RESUMEN
Urothelial cell carcinoma (UCC) is the second most common genitourinary malignant disease in the USA, and tobacco smoking is the major known risk factor for UCC development. Exposure to carcinogens, such as those contained in tobacco smoke, is known to directly or indirectly damage DNA, causing mutations, chromosomal deletion events and epigenetic alterations in UCC. Molecular studies have shown that chromosome 9 alterations and P53, RAS, RB and PTEN mutations are among the most frequent events in UCC. Recent studies suggested that continuous tobacco carcinogen exposure drives and enhances the selection of epigenetically altered cells in UCC, predominantly in the invasive form of the disease. However, the sequence of molecular events that leads to UCC after exposure to tobacco smoke is not well understood. To elucidate molecular events that lead to UCC oncogenesis and progression after tobacco exposure, we developed an in vitro cellular model for smoking-induced UCC. SV-40 immortalized normal HUC1 human bladder epithelial cells were continuously exposed to 0.1% cigarette smoke extract (CSE) until transformation occurred. Morphological alterations and increased cell proliferation of non-malignant urothelial cells were observed after 4 months (mo) of treatment with CSE. Anchorage-independent growth assessed by soft agar assay and increase in the migratory and invasive potential was observed in urothelial cells after 6 mo of CSE treatment. By performing a PCR mRNA expression array specific to the PI3K-AKT pathway, we found that 26 genes were upregulated and 22 genes were downregulated after 6 mo of CSE exposure of HUC1 cells. Among the altered genes, PTEN, FOXO1, MAPK1 and PDK1 were downregulated in the transformed cells, while AKT1, AKT2, HRAS, RAC1 were upregulated. Validation by RT-PCR and western blot analysis was then performed. Furthermore, genome-wide methylation analysis revealed MCAM, DCC and HIC1 are hypermethylated in CSE-treated urothelial cells when compared with non-CSE exposed cells. The methylation status of these genes was validated using quantitative methylation-specific PCR (QMSP), confirming an increase in methylation of CSE-treated urothelial cells compared to untreated controls. Therefore, our findings suggest that a tobacco signature could emerge from distinctive patterns of genetic and epigenetic alterations and can be identified using an in vitro cellular model for the development of smoking-induced cancer.
Asunto(s)
Metilación de ADN , Genoma Humano , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fumar , Carcinoma de Células Transicionales/metabolismo , Carcinoma de Células Transicionales/patología , Proteínas de Ciclo Celular , Línea Celular , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica/efectos de los fármacos , Receptor DCC , Regulación hacia Abajo/efectos de los fármacos , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/metabolismo , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Extractos Vegetales/farmacología , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Nicotiana/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
A fully human monoclonal antibody (CS-D7, IgG1) specific for the iron regulated surface determinant B (IsdB) of Staphylococcus aureus was isolated from the Cambridge Antibody Technology (CAT) scFv antibody library. As compared to previously described IsdB specific murine monoclonals, CS-D7 has a unique, non-overlapping binding site on IsdB, and exhibits increased in vivo activity. The antibody recognizes a conformational epitope spanning amino acids 50 to 285 and has a binding affinity of 340 (± 75) pM for IsdB. CS-D7 bound to a wide variety of S. aureus strains, but not to an isdB deletion mutant. The antibody mediated opsonophagocytic (OP) killing in vitro and mediated significant protection in vivo. In a murine lethal sepsis model, the antibody conferred protection from death when dosed prior to challenge, but not when dosed after challenge. Importantly, in a central venous catheter (CVC) model in rats, the antibody reduced bacteremia and prevented colonization of indwelling catheters. Protection was observed when rats were dosed with CS-D7 prior to challenge as well as post challenge. IsdB is currently being investigated for clinical efficacy against S. aureus infection, and the activity of this human IsdB specific antibody supplements the growing body of evidence to support targeting this antigen for vaccine development.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Proteínas de Transporte de Catión/inmunología , Infecciones Estafilocócicas/mortalidad , Infecciones Estafilocócicas/prevención & control , Staphylococcus aureus/inmunología , Animales , Anticuerpos Antibacterianos/inmunología , Anticuerpos Antibacterianos/metabolismo , Anticuerpos Monoclonales/metabolismo , Especificidad de Anticuerpos , Bacteriemia/inmunología , Bacteriemia/microbiología , Bacteriemia/mortalidad , Bacteriemia/prevención & control , Cateterismo Venoso Central/efectos adversos , Proteínas de Transporte de Catión/genética , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Proteínas Opsoninas/metabolismo , Fagocitosis , Ratas , Ratas Sprague-Dawley , Sepsis/microbiología , Sepsis/mortalidad , Sepsis/prevención & control , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidad , Tasa de Supervivencia , Resultado del TratamientoRESUMEN
Staphylococcus aureus is a major cause of nosocomial infections worldwide, and the rate of resistance to clinically relevant antibiotics, such as methicillin, is increasing; furthermore, there has been an increase in the number of methicillin-resistant S. aureus community-acquired infections. Effective treatment and prevention strategies are urgently needed. We investigated the potential of the S. aureus surface protein iron surface determinant B (IsdB) as a prophylactic vaccine against S. aureus infection. IsdB is an iron-sequestering protein that is conserved in diverse S. aureus clinical isolates, both methicillin resistant and methicillin sensitive, and it is expressed on the surface of all isolates tested. The vaccine was highly immunogenic in mice when it was formulated with amorphous aluminum hydroxyphosphate sulfate adjuvant, and the resulting antibody responses were associated with reproducible and significant protection in animal models of infection. The specificity of the protective immune responses in mice was demonstrated by using an S. aureus strain deficient for IsdB and HarA, a protein with a high level of identity to IsdB. We also demonstrated that IsdB is highly immunogenic in rhesus macaques, inducing a more-than-fivefold increase in antibody titers after a single immunization. Based on the data presented here, IsdB has excellent prospects for use as a vaccine against S. aureus disease in humans.