after infection resulting in an 85% reduction in cell viability. Importantly, LNCaP cells stably transfected to overexpress Bcl-2 showed similar patterns of cell death when infected with Ad/ARR(2)PB.Bax.GFP, an 82% reduction in cell viability seen 48 h after infection. Apoptosis was confirmed by measuring caspase activation and using the TUNEL assay. Tissue specificity was evaluated using A549 cells (lung adenocarcinoma), SK-Hep-1 (liver cancer) cells, and Hela (cervical cancer) cells which did not show detectable expression of virally delivered Bax protein or any increase in cell death. Systemic administration of Ad/ARR2PB. Bax.GFP in nude mice revealed no toxicity in liver, lung, kidney, or spleen. This study shows that infection with the second-generation adenovirus, ARR2PB.Bax.GFP, results in highly specific cytotoxicity in LNCaP cells, and that consequent overexpression of Bax in prostate carcinoma, even in the context of high levels of Bcl-2 protein, resulted in apoptosis. These results suggest that a second-generation adenovirus-mediated, prostate-specific Bax gene therapy is a promising approach for the treatment of prostate cancer.
Asunto(s)
Adenoviridae/genética , Apoptosis , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Neoplasias de la Próstata/terapia , Proteínas Proto-Oncogénicas/genética , Animales , Western Blotting/métodos , Caspasa 3 , Caspasas/metabolismo , Activación Enzimática , Expresión Génica , Vectores Genéticos/toxicidad , Humanos , Etiquetado Corte-Fin in Situ , Masculino , Ratones , Ratones Desnudos , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas c-bcl-2/genética , Factores de Tiempo , Células Tumorales Cultivadas , Proteína X Asociada a bcl-2RESUMEN
Anaplastic large-cell lymphoma (ALCL) of T- or null-cell lineage, as defined in the revised European-American lymphoma classification, includes a subset of tumors that carry the t(2;5)(p23;q35) resulting in overexpression of anaplastic lymphoma kinase (ALK). Patients with ALK+ ALCL are reported to have a better prognosis than patients with ALK- ALCL. Because the mechanisms for this survival difference are unknown, we investigated the hypothesis that apoptotic pathways may be involved. We therefore assessed expression levels of the anti-apoptotic proteins BCL-2 and BCL-XL and the pro-apoptotic proteins BAX and BCL-XS in T/null-cell ALCL using immunohistochemical methods and correlated the findings with ALK expression and apoptotic rate (AR), the latter assessed by a modified Tdt-mediated dUTP nick-end labeling assay. ALK was detected in 21 of 66 (31.8%) ALCLs. BCL-2 was not detected in 21 ALK+ ALCLs but was present in 26 of 45 (57.8%) ALK- ALCLs (P < 0.0001). ALK+ and ALK- ALCLs also showed significant differences in expression of BCL-XL, BAX, and BCL-XS. ALK+ tumors less commonly had a high level of BCL-XL (1 of 17 versus 14 of 35, P = 0.01), and more commonly had high levels of BAX (13 of 18 versus 15 of 36, P = 0.05), and BCL-XS (11 of 16 versus 12 of 31, P = 0.05) compared with ALK- tumors. ALK+ tumors also had a higher mean AR than ALK- tumors (3.4% versus 1.1%, P = 0.0002). Differential expression of BCL-2 family proteins may be responsible for the higher AR observed in ALK+ ALCL and provides a possible biological explanation for the better prognosis reported for patients with ALK+ ALCL.
Asunto(s)
Genes bcl-2 , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/patología , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Adolescente , Adulto , Anciano , Quinasa de Linfoma Anaplásico , Apoptosis , División Celular , Niño , Preescolar , Mapeo Cromosómico , Cromosomas Humanos Par 2 , Cromosomas Humanos Par 5 , Femenino , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Masculino , Persona de Mediana Edad , Índice Mitótico , Estadificación de Neoplasias , Pronóstico , Proteínas Tirosina Quinasas/análisis , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Proteínas Tirosina Quinasas Receptoras , Translocación Genética , Proteína X Asociada a bcl-2 , Proteína bcl-XRESUMEN
To determine if TRAIL-induced apoptosis in human prostate tumor cells was suppressed by bcl-2, we compared the levels of apoptosis induced by recombinant human TRAIL in pairs of isogenic cell lines that do or do not express bcl-2. Three human prostate tumor cell lines (PC3, DU145 and LNCaP) and their bcl-2-expressing counterparts were tested for their susceptibility to TRAIL. Cells were exposed to TRAIL in the presence of cycloheximide which acted as a sensitizer. Apoptosis was induced rapidly in PC3 and DU145 neo-control transfected cells, whereas induction in LNCaP required 24 h. All three cell line variants expressing bcl-2 were resistant to the apoptotic effects of TRAIL. Caspase 3 and 8 activation was also detected in the neo control cells after treatment with TRAIL, demonstrating the rapid activation of the caspase cascade similar to that seen with other death receptors. Bcl-2 overexpression in these cells blocked activation of these caspases, suggesting that bcl-2 expression of human cancer cells may be a critical factor in the therapeutic efficacy of TRAIL.