RESUMEN
The human fetal osteoblast cell line (hFOB 1.19) has been proposed as an accessible experimental model for study of osteoblast biology relating to drug development and biomaterial engineering. For their multilineage differentiation potential, hFOB has been compared to human mesenchymal progenitor cells and used to investigate bone-metabolism in vitro. Hereby, we studied whether and to what extent the conditionally immortalized cell line hFOB 1.19 can serve as a surrogate model for bone-marrow derived mesenchymal stromal cells (bmMSC). hFOB indeed exhibit specific characteristics reminiscent of bmMSC, as colony formation, migration capacity and the propensity to grow as multicellular aggregates. After prolonged culture, in contrast to the expected effect of immortalization, hFOB acquired a delayed growth rate. In close resemblance to bmMSC at increasing passages, also hFOB showed morphological abnormalities, enlargement and finally reduced proliferation rates together with enhanced expression of the cell cycle inhibitors p21 and p16. hFOB not only have the ability to undergo multilineage differentiation but portray several important aspects of human bone marrow mesenchymal stromal cells. Superior to primary MSC and osteoblasts, hFOB enabled the generation of continuous cell lines. These provide an advanced basis for investigating age-related dysfunctions of MSCs in an in vitro 3D-stem cell microenvironment.
Asunto(s)
Huesos/embriología , Técnicas de Cultivo de Célula/métodos , Células Madre Mesenquimatosas/citología , Osteoblastos/citología , Huesos/citología , Huesos/metabolismo , Diferenciación Celular , Línea Celular , Movimiento Celular , Proliferación Celular , Medios de Cultivo Condicionados , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Humanos , Células Madre Mesenquimatosas/metabolismo , Osteoblastos/metabolismoRESUMEN
On the 18th of March 2020, the Malaysia government declared a movement control order (MCO) due to the unprecedented COVID-19 pandemic. Although the majority of patients presented with respiratory-related symptoms, COVID-19 patients may present atypically with neurological manifestations and may even have an increased risk of stroke. The Malaysia Stroke Council is concerned regarding the level of care given to stroke patients during this pandemic. During the recent National Stroke Workflow Steering Committee meeting, a guide was made based on the currently available evidences to assist Malaysian physicians providing acute stroke care in the hospital setting in order to provide the best stroke care while maintaining their own safety. The guide comprises of prehospital stroke awareness, hyperacute stroke care, stroke care unit and intensive care unit admission, post-stroke rehabilitation and secondary prevention practice. We urge continuous initiative to provide the best stroke care possible and ensure adequate safety for both patients and the stroke care team.
Asunto(s)
Betacoronavirus , Infecciones por Coronavirus/complicaciones , Pandemias , Neumonía Viral/complicaciones , Accidente Cerebrovascular/terapia , COVID-19 , Humanos , Unidades de Cuidados Intensivos , Malasia , Guías de Práctica Clínica como Asunto , SARS-CoV-2 , Accidente Cerebrovascular/diagnóstico por imagen , Rehabilitación de Accidente Cerebrovascular , Tomografía Computarizada por Rayos XRESUMEN
Tumor-stromal interaction is a dynamic process that promotes tumor growth and metastasis via cell-cell interaction and extracellular vesicles. Recent studies demonstrate that stromal fibroblast-derived molecular signatures can be used to predict disease progression and drug resistance. To identify the epigenetic role of stromal noncoding RNAs in tumor-stromal interactions in the tumor microenvironment, we performed microRNA profiling of patient cancer-associated prostate stromal fibroblasts isolated by laser capture dissection microscopy and in bone-associated stromal models. We found specific upregulation of miR-409-3p and miR-409-5p located within the embryonically and developmentally regulated DLK1-DIO3 (delta-like 1 homolog-deiodinase, iodothyronine 3) cluster on human chromosome 14. The findings in cell lines were further validated in human prostate cancer tissues. Strikingly, ectopic expression of miR-409 in normal prostate fibroblasts conferred a cancer-associated stroma-like phenotype and led to the release of miR-409 via extracellular vesicles to promote tumor induction and epithelial-to-mesenchymal transition in vitro and in vivo. miR-409 promoted tumorigenesis through repression of tumor suppressor genes such as Ras suppressor 1 and stromal antigen 2. Thus, stromal fibroblasts derived miR-409-induced tumorigenesis, epithelial-to-mesenchymal transition and stemness of the epithelial cancer cells in vivo. Therefore, miR-409 appears to be an attractive therapeutic target to block the vicious cycle of tumor-stromal interactions that plagues prostate cancer patients.
Asunto(s)
Carcinogénesis/genética , Transformación Celular Neoplásica/genética , Transición Epitelial-Mesenquimal/genética , Fibroblastos/patología , MicroARNs/genética , Próstata/patología , Neoplasias de la Próstata/genética , Animales , Carcinogénesis/patología , Línea Celular Tumoral , Transformación Celular Neoplásica/patología , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Masculino , Ratones , Ratones Desnudos , Neoplasias de la Próstata/patología , Microambiente Tumoral/genética , Regulación hacia Arriba/genéticaRESUMEN
Perlecan/HSPG2, a large heparan sulfate (HS) proteoglycan, normally is expressed in the basement membrane (BM) underlying epithelial and endothelial cells. During prostate cancer (PCa) cell invasion, a variety of proteolytic enzymes are expressed that digest BM components including perlecan. An enzyme upregulated in invasive PCa cells, matrilysin/matrix metalloproteinase-7 (MMP-7), was examined as a candidate for perlecan proteolysis both in silico and in vitro. Purified perlecan showed high sensitivity to MMP-7 digestion even when fully decorated with HS or when presented in native context connected with other BM proteins. In both conditions, MMP-7 produced discrete perlecan fragments corresponding to an origin in immunoglobulin (Ig) repeat region domain IV. While not predicted by in silico analysis, MMP-7 cleaved every subpart of recombinantly generated perlecan domain IV. Other enzymes relevant to PCa that were tested had limited ability to cleave perlecan including prostate specific antigen, hepsin, or fibroblast activation protein α. A long C-terminal portion of perlecan domain IV, Dm IV-3, induced a strong clustering phenotype in the metastatic PCa cell lines, PC-3 and C4-2. MMP-7 digestion of Dm IV-3 reverses the clustering effect into one favoring cell dispersion. In a C4-2 Transwell® invasion assay, perlecan-rich human BM extract that was pre-digested with MMP-7 showed loss of barrier function and permitted a greater level of cell penetration than untreated BM extract. We conclude that enzymatic processing of perlecan in the BM or territorial matrix by MMP-7 as occurs in the invasive tumor microenvironment acts as a molecular switch to alter PCa cell behavior and favor cell dispersion and invasiveness.
Asunto(s)
Proteoglicanos de Heparán Sulfato/genética , Metaloproteinasa 7 de la Matriz/genética , Invasividad Neoplásica/genética , Neoplasias de la Próstata/genética , Membrana Basal/metabolismo , Membrana Basal/patología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Proteínas de la Matriz Extracelular/genética , Regulación Neoplásica de la Expresión Génica , Proteoglicanos de Heparán Sulfato/metabolismo , Humanos , Masculino , Metaloproteinasa 7 de la Matriz/metabolismo , Invasividad Neoplásica/patología , Neoplasias de la Próstata/patología , Proteolisis , Activación Transcripcional , Microambiente TumoralRESUMEN
Epithelial-mesenchymal transition (EMT) is a crucial mechanism for the acquisition of migratory and invasive capabilities by epithelial cancer cells. By conducting quantitative proteomics in experimental models of human prostate cancer (PCa) metastasis, we observed strikingly decreased expression of EPLIN (epithelial protein lost in neoplasm; or LIM domain and actin binding 1, LIMA-1) upon EMT. Biochemical and functional analyses demonstrated that EPLIN is a negative regulator of EMT and invasiveness in PCa cells. EPLIN depletion resulted in the disassembly of adherens junctions, structurally distinct actin remodeling and activation of ß-catenin signaling. Microarray expression analysis identified a subset of putative EPLIN target genes associated with EMT, invasion and metastasis. By immunohistochemistry, EPLIN downregulation was also demonstrated in lymph node metastases of human solid tumors including PCa, breast cancer, colorectal cancer and squamous cell carcinoma of the head and neck. This study reveals a novel molecular mechanism for converting cancer cells into a highly invasive and malignant form, and has important implications in prognosis and treating metastasis at early stages.
Asunto(s)
Proteínas del Citoesqueleto/biosíntesis , Regulación hacia Abajo , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/biosíntesis , Neoplasias de la Próstata/metabolismo , Uniones Adherentes/metabolismo , Uniones Adherentes/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Femenino , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Humanos , Ganglios Linfáticos/patología , Metástasis Linfática , Masculino , Invasividad Neoplásica , Neoplasias de la Próstata/patología , Transducción de Señal , beta Catenina/metabolismoRESUMEN
Semiconductor quantum dots (QDs) are bright fluorescent nanoparticles that have been successfully used for the detection of biomarker expression in cells. The objective of the present study is to use this technology in a multiplexing manner to determine at a single cell level the expression of a cell-specific bio-marker, prostate-specific antigen (PSA) expressed by human prostate cancer LNCaP and ARCaP cell lines. Here we compared the sensitivity of immunohistochemistry (IHC) and QD-based detection of AR and PSA expression in these cell lines. Further, we conducted multiplexing QD-based detection of PSA and androgen receptor (AR) expression in LNCaP cells subjecting to androgen (R1881) stimulation. The involvement of AR in PSA regulation in LNCaP cells, at a single cell level, was confirmed by the co-incubation of LNCaP cells in the presence of both R1881 and its receptor antagonist, bicalutamide (Casodex). We showed here the superior quality of QDs, in comparison to IHC, for the detection of AR and PSA in cultured LNCaP and ARCaP cells. Multiplexing QDs technique can be used to detect simultaneously AR and PSA expression induced by R1881 which promoted AR translocation from its cytosolic to the nuclear compartment. We observed AR antagonist, bicalutamide, inhibited AR nuclear translocation and PSA, but not AR expression in LNCaP cells.
Asunto(s)
Antígeno Prostático Específico/biosíntesis , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/biosíntesis , Antagonistas de Receptores Androgénicos , Andrógenos/farmacología , Anilidas/farmacología , Línea Celular Tumoral , Humanos , Inmunohistoquímica , Masculino , Metribolona/farmacología , Nitrilos/farmacología , Transporte de Proteínas , Puntos Cuánticos , Compuestos de Tosilo/farmacologíaRESUMEN
Aberrant expression of vascular endothelial growth factor (VEGF) is associated with human prostate cancer (PCa) metastasis and poor clinical outcome. We found that both phosphorylation of cyclic AMP-responsive element-binding protein (CREB) and VEGF levels were significantly elevated in patient bone metastatic PCa specimens. A PCa ARCaP progression model demonstrating epithelial-to-mesenchymal transition exhibited increased CREB phosphorylation and VEGF expression as ARCaP cells became progressively more mesenchymal and bone-metastatic. Activation of CREB induced, whereas inhibition of CREB blocked, VEGF expression in ARCaP cells. CREB may regulate VEGF transcription via a hypoxia-inducible factor-dependent mechanism in normoxic conditions. Activation of CREB signaling is involved in the coordinated regulation of VEGF and may pre-dispose to PCa bone metastasis.
Asunto(s)
Neoplasias Óseas/secundario , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Próstata/patología , Factor A de Crecimiento Endotelial Vascular/genética , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/análisis , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Masculino , Regiones Promotoras Genéticas , Neoplasias de la Próstata/química , Neoplasias de la Próstata/metabolismo , Transcripción Genética , Factor A de Crecimiento Endotelial Vascular/sangreRESUMEN
We studied the psychological impact of the outbreak of Severe Acute Respiratory Syndrome (SARS) to understand if age and residential location were risk factors associated with post-traumatic disturbance, namely intrusion, avoidance, and hyperarousal. One hundred and forty-six volunteers belonging to four groups classified along the dimensions of age (middle-aged versus older-aged) and location (high SARS-prevalent regions versus low SARS-prevalent regions), participated in this study. After controlling for depression, residents in high SARS-prevalent regions, regardless of age, consistently developed more intense post-traumatic disturbance than residents in low SARS-prevalent regions. Furthermore, the prevalence of probable post-traumatic stress disorder (PTSD) cases was significantly higher in older people and in residents of SARS-prevalent regions. Our findings suggest the importance of mental health aftercare in the post-epidemic period of disease epidemics.
Asunto(s)
Envejecimiento/psicología , Síndrome Respiratorio Agudo Grave/psicología , Trastornos por Estrés Postraumático/epidemiología , Trastornos por Estrés Postraumático/psicología , Adaptación Psicológica/fisiología , Factores de Edad , Anciano , Actitud Frente a la Salud , Trastorno Depresivo/epidemiología , Trastorno Depresivo/psicología , Brotes de Enfermedades , Humanos , Persona de Mediana Edad , Trastornos de la Personalidad/epidemiología , Trastornos de la Personalidad/psicología , Prevalencia , Escalas de Valoración Psiquiátrica/estadística & datos numéricos , Características de la Residencia , Factores de Riesgo , Índice de Severidad de la EnfermedadRESUMEN
Numerous mouse models of prostate carcinogenesis have been developed, but hitherto there has been no model in which the prostate gland could be imaged in live animals. The transgenic model generated here targeted mouse prostate gland using a firefly luciferase enzyme under the control of a small but highly active and specific supra prostate-specific antigen (sPSA) promoter. We evaluated postnatal prostate development, involution and androgen-induced restoration of prostate growth in adult transgenic mice using bioluminescence imaging. Results of our study showed that: (i) the prostate gland of male offspring did not yield a significant bioluminescence signal until after sexual maturity. Luciferase was detected in the luminal epithelial cells of the ventral and dorsolateral lobes of the prostate gland and caput epididymis, with little or no activity in 18 other organs evaluated. (ii) While a constant high level of bioluminescence was detected in the mouse prostate from 5 to 35 weeks of age, a slight drop in bioluminescence was detected at 36 to 54 weeks. (iii) Upon castration, the luciferase activity signal associated with mouse prostate detected by a cooled charge-coupled device camera was dramatically reduced. This signal could be rapidly restored to pre-castration levels after androgen administration. Androgen-induced luciferase activity subsided to nearly basal levels 5 days following the last injection. These data demonstrate that a bioluminescent mouse model with luciferase activity restricted to the prostate gland under the control of a (sPSA) promoter can be used on a real-time basis in live animals to investigate the development and responsiveness of the prostate gland to exogenously administered androgen. This model can be extended to detect the responsiveness of the prostate gland to therapy and used as a founder strain to visualize tumors in hosts with different genetic backgrounds.
Asunto(s)
Andrógenos/metabolismo , Luciferasas/metabolismo , Ratones Transgénicos , Próstata/crecimiento & desarrollo , Próstata/metabolismo , Animales , Castración , Femenino , Luciferasas/genética , Masculino , Ratones , Microscopía/métodos , Regiones Promotoras Genéticas , Antígeno Prostático Específico/genética , Antígeno Prostático Específico/metabolismo , Distribución TisularRESUMEN
BACKGROUND: Prostate cancer (PC) has a propensity to metastasize to the skeleton, inducing an osteoblastic response in the host. Recent epidemiological studies have suggested that circulating IGF-I may be important for both the pathogenesis and dissemination of PC. We have postulated that tumor secreted IGF-I in conjunction with endogenous IGF-I contributes to the osteoblastic phenotype characteristic of metastatic PC. METHODS: To test this thesis we studied the established LNCaP PC progression model consisting of three genetically related human PC cell lines. RESULTS: Using RIA, we found serum-free conditioned media (CM) of LNCaP and C4-2 had no measurable IGF-I, whereas IGF-I was easily detected in CM from C4-2B cells at 24 hr (i.e., 1.8 +/- 0.53 ng/mg cell protein). Real-time PCR of IGF-I mRNA showed that C4-2B expressed 100-fold more IGF-I mRNA than LNCaP cells. In addition, C4-2B expression of IGF-I mRNA was substantially increased in the presence of exogenous IGF-I to nearly twofold. While IGFBP-3 and IGFBP-1 were not detectable in the CM of any PC line, all cells secreted IGFBP-2. C4-2B cells produced 40% more IGFBP-2 than LNCaP or C4-2 cells (C4-2B at 167 +/- 43 ng/mg cell protein). RANKL, a product of bone stromal cells, was also differentially expressed: LNCaP had threefold higher RANKL mRNA compared to C4-2 and C4-2B and at least equivalent protein expression. CONCLUSIONS: Our results suggest that PC cells that have metastasized to bone have an upregulated IGF-I regulatory system. This suggests an activated IGF-I axis contributes to the host-PC interaction in promoting osteoblastic metastases.
Asunto(s)
Neoplasias Óseas/secundario , Factor I del Crecimiento Similar a la Insulina/metabolismo , Neoplasias de la Próstata/patología , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Medios de Cultivo Condicionados , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/genética , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Ligando RANK , ARN Mensajero/química , ARN Mensajero/genética , ARN Ribosómico 18S/genética , ARN Ribosómico 18S/metabolismo , Receptor Activador del Factor Nuclear kappa-B , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia ArribaRESUMEN
Prostate cancer is the most common cancer and the second leading cause of cancer-related death among North American men. The low cure rate for prostate cancer is associated with the fact that many patients have metastatic disease at the time of disease presentation. Currently available therapeutic modalities for prostate cancer, such as surgery, radiation, hormone therapy, and chemotherapy, have failed to cure patients because these therapies are not sufficiently tumor-specific, resulting in dose-limiting toxicity. Therefore, gene therapy may offer great promise in this regard. In this article, we summarize current advances in gene therapy technologies for the treatment of cancer in general, and future prospects for treatment of human prostate cancer metastasis. We specifically emphasize current studies for improvement, both in the efficiency and the specificity of viral and nonviral vectors, and restricted transgene expression in tumors, to achieve selective targeting with minimized host organ toxicity, based on the molecular understanding of potential regulatory differences between normal and tumor cells. Cell and animal models used to study prostate cancer growth, invasion, and metastasis, and their usefulness in preclinical evaluation of therapeutic vectors in the treatment of prostate cancer skeletal metastasis are also discussed.
Asunto(s)
Adenocarcinoma/terapia , Neoplasias de la Próstata/terapia , Adenocarcinoma/genética , Adenocarcinoma/patología , Proteínas E1A de Adenovirus/fisiología , Animales , Biolística , Vacunas contra el Cáncer/uso terapéutico , Ciclo Celular/genética , Terapia Combinada , Citocinas/uso terapéutico , ADN Recombinante/administración & dosificación , ADN Recombinante/uso terapéutico , Células Dendríticas/trasplante , Portadores de Fármacos , Predicción , Regulación Neoplásica de la Expresión Génica , Vectores Genéticos/administración & dosificación , Vectores Genéticos/uso terapéutico , Humanos , Fragmentos de Inmunoglobulinas/uso terapéutico , Inmunoterapia/métodos , Inyecciones Intralesiones , Liposomas/administración & dosificación , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Modelos Animales , Invasividad Neoplásica , Metástasis de la Neoplasia , Trasplante de Neoplasias , Oligonucleótidos Antisentido/uso terapéutico , Especificidad de Órganos , Regiones Promotoras Genéticas , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , ARN Catalítico/uso terapéutico , Ratas , Trasplante HeterólogoRESUMEN
The growth and development of the prostate gland are regulated by the androgen and the androgen receptor (AR). Despite our molecular understanding of the roles of the AR regulating; a downstream target gene transcription, the direct or indirect (stromally mediated) actions of the androgen in controlling prostate cell growth and differentiation are still unclear. In this report, an invasive; and metastatic human prostate tumor cell line, androgen-repressed human prostate cancer cell line (ARCaP), either transduced with wild-type human AR (hAR) or a control neomycin-resistant plasmid DNA, was used to evaluate the direct role of AR in regulating prostate tumor cell growth and gene transcription. Results showed that: (a) introduction of wild-type hAR to ARCaP cells restored positive androgen regulation of prostate tumor cell growth in vitro through an enhanced cell-cycle progression from G(0)/G(1) to S and G(2)-M phases; (b) hAR was shown to transactivate glucocorticoid-responsive element but not prostate-specific antigen promoter-directed reporter gene expression; and (c) hAR-transduced ARCaP cells exhibited reduced growth, invasion, and migratory behavior in vitro and tumor growth in vivo. These results suggest that the introduction of hAR into the invasive human prostate cancer ARCaP cell line restored its androgen-regulated cell growth, decreased the rate of tumor growth, and selectively activated AR target gene expression. These cellular functions in response to androgen are commonly associated with increased differentiation of prostate epithelial cells.
Asunto(s)
Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Receptores Androgénicos/fisiología , Activación Transcripcional/fisiología , Andrógenos/fisiología , Animales , Adhesión Celular/fisiología , División Celular/fisiología , Movimiento Celular/fisiología , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Metribolona/farmacología , Ratones , Ratones Desnudos , Receptores Androgénicos/genética , Congéneres de la Testosterona/farmacología , Transfección , Células Tumorales CultivadasRESUMEN
Prostate cancer has a high propensity to metastasize to bone, which often resists hormone, radiation, and chemotherapies. Because of the reciprocal nature of the prostate cancer and bone stroma interaction, we designed a cotargeting strategy using a conditional replication-competent adenovirus to target the growth of tumor cells and their associated osteoblasts. The recombinant Ad-OC-E1a was constructed using a noncollagenous bone matrix protein osteocalcin (OC) promoter to drive the viral early E1a gene with restricted replication in cells that express OC transcriptional activity. Unlike Ad-PSE-E1a, Ad-OC-E1a was highly efficient in inhibiting the growth of PSA-producing (LNCaP, C4-2, and ARCaP) and nonproducing (PC-3 and DU145) human prostate cancer cell lines. This virus was also found to effectively inhibit the growth of human osteoblasts and human prostate stromal cells in vitro. Athymic mice bearing s.c. androgen receptor-negative and PSA-negative PC-3 xenografts responded to a single intratumoral administration of 2 x 10(9) plaque-forming unit(s) of Ad-OC-E1a. In SCID/bg mice, intraosseous growth of androgen receptor-positive and PSA-producing C4-2 xenografts responded markedly to i.v. administrations of a single dose of Ad-OC-E1a. One hundred percent of the treated mice responded to this systemic Ad-OC-E1a therapy with a decline of serum PSA to an undetectable level, and 80% of the mice with PSA rebound responded to the second dose of systemic Ad-OC-E1a. Forty percent of the mice were found to be cured by systemic Ad-OC-E1a without subsequent PSA rebound or tumor cells found in the skeleton. This cotargeting strategy shows a broader spectrum and appears to be more effective than systemic Ad-PSE-E1a in preclinical models of human prostate cancer skeletal metastasis.
Asunto(s)
Proteínas E1A de Adenovirus/genética , Neoplasias Óseas/secundario , Neoplasias Óseas/terapia , Terapia Genética/métodos , Osteocalcina/genética , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/terapia , Adenovirus Humanos/genética , Adenovirus Humanos/fisiología , Animales , Neoplasias Óseas/genética , División Celular , Inhibidores de Crecimiento/biosíntesis , Inhibidores de Crecimiento/genética , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Desnudos , Ratones SCID , Neoplasias Hormono-Dependientes/patología , Neoplasias Hormono-Dependientes/terapia , Osteocalcina/biosíntesis , Osteoclastos/patología , Regiones Promotoras Genéticas , Antígeno Prostático Específico/fisiología , Neoplasias de la Próstata/genética , Receptores Androgénicos/fisiología , Células del Estroma/patología , Replicación Viral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A three-dimensional (3D) integrated rotating-wall vessel cell-culture system was used to evaluate the interaction between a human prostate cancer cell line, LNCaP, and microcarrier beads alone, or microcarrier beads previously seeded with either prostate or bone stromal cells. Upon coculture of LNCaP cells with microcarrier beads either in the presence or in the absence of prostate or bone stromal cells, 3D prostate organoids were formed with the expected hormonal responsiveness to androgen, increased cell growth, and prostate-specific antigen production. In this communication, we define permanent phenotypic and genotypic changes of LNCaP cells upon coculture with microcarrier beads alone, or with microcarrier beads previously seeded with either prostate or bone stromal cells. Most notably, we observed selective genetic changes, i.e., chromosomal losses or gains, as evaluated by both conventional cytogenetic and comparative genomic hybridization, in LNCaP sublines derived from the prostate organoids. Moreover, the derivative LNCaP cells appear to have altered growth profiles, and exhibit permanent and stable changes in response to androgen, estrogen, and growth factors. The derivative LNCaP sublines showed increased anchorage-independent growth rate, and enhanced tumorigenicity and metastatic potential when inoculated orthotopically in castrated athymic mice. Our results support the hypothesis that further nonrandom genetic and phenotypic changes in prostate cancer epithelial cells can occur through an event that resembles "adaptive mutation" such as has been described in bacteria subjected to nutritional starvation. The occurrence of such permanent changes may be highly contact dependent, and appears to be driven by specific microenvironmental factors surrounding the tumor cell epithelium grown as 3D prostate organoids.
Asunto(s)
Técnicas de Cultivo de Célula , Genotipo , Fenotipo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Animales , División Celular/efectos de los fármacos , Aberraciones Cromosómicas , Bandeo Cromosómico , Técnicas de Cocultivo , Análisis Citogenético , Estradiol/farmacología , Sustancias de Crecimiento/farmacología , Humanos , Masculino , Metribolona/farmacología , Ratones , Ratones Desnudos , Microesferas , Metástasis de la Neoplasia , Trasplante de Neoplasias , Antígeno Prostático Específico , Rotación , Células del Estroma , Células Tumorales CultivadasRESUMEN
BACKGROUND: The development and progression of prostate neoplasia may recapitulate the early developmental pattern of expression of genes in the prostate. The study of prostate development may, therefore, provide insights into the molecular mechanisms important in prostate neoplasia and reveal new markers. METHODS: We compared postnatal expression of four genes: neu and epidermal growth factor receptor genes (EGFR), androgen-upregulated in the ventral prostate of adult rats (C-3), and androgen-repressed (CK8) in Sprague-Dawley rats. In situ hybridization was performed on prostate frozen sections collected on postnatal days 1, 5, 10, 15, 20, 30, and 60 from five rats per day. Staining intensities for antisense probes specific for each gene were determined relative to day 1 intensity. RESULTS: Growth factor receptors including neu and EGFR may be coordinately regulated in the basal-cell population during prostate development. CK8 and C-3 show evidence of similar androgen regulation during development. CONCLUSIONS: CK8 and C-3 have distinct patterns of expression in the postnatal period of development and these genes may be good markers of differentiation. Both neu and EGFR may be involved in androgen-independent growth of basal cell population in prostate. Prostate 47:164-171, 2001.
Asunto(s)
Proteína de Unión a Andrógenos/genética , Receptores ErbB/genética , Genes erbB-2/fisiología , Queratinas/genética , Próstata/fisiología , Proteína de Unión a Andrógenos/biosíntesis , Proteína de Unión a Andrógenos/fisiología , Animales , Receptores ErbB/biosíntesis , Receptores ErbB/fisiología , Regulación de la Expresión Génica , Genes erbB-2/genética , Hibridación in Situ , Queratinas/sangre , Queratinas/fisiología , Masculino , Próstata/crecimiento & desarrollo , Próstata/metabolismo , Prostateína , Ratas , Ratas Sprague-Dawley , Secretoglobinas , UteroglobinaRESUMEN
During prostate cancer progression, invasive glandular epithelial cells move out of the ductal-acinar architecture and through the surrounding basement membrane. Extracellular matrix proteins and associated soluble factors in the basal lamina and underlying stroma are known to be important regulators of prostate cell behaviors in both normal and malignant tissues. In this study, we assessed cell interactions with extracellular matrix and stromal factors during disease progression by characterizing integrin usage and expression in a series of parental and lineage-derived LNCaP human prostate cancer cell lines. Although few shifts in integrin expression were found to accompany disease progression, integrin heterodimer usage did change significantly. The more metastatic sublines were distinct in their use of alphavbeta3 and, when compared with parental LNCaP cells, showed a shift in alpha6 heterodimerization, a subunit critical not only for interaction with prostate basal lamina but also for interaction with the bone matrix, a favored site of prostate cancer metastases.
Asunto(s)
Adenocarcinoma/secundario , Movimiento Celular/fisiología , Integrinas/fisiología , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/fisiopatología , Adenocarcinoma/patología , Adenocarcinoma/fisiopatología , Adhesión Celular/fisiología , Línea Celular , Medios de Cultivo Condicionados , Progresión de la Enfermedad , Matriz Extracelular/fisiología , Humanos , Técnicas In Vitro , Laminina/fisiología , Masculino , Invasividad Neoplásica , Subunidades de ProteínaRESUMEN
A novel method for the production of adenoviral vectors on a scale sufficient to support most research applications and early phase clinical trials is presented. This method utilizes serum-free cell culture medium and a hollow fiber cell culture apparatus. Significantly less time and space are required than in conventional methods, and the resulting adenovirus is collected in a much smaller volume, simplifying the purification steps. The protocol described is a reproducible, convenient, biologically safe, and environmentally sound method for the production of adenoviral vectors for laboratory use and has the potential to scale-up the adenovirus production for clinical use.
Asunto(s)
Adenoviridae/crecimiento & desarrollo , Vectores Genéticos , Adenoviridae/genética , Técnicas de Cultivo de Célula , Medio de Cultivo Libre de Suero , Recombinación GenéticaRESUMEN
The estrogen receptor-alpha (ER-alpha) is a ligand-dependent transcription factor that regulates the growth, differentiation, and development of hormone-responsive target organs. While ER-alpha has been reported to play critical role in the pathogenesis and prognosis of breast and prostate cancers, its possible functional role in regulating prostate cancer cell growth in a ligand-dependent or -independent manner is poorly understood. We addressed this question by stably transfecting wild type (wt) ER-alpha cDNA into an invasive estrogen receptor-negative human prostate cancer cell line ARCaP. We isolated several clonal lines of transfected cells expressing varying levels of ER-alpha. The ectopic expression of wt ER-a markedly inhibited the growth of ARCaP cells in vitro in an ER-a dose-dependent but ligand-independent manner. Flow cytometric analysis of the wt ER-alpha-transfected ARCaP cells revealed that wt ER-alpha expression arrested cell growth in G1 phase. Our results suggest that ER-alpha may regulate prostate cell growth and participate in the pathogenesis of prostate cancer. ER-alpha may be delivered and expressed ectopically to target prostate cancer progression.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Receptores de Estrógenos/genética , Western Blotting , Ciclo Celular/genética , División Celular/genética , Cartilla de ADN/química , Receptor alfa de Estrógeno , Estrógenos/farmacología , Estrógenos/fisiología , Citometría de Flujo , Humanos , Masculino , Neoplasias Hormono-Dependientes , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Células Tumorales CultivadasRESUMEN
PURPOSE: The goal of this research is to determine the feasibility of an immunotherapeutic approach based on the use of monoclonal antibodies (mAb) to target complement activation fragments on opsonized cancer cells. METHODS: We investigated whether treatment of LNCaP and C4-2 human prostate cancer cell lines with normal human serum would allow for deposition of sufficient amounts of the complement-activation protein C3b and its fragments [collectively referred to as C3b(i)] such that these proteins could serve as cancer-cell-associated antigens for targeting by mAb. Radioimmunoassays, flow cytometry, and magnetic purging with specific immunomagnetic beads were used for the analyses. RESULTS: In vitro opsonization of human prostate cancer cells with normal human serum resulted in deposition of C3b(i) in sufficient quantity (approx. 100,000 molecules/cell) for the cells to be targeted in a variety of protocols. We found that 51Cr-labeled and C3b(i)-opsonized cancer cells could be specifically purged at high efficiency (95%-99%) using anti-C3b(i) mAb covalently coupled to magnetic beads. Flow-cytometry experiments indicated that most normal white cells were not removed under similar conditions. Opsonization of cancer cells with sera from men with prostate cancer led to lower levels of cell-associated IgM and, subsequently, lower amounts of C3b(i) deposited than in normal subjects. Prototype experiments suggested that this deficiency could be corrected by addition of IgM from normal donor plasma. CONCLUSION: mAb directed against complement-activation products may provide new opportunities to deliver diagnostic and therapeutic agents selectively to cancer cells and tumor deposits. These opportunities may include ex vivo purging of C3b(i)-opsonized cancer cells prior to autologous bone marrow or stem cell transplantation.
Asunto(s)
Adenocarcinoma/patología , Anticuerpos Monoclonales/inmunología , Anticuerpos Antineoplásicos/inmunología , Antígenos de Neoplasias/inmunología , Complemento C3b/inmunología , Neoplasias de la Próstata/patología , Adenocarcinoma/sangre , Adenocarcinoma/inmunología , Andrógenos , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Antineoplásicos/uso terapéutico , Especificidad de Anticuerpos , Purgación de la Médula Ósea , Estudios de Factibilidad , Citometría de Flujo , Humanos , Inmunoglobulina M/inmunología , Pruebas Inmunológicas , Separación Inmunomagnética , Inmunoterapia , Masculino , Neoplasias Hormono-Dependientes/inmunología , Neoplasias Hormono-Dependientes/patología , Proteínas Opsoninas/sangre , Proteínas Opsoninas/inmunología , Neoplasias de la Próstata/sangre , Neoplasias de la Próstata/inmunología , Formación de Roseta , Células Tumorales Cultivadas/inmunologíaRESUMEN
A semi-nested reverse transcriptase-polymerase chain reaction (RT-PCR) was applied to evaluate the presence of estrogen receptor-alpha (ER-alpha) in human prostate cancer cells. Unexpectedly, a novel fatty acid synthase (FAS)/ER-alpha fusion transcript was identified, in which the N-terminus of FAS was fused in-frame with the C-terminus of ER-alpha. The existence of the FAS/ER-alpha transcript was further confirmed by RT-PCR analysis using various sets of amplification primers and different reverse-transcribed primers in the presence of dimethyl sulfoxide to eliminate the secondary structure of RNA. The predicted FAS/ER-alpha protein would contain largely domain I of FAS and the entire ligand binding domain of ER-alpha. The FAS/ER-alpha was expressed in a variety of human cancer cell lines including prostate, breast, cervical and bladder cancer cell lines. Our data suggest that the presence of FAS/ER-alpha may complicate the FAS and the ER-alpha signalling pathway.