RESUMEN
Free intracellular calcium ([Ca2+]i), in addition to being an important second messenger, is a key regulator of many cellular processes including cell membrane potential, proliferation, and apoptosis. In many cases, the mobilization of [Ca2+]i is controlled by intracellular store activation and calcium influx. We have investigated the effect of several ion channel modulators, which have been used to treat a range of human diseases, on [Ca2+]i release, by ratiometric calcium imaging. We show that six such modulators [amiodarone (Ami), dofetilide, furosemide (Fur), minoxidil (Min), loxapine (Lox), and Nicorandil] initiate release of [Ca2+]i in prostate and breast cancer cell lines, PC3 and MCF7, respectively. Whole-cell currents in PC3 cells were inhibited by the compounds tested in patch-clamp experiments in a concentration-dependent manner. In all cases [Ca2+]i was increased by modulator concentrations comparable to those used clinically. The increase in [Ca2+]i in response to Ami, Fur, Lox, and Min was reduced significantly (P < 0.01) when the external calcium was reduced to nM concentration by chelation with EGTA. The data suggest that many ion channel regulators mobilize [Ca2+]i We suggest a mechanism whereby calcium-induced calcium release is implicated; such a mechanism may be important for understanding the action of these compounds.
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Calcio/metabolismo , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Canales Iónicos/metabolismo , Línea Celular Tumoral , Fenómenos Electrofisiológicos/efectos de los fármacos , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Humanos , CinéticaRESUMEN
Feline oral squamous cell carcinoma (FOSCC) is an aggressive neoplasm in cats. Little is known about the possible molecular mechanisms that may be involved in the initiation, maintenance and progression of FOSCC. Wnt signalling is critical in development and disease, including many mammalian cancers. In this study, we have investigated the expression of Wnt signalling related proteins using quantitative immunohistochemical techniques on tissue arrays. We constructed tissue arrays with 58 individual replicate tissue samples. We tested for the expression of four key Wnt/ß-catenin transcription targets, namely Cyclin D1 (CCND1 or CD1), FRA1, c-Myc and MMP7. All antibodies showed cross reactivity in feline tissue except MMP7. Quantitative immunohistochemical analysis of single proteins (expressed as area fraction / amount of tissue for normal vs tumor, mean ± SE) showed that the expression of CD1 (3.9 ± 0.5 vs 12.2 ± 0.9), FRA1 (5.5 ± 0.6 vs 16.8 ± 1.1) and c-Myc (5.4 ± 0.5 vs 12.5 ± 0.9) was increased in FOSCC tissue by 2.3 to 3 fold compared to normal controls (p<0.0001). By using a multilabel, quantitative fluorophore technique we further investigated if the co-localization of these proteins (all transcription factors) with each other and in the nucleus (stained with 4',6-diamidino-2-phenylindole, DAPI) was altered in FOSCC compared to normal tissue. The global intersection coefficients, a measure of the proximity of two fluorophore labeled entities, showed that there was a significant change (p < 0.01) in the co-localization for all permutations (e.g. CD1/FRA1 etc), except for the nuclear localization of CD1. Our results show that putative targets of Wnt signalling transcription are up-regulated in FOSCC with alterations in the co-localization of these proteins and could serve as a useful marker for the disease.
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Carcinoma de Células Escamosas/metabolismo , Enfermedades de los Gatos/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Boca/metabolismo , Vía de Señalización Wnt , Animales , Carcinoma de Células Escamosas/veterinaria , Gatos , Ciclina D1/metabolismo , Concentración de Iones de Hidrógeno , Metaloproteinasa 7 de la Matriz/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Curva ROC , Factores de Transcripción/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMEN
Penile squamous cell carcinoma (PeCa) is a rare malignancy and little is known regarding the molecular mechanisms involved in carcinogenesis of PeCa. The Wnt signaling pathway, with the transcription activator ß-catenin as a major transducer, is a key cellular pathway during development and in disease, particularly cancer. We have used PeCa tissue arrays and multi-fluorophore labelled, quantitative, immunohistochemistry to interrogate the expression of WNT4, a Wnt ligand, and three targets of Wnt-ß-catenin transcription activation, namely, MMP7, cyclinD1 (CD1) and c-MYC in 141 penile tissue cores from 101 unique samples. The expression of all Wnt signaling proteins tested was increased by 1.6 to 3 fold in PeCa samples compared to control tissue (normal or cancer adjacent) samples (p<0.01). Expression of all proteins, except CD1, showed a significant decrease in grade II compared to grade I tumors. High magnification, deconvolved confocal images were used to measure differences in co-localization between the four proteins. Significant (p<0.04-0.0001) differences were observed for various permutations of the combinations of proteins and state of the tissue (control, tumor grades I and II). Wnt signaling may play an important role in PeCa and proteins of the Wnt signaling network could be useful targets for diagnosis and prognostic stratification of disease.
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Antígenos CD1/metabolismo , Metaloproteinasa 7 de la Matriz/metabolismo , Neoplasias del Pene/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transcripción Genética , Proteína Wnt4/genética , beta Catenina/genética , Estudios de Casos y Controles , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Clasificación del Tumor , Proteínas de Neoplasias/metabolismo , Neoplasias del Pene/patología , Proteína Wnt4/metabolismo , beta Catenina/metabolismoRESUMEN
When primary cultures of normal cells are cloned, three types of colony grow, called holoclones, meroclones and paraclones. These colonies are believed to be derived from stem cells, transit-amplifying cells and differentiated cells respectively. More recently, this approach has been extended to cancer cell lines. However, we observed that meroclones from the prostate cancer cell line DU145 produce holoclones, a paradoxical observation as meroclones are thought to be derived from transit-amplifying cells. The purpose of this study was to confirm this observation and determine if both holoclones and meroclones from cancer cell lines contain stem cells. We demonstrated that both holoclones and meroclones can be serially passaged indefinitely, are highly proliferative, can self-renew to form spheres, are serially tumorigenic and express stem cell markers. This study demonstrates that the major difference between holoclones and meroclones derived from a cancer cell line is the proportion of stem cells within each colony, not the presence or absence of stem cells. These findings may reflect the properties of cancer as opposed to normal cells, perhaps indicating that the hierarchy of stem cells is more extensive in cancer.
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Diferenciación Celular/fisiología , Células Clonales/citología , Neoplasias de la Próstata/fisiopatología , Células Madre/citología , Análisis de Varianza , Biomarcadores/metabolismo , Línea Celular Tumoral , Células Clonales/clasificación , Ensayo de Unidades Formadoras de Colonias , Fluorescencia , Humanos , Inmunohistoquímica , MasculinoRESUMEN
BACKGROUND: Semaphorins act as chemotactic cues for cell movement via their transmembrane receptors, plexins. Somatic missense mutations in the plexinB1 gene coupled with overexpression of the protein frequently occur in prostate tumors, indicating a role for plexinB1 in the pathogenesis of prostate cancer. However, the effect of semaphorin/plexin signaling is highly context dependent and whether plexinB1 acts as an inducer or inhibitor of prostate tumor progression in this context is not known. METHODS: The response of prostate cancer cell lines to plexinB1 activation was assessed in migration, invasion, proliferation and protein phosphorylation assays. Expression was assessed by quantitative RTPCR and immunoblotting. RESULTS: Different prostate cancer cell lines respond to Sema4D (the ligand for plexinB1) in diverse ways. Activation of endogenous plexinB1 enhances migration, invasion and anchorage-independent growth of LNCaP prostate cancer cells via activation of ErbB2 and Akt. In contrast, Sema4D-stimulation decreased the motility and proliferative capacity of PC3 cells. LNCaP has a missense mutation (Thr1697Ala) in the plexinB1 gene while LNCaP-LN3, a derivative of LNCaP, expresses high levels of wild-type plexinB1 only. Sema4D stimulation increases the motility and anchorage independent growth of both cell lines, showing that these responses are not dependent on the presence of the Thr1697Ala form of plexinB1. ErbB2 and plexinB1 are expressed in primary prostate epithelial cells. CONCLUSIONS: PlexinB1 signals via ErbB2 to increase the invasive phenotype of prostate cancer cells. Both wild-type and mutant forms of plexinB1 are potential targets for anti-cancer therapy in prostate tumors that express ErbB2.
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Mutación , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/fisiología , Animales , Células COS , Chlorocebus aethiops , Humanos , Masculino , Neoplasias de la Próstata/fisiopatología , Células Tumorales CultivadasRESUMEN
Prostate carcinoma is the most common cancer in men with few, quantifiable, biomarkers. Prostate cancer biomarker discovery has been hampered due to subjective analysis of protein expression in tissue sections. An unbiased, quantitative immunohistochemical approach provided here, for the diagnosis and stratification of prostate cancer could overcome this problem. Antibodies against four proteins BTF3, HINT1, NDRG1 and ODC1 were used in a prostate tissue array (> 500 individual tissue cores from 82 patients, 41 case pairs matched with one patient in each pair had biochemical recurrence). Protein expression, quantified in an unbiased manner using an automated analysis protocol in ImageJ software, was increased in malignant vs non-malignant prostate (by 2-2.5 fold, p<0.0001). Operating characteristics indicate sensitivity in the range of 0.68 to 0.74; combination of markers in a logistic regression model demonstrates further improvement in diagnostic power. Triple-labeled immunofluorescence (BTF3, HINT1 and NDRG1) in tissue array showed a significant (p<0.02) change in co-localization coefficients for BTF3 and NDRG1 co-expression in biochemical relapse vs non-relapse cancer epithelium. BTF3, HINT1, NDRG1 and ODC1 could be developed as epithelial specific biomarkers for tissue based diagnosis and stratification of prostate cancer.
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Proteínas de Ciclo Celular/metabolismo , Regulación Neoplásica de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Ornitina Descarboxilasa/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Factores de Transcripción/metabolismo , Biomarcadores de Tumor/metabolismo , Carcinogénesis , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Continuous human cell lines have been used extensively as models for biomedical research. In working with these cell lines, researchers are often unaware of the risk of cross-contamination and other causes of misidentification. To reduce this risk, there is a pressing need to authenticate cell lines, comparing the sample handled in the laboratory to a previously tested sample. The American Type Culture Collection Standards Development Organization Workgroup ASN-0002 has developed a Standard for human cell line authentication, recommending short tandem repeat (STR) profiling for authentication of human cell lines. However, there are known limitations to the technique when applied to cultured samples, including possible genetic drift with passage. In our study, a dataset of 2,279 STR profiles from four cell banks was used to assess the effectiveness of the match criteria recommended within the Standard. Of these 2,279 STR profiles, 1,157 were grouped into sets of related cell lines-duplicate holdings, legitimately related samples or misidentified cell lines. Eight core STR loci plus amelogenin were used to unequivocally authenticate 98% of these related sets. Two simple match algorithms each clearly discriminated between related and unrelated samples, with separation between related samples at ≥80% match and unrelated samples at <50% match. A small degree of overlap was noted at 50-79% match, mostly from cell lines known to display variable STR profiles. These match criteria are recommended as a simple and effective way to interpret results from STR profiling of human cell lines.
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Algoritmos , Perfilación de la Expresión Génica/métodos , Técnicas de Genotipaje/normas , Repeticiones de Microsatélite/genética , Línea Celular , Humanos , Reacción en Cadena de la PolimerasaAsunto(s)
Línea Celular , Comunicación , Identificación Biométrica , Humanos , Repeticiones de MicrosatéliteRESUMEN
Stem cells may play a role in the development and maintenance of proliferative diseases of the prostate such as prostate cancer and benign prostatic hyperplasia. Cell membrane protein markers, CD49f, CD133 and CD44, have been shown to identify putative prostate stem cells, but a lack of consensus exists with regards to the most efficient marker(s) for stem-like cell identification. This study aimed to determine whether previously reported markers had equal capacity to select monolayer and spheroid colony-forming cells (CFCs), which were used as surrogate readouts of stem-like cells, and to characterize the expression of CD49f, CD44 and CD133 by flow cytometry and immunohistochemistry.In benign prostate cells, CD49f+, CD44+, and CD133+ cells represented 5.6±3.1%, 28.2±4.1% and 0.10±0.06% of total cells. Both monolayer- and spheroid-CFCs existed at a frequency of approximately 0.5% of total cells. CD49f+, CD44+, and CD133+ subpopulations differed significantly in their ability to select benign CFCs. The highest recovery of CFCs was achieved by CD49f+ selection (98%), whereas CD44+ or CD133+ selection led to poor CFC-recovery (17% and 3%, respectively). For the first time, we show highly efficient recovery of CFCs from advanced prostate cancer by CD49f+, but not by CD44+ or CD133+ selection. Furthermore, CD133 expression (AC133 clone) could not be detected in benign prostate cells by either immunohistochemistry or flow cytometry. We conclude that CD49f, but not previously described stem cell markers CD133 and CD44, to be optimal for selection of monolayer- and spheroid-CFCs in the benign and malignant prostate.
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Biomarcadores de Tumor/metabolismo , Integrina alfa6/metabolismo , Próstata/patología , Neoplasias de la Próstata/patología , Esferoides Celulares/metabolismo , Células Madre/metabolismo , Antígeno AC133 , Antígenos CD/metabolismo , Biomarcadores de Tumor/inmunología , Biopsia , Células Cultivadas , Epítopos/análisis , Citometría de Flujo/métodos , Glicoproteínas/metabolismo , Humanos , Receptores de Hialuranos/metabolismo , Inmunohistoquímica , Masculino , Células Madre Neoplásicas/inmunología , Células Madre Neoplásicas/metabolismo , Péptidos/metabolismo , Esferoides Celulares/patología , Células Madre/inmunologíaRESUMEN
BACKGROUND: Semaphorins act as chemotactic cues for cell movement via their transmembrane receptors, plexins. Somatic missense mutations in the plexinB1 gene coupled with overexpression of the protein frequently occur in prostate tumours, indicating a role for plexinB1 in the pathogenesis of prostate cancer. RESULTS: Two specific mutations found in prostate cancer enhance RhoD binding and one other mutation results in loss of inhibition of Rac-dependent Pak1 phosphorylation and lamellipodia formation and in impairment of trafficking of plexinB1 to the membrane. None of the three characterised mutations affect PDZRhoGEF binding, RhoA activity, the interaction of plexinB1 with the oncogenes ErbB2 or c-Met or ErbB2 phosphorylation. The mutations have the net effect of increasing cell motility by blocking plexinB1-mediated inhibition of Rac while enhancing the interaction with RhoD, an anti-migratory factor. CONCLUSIONS: PlexinB1 mutations block plexinB1-mediated signalling pathways that inhibit cell motility.
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Mutación , Proteínas del Tejido Nervioso/genética , Neoplasias de la Próstata/genética , Receptores de Superficie Celular/genética , Animales , Línea Celular , Membrana Celular/metabolismo , Chlorocebus aethiops , Activación Enzimática/genética , Humanos , Masculino , Fosforilación , Neoplasias de la Próstata/metabolismo , Unión Proteica , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-met/metabolismo , Receptor ErbB-2/metabolismo , Transducción de Señal , Quinasas p21 Activadas/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
BACKGROUND: Radical prostatectomy cures the majority of men with clinically localized disease, but up to 30% of men relapse with rising serum PSA levels. Stage, Gleason grade, and pre-operative PSA levels are associated with outcome but do not accurately predict which individuals will relapse. MicroRNA (miRNA) levels are altered in cancer and are associated with progression of disease. The miR-200 family has roles in prostate cancer. METHODS: miR-200a levels were measured in 18 radical prostatectomy samples from men who did not relapse and from 18 who did relapse, matched for stage (all T3), grade, and PSA levels. A pair of cancer and normal prostate cell lines derived from the same radical prostatectomy specimen were transfected with miR-200a to determine the effects on growth, wound healing, and invasion. RESULTS: Comparing the matched samples, 11 of the relapsers contained lower, 2 higher and 5 similar levels to the non-relapsers. Transient transfection of miR-200a significantly reduced cell proliferation in prostate cancer cell lines but did not affect invasiveness. CONCLUSION: miR-200a overexpression reduced prostate cancer cell growth and may have potential, in combination with other markers, in stratifying prostate cancer patients for more intensive monitoring and therapy.
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MicroARNs/genética , Recurrencia Local de Neoplasia/genética , Antígeno Prostático Específico/sangre , Próstata/metabolismo , Neoplasias de la Próstata/genética , Línea Celular , Línea Celular Tumoral , Proliferación Celular , Humanos , Masculino , MicroARNs/biosíntesis , Recurrencia Local de Neoplasia/sangre , Prostatectomía , Neoplasias de la Próstata/sangre , Neoplasias de la Próstata/cirugía , TransfecciónRESUMEN
Proteomics has offered the hope of biomarker discovery to improve the management of prostate cancer. Markers are needed for screening and diagnosis, distinguishing latent from aggressive disease, defining the men who will benefit from therapy, differentiating localized from metastatic disease, predicting outcome and identifying new targets for therapy. There are many potential sources of proteins derived from the prostate, including urine, prostatic fluid (expressed or ejaculate), serum, and plasma or tissue, each with distinct advantages and limitations. Equally, there are many methodological platforms for proteomic studies of the prostate. Despite the promise, protoemics has yielded little of relevance to the management of prostate cancer, and most of the work that has been published is either irreproducible or of no clinical value.
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Neoplasias de la Próstata/metabolismo , Proteómica , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/orina , Humanos , Masculino , Neoplasias de la Próstata/sangre , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/orina , Unión ProteicaRESUMEN
Cell misidentification and cross-contamination have plagued biomedical research for as long as cells have been employed as research tools. Examples of misidentified cell lines continue to surface to this day. Efforts to eradicate the problem by raising awareness of the issue and by asking scientists voluntarily to take appropriate actions have not been successful. Unambiguous cell authentication is an essential step in the scientific process and should be an inherent consideration during peer review of papers submitted for publication or during review of grants submitted for funding. In order to facilitate proper identity testing, accurate, reliable, inexpensive, and standardized methods for authentication of cells and cell lines must be made available. To this end, an international team of scientists is, at this time, preparing a consensus standard on the authentication of human cells using short tandem repeat (STR) profiling. This standard, which will be submitted for review and approval as an American National Standard by the American National Standards Institute, will provide investigators guidance on the use of STR profiling for authenticating human cell lines. Such guidance will include methodological detail on the preparation of the DNA sample, the appropriate numbers and types of loci to be evaluated, and the interpretation and quality control of the results. Associated with the standard itself will be the establishment and maintenance of a public STR profile database under the auspices of the National Center for Biotechnology Information. The consensus standard is anticipated to be adopted by granting agencies and scientific journals as appropriate methodology for authenticating human cell lines, stem cells, and tissues.
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Biología Celular/normas , Perfilación de la Expresión Génica/métodos , Repeticiones de Microsatélite/genética , Manejo de Especímenes/métodos , Bancos de Tejidos/normas , Línea Celular , Humanos , Células Madre , Estados UnidosRESUMEN
Mycoplasma is a prokaryotic organism that is a frequent and occult contaminant of cell cultures. This organism can modify many aspects of cell physiology, rendering experiments that are conducted with contaminated cells worthless. Because of their small size, Mycoplasmas can pass through filters used to prevent bacterial and fungal contamination and potentially spread to all the cultures in a laboratory. It is essential that all new cell cultures entering a laboratory and all cell banks are tested for the presence of Mycoplasma. It is recommended that two techniques be used, selected from a PCR-based method, indirect staining and an agar and broth culture. This protocol describes these three tests for detecting Mycoplasma, which take from 1 d to 3-4 weeks, and such tests should be an obligatory component of quality control in every tissue culture laboratory.
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Técnicas Bacteriológicas/métodos , Mycoplasma/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Coloración y Etiquetado/métodos , ADN Bacteriano , Electroforesis en Gel de Agar , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
Wnt signaling is a critical regulatory pathway in development and disease. Very little is known about the mechanisms of Wnt signaling in prostate cancer, a leading cause of death in men. A quantitative analysis of the expression of Wnt5A protein in human tissue arrays, containing 600 prostate tissue cores, showed >50% increase in malignant compared to benign cores (p<0.0001). In a matched pair of prostate cancer and normal cell line, expression of Wnt5A protein was also increased. Calcium waves were induced in prostate cells in response to Wnt5A with a 3 fold increase in Flou-4 intensity. The activity of Ca(2+)/calmodulin dependent protein kinase (CaMKII), a transducer of the non-canonical Wnt/Ca(2+) signaling, increased by 8 fold in cancer cells; no change was observed in beta-catenin expression, known to activate the canonical Wnt/beta-catenin pathway. Mining of publicly available human prostate cancer oligoarray datasets revealed that the expression of numerous genes (e.g., CCND1, CD44) under the control of beta-catenin transcription is down-regulated. Confocal and quantitative electron microscopy showed that specific inhibition of CaMKII in cancer cells causes remodeling of the actin cytoskeleton, irregular wound edges and loose intercellular architecture and a 6 and 8 fold increase in the frequency and length of filopodia, respectively. Conversely, untreated normal prostate cells showed an irregular wound edge and loose intercellular architecture; incubation of normal prostate cells with recombinant Wnt5A protein induced actin remodeling with a regular wound edge and increased wound healing capacity. Live cell imaging showed that a functional consequence of CaMKII inhibition was 80% decrease in wound healing capacity and reduced cell motility in cancer cells. We propose that non-canonical Wnt/Ca(2+) signaling via CaMKII acts as a novel regulator of structural plasticity and cell motility in prostate cancer.
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Actinas/metabolismo , Señalización del Calcio , Movimiento Celular , Citoesqueleto/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Wnt/metabolismo , Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Microscopía Confocal , Próstata/enzimología , Próstata/patología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/ultraestructura , Proteínas Proto-Oncogénicas/genética , Seudópodos/metabolismo , Seudópodos/ultraestructura , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Proteínas Wnt/genética , Proteína Wnt-5a , Cicatrización de HeridasRESUMEN
Hepatocyte growth factor (HGF) is associated with tumour progression and increases the invasiveness of prostate carcinoma cells. Migration and invasion require coordinated reorganisation of the actin cytoskeleton and regulation of cell-adhesion dynamics. Rho-family GTPases orchestrate both of these cellular processes. p21-activated kinase 4 (PAK4), a specific effector of the Rho GTPase Cdc42, is activated by HGF, and we have previously shown that activated PAK4 induces a loss of both actin stress fibres and focal adhesions. We now report that DU145 human prostate cancer cells with reduced levels of PAK4 expression are unable to successfully migrate in response to HGF, have prominent actin stress fibres, and an increase in the size and number of focal adhesions. Moreover, these cells have a concomitant reduction in cell-adhesion turnover rates. We find that PAK4 is localised at focal adhesions, is immunoprecipitated with paxillin and phosphorylates paxillin on serine 272. Furthermore, we demonstrate that PAK4 can regulate RhoA activity via GEF-H1. Our results suggest that PAK4 is a pluripotent kinase that can regulate both actin cytoskeletal rearrangement and focal-adhesion dynamics.
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Adhesiones Focales/metabolismo , Neoplasias de la Próstata/metabolismo , Quinasas p21 Activadas/farmacología , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Adhesiones Focales/patología , Regulación Neoplásica de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Masculino , Paxillin/metabolismo , Fosforilación/efectos de los fármacos , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Unión Proteica/efectos de los fármacos , Factores de Intercambio de Guanina Nucleótido Rho , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Continuous cell lines consist of cultured cells derived from a specific donor and tissue of origin that have acquired the ability to proliferate indefinitely. These cell lines are well-recognized models for the study of health and disease, particularly for cancer. However, there are cautions to be aware of when using continuous cell lines, including the possibility of contamination, in which a foreign cell line or microorganism is introduced without the handler's knowledge. Cross-contamination, in which the contaminant is another cell line, was first recognized in the 1950s but, disturbingly, remains a serious issue today. Many cell lines become cross-contaminated early, so that subsequent experimental work has been performed only on the contaminant, masquerading under a different name. What can be done in response-how can a researcher know if their own cell lines are cross-contaminated? Two practical responses are suggested here. First, it is important to check the literature, looking for previous work on cross-contamination. Some reports may be difficult to find and to make these more accessible, we have compiled a list of known cross-contaminated cell lines. The list currently contains 360 cell lines, drawn from 68 references. Most contaminants arise within the same species, with HeLa still the most frequently encountered (29%, 106/360) among human cell lines, but interspecies contaminants account for a small but substantial minority of cases (9%, 33/360). Second, even if there are no previous publications on cross-contamination for that cell line, it is essential to check the sample itself by performing authentication testing.
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Técnicas de Cultivo de Célula , Línea Celular , Modelos Biológicos , Animales , HumanosRESUMEN
The primary hyperoxalurias, PH1 and PH2, are inherited disorders caused by deficiencies of alanine:glyoxylate aminotransferase and glyoxylate reductase, respectively. Mutations in either of these enzymes leads to endogenous oxalate overproduction primarily in the liver, but most pathological effects are exhibited in the kidney ultimately leading to end-stage renal failure and systemic oxalosis. To provide a non-invasive means of accessing kidney cells from individuals with primary hyperoxaluria, we have derived primary cultures of renal proximal tubule cells from the urine of these patients. The cells stain positively for the epithelial markers pan-cytokeratin and zonula occludens 1 and the proximal tubule marker gamma-glutamyl transpeptidase. Mutation analysis confirmed that the cultured cells had the same genotype as the leucocytes of the patients and also expressed glyoxylate reductase at the mRNA level, illustrating their potential value as a source of renal material from these individuals.