RESUMO
OBJECTIVES: The aim of this study was to investigate the distribution of primary cilia on secretory cells in normal fallopian tube (FT) and serous tubal intraepithelial carcinoma (STIC). METHODS: Fallopian tube tissue samples were obtained from 4 females undergoing prophylactic hysterectomies and 6 patients diagnosed with STIC. A mogp-TAg transgenic mouse STIC sample was also compared with a wild-type mouse FT sample. Serous tubal intraepithelial carcinoma was identified by hematoxylin and eosin staining and confirmed by positive Ki-67 and p53 immunohistochemical staining of tissue sections. We assessed the relative distribution of primary cilia on secretory cells and motile cilia on multiple ciliated cells by immunofluorescence and immunohistochemical staining. Ciliary function was assessed by immunofluorescence staining of specific ciliary marker proteins and responsiveness to Sonic Hedgehog signaling. RESULTS: Primary cilia are widespread on secretory cells in the ampulla, isthmus, and in particular, the fimbriae of human FT where they may appear to mediate ciliary-mediated Sonic Hedgehog signaling. A statistically significant reduction in the number of primary cilia on secretory cells was observed in human STIC samples compared with normal controls (P < 0.0002, Student t test), supported by similar findings in a mouse STIC sample. Immunohistochemical staining for dynein axonemal heavy chain 5 discriminated multiple motile cilia from primary cilia in human FT. CONCLUSIONS: Primary cilia are widespread on secretory cells in the ampulla, isthmus, and in particular, the fimbriae of the human FT but are significantly reduced in both human and mouse STIC samples. Immunohistochemical staining for ciliary proteins may have clinical utility for early detection of STIC.
Assuntos
Carcinoma in Situ/patologia , Cílios/fisiologia , Cistadenocarcinoma Seroso/patologia , Neoplasias das Tubas Uterinas/patologia , Tubas Uterinas/citologia , Animais , Carcinoma in Situ/metabolismo , Cílios/metabolismo , Cistadenocarcinoma Seroso/metabolismo , Neoplasias das Tubas Uterinas/metabolismo , Tubas Uterinas/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Antígeno Ki-67/metabolismo , Camundongos , Camundongos Transgênicos , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Cultura Primária de Células , Proteína Supressora de Tumor p53/metabolismoRESUMO
Joubert syndrome related disorders (JSRDs) have broad but variable phenotypic overlap with other ciliopathies. The molecular etiology of this overlap is unclear but probably arises from disrupting common functional module components within primary cilia. To identify additional module elements associated with JSRDs, we performed homozygosity mapping followed by next-generation sequencing (NGS) and uncovered mutations in TMEM237 (previously known as ALS2CR4). We show that loss of the mammalian TMEM237, which localizes to the ciliary transition zone (TZ), results in defective ciliogenesis and deregulation of Wnt signaling. Furthermore, disruption of Danio rerio (zebrafish) tmem237 expression produces gastrulation defects consistent with ciliary dysfunction, and Caenorhabditis elegans jbts-14 genetically interacts with nphp-4, encoding another TZ protein, to control basal body-TZ anchoring to the membrane and ciliogenesis. Both mammalian and C. elegans TMEM237/JBTS-14 require RPGRIP1L/MKS5 for proper TZ localization, and we demonstrate additional functional interactions between C. elegans JBTS-14 and MKS-2/TMEM216, MKSR-1/B9D1, and MKSR-2/B9D2. Collectively, our findings integrate TMEM237/JBTS-14 in a complex interaction network of TZ-associated proteins and reveal a growing contribution of a TZ functional module to the spectrum of ciliopathy phenotypes.
Assuntos
Doenças Cerebelares/genética , Cílios/genética , Anormalidades do Olho/genética , Doenças Renais Císticas/genética , Proteínas de Membrana/genética , Mutação , Anormalidades Múltiplas , Adulto , Animais , Síndrome de Bardet-Biedl/genética , Caenorhabditis elegans/genética , Caenorhabditis elegans/ultraestrutura , Estudos de Casos e Controles , Linhagem Celular , Cerebelo/anormalidades , Criança , Pré-Escolar , Mapeamento Cromossômico , Cílios/metabolismo , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Estudos de Associação Genética , Haplótipos , Humanos , Lactente , Recém-Nascido , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Microscopia Eletrônica de Transmissão , Complexos Multiproteicos/metabolismo , Polimorfismo de Nucleotídeo Único , Retina/anormalidades , Análise de Sequência de DNA , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , Peixe-Zebra/embriologia , Peixe-Zebra/genéticaRESUMO
Monoclonal antibodies have revolutionized therapies, but non-immunoglobulin scaffolds are becoming compelling alternatives owing to their adaptability. Their ability to be labeled with imaging or cytotoxic compounds and to create multimeric proteins is an attractive strategy for therapeutics. Focusing on HER2, a frequently overexpressed receptor in breast cancer, this study addresses some limitations of conventional targeting moieties by harnessing the potential of these scaffolds. HER2-binding Affimers were isolated and characterized, demonstrating potency as binding reagents and efficient internalization by HER2-overexpressing cells. Affimers conjugated with cytotoxic agent achieved dose-dependent reductions in cell viability within HER2-overexpressing cell lines. Bispecific Affimers, targeting HER2 and virus-like particles, facilitated efficient internalization of virus-like particles carrying enhanced green fluorescent protein (eGFP)-encoding RNA, leading to protein expression. Anti-HER2 affibody or designed ankyrin repeat protein (DARPin) fusion constructs with the anti-VLP Affimer further underscore the adaptability of this approach. This study demonstrates the versatility of scaffolds for precise delivery of cargos into cells, advancing biotechnology and therapeutic research.
RESUMO
The MCPH1 gene, also known as BRCT-repeat inhibitor of hTERT expression (BRIT1), has three BRCA1 carboxyl-terminal domains which is an important regulator of DNA repair, cell cycle checkpoints and chromosome condensation. MCPH1/BRIT1 is also known as a tumour suppressor in different types of human cancer. The expression level of the MCPH1/BRIT1 gene is decreased at the DNA, RNA or protein level in a number of types of cancers including breast cancer, lung cancer, cervical cancer, prostate cancer and ovarian cancer compared to normal tissue. This review also showed that deregulation of MCPH1/BRIT1 is significantly associated with reduced overall survival in 57% (12/21) and relapsed free survival in 33% (7/21) of cancer types especially in oesophageal squamous cell carcinoma and renal clear cell carcinoma. A common finding of this study is that the loss of MCPH1/BRIT1 gene expression plays a key role in promoting genome instability and mutations supporting its function as a tumour suppressor gene.
RESUMO
Kinases are important therapeutic targets, and their inhibitors are classified according to their mechanism of action, which range from blocking ATP binding to covalent inhibition. Here, a mechanism of inhibition is highlighted by capturing p21-activated kinase 5 (PAK5) in an intermediate state of activation using an Affimer reagent that binds in the P+1 pocket. PAK5 was identified from a non-hypothesis-driven high-content imaging RNAi screen in urothelial cancer cells. Silencing of PAK5 resulted in reduced cell number, G1/S arrest, and enlargement of cells, suggesting it to be important in urothelial cancer cell line survival and proliferation. Affimer reagents were isolated to identify mechanisms of inhibition. The Affimer PAK5-Af17 recapitulated the phenotype seen with siRNA. Co-crystallization revealed that PAK5-Af17 bound in the P+1 pocket of PAK5, locking the kinase into a partial activation state. This mechanism of inhibition indicates that another class of kinase inhibitors is possible.
Assuntos
Neoplasias , Quinases Ativadas por p21 , Humanos , Quinases Ativadas por p21/genética , Quinases Ativadas por p21/metabolismo , Fosforilação , Ligação ProteicaRESUMO
CUB and Sushi Multiple Domains 1 (CSMD1), a tumour suppressor gene, encodes a large membrane-bound protein including a single transmembrane domain. This transmembrane region has a potential tyrosine phosphorylation site, suggesting that CSMD1 is involved in controlling cellular functions. Although the specific mechanisms of action for CSMD1 have not yet been uncovered, it has been linked to a number of processes including development, complement control, neurodevelopment, and cancer progression. In this review, we summarise CSMD1 functions in the cellular processes involved in the complement system, metastasis, and Epithelial mesenchymal transition (EMT) and also in the diseases schizophrenia, Parkinson's disease, and cancer. Clarifying the association between CSMD1 and the aforementioned diseases will contribute to the development of new diagnosis and treatment methods for these diseases. Recent studies in certain cancer types, e.g., gastric cancer, oesophageal cancer, and head and neck squamous cell carcinomas, have indicated the involvement of CSMD1 in response to immunotherapy.
Assuntos
Neoplasias de Cabeça e Pescoço , Esquizofrenia , Humanos , Proteínas Supressoras de Tumor/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço , Proteínas de Membrana/genéticaRESUMO
The authors have investigated the expression of the microcephalin (MCPH1) protein to evaluate its prognostic importance in breast cancer. Microcephalin is a damage response protein involved in the regulation of BRCA1 and BRCA2. BRCA1 mutations are often associated with basal-like breast cancer, which are also often negative for oestrogen receptor (ER), progesterone receptor (PR) and HER2. MCPH1 immunohistochemistry was performed on 319 breast cancers prepared as tissue microarray and correlated with pathology, survival, ER, PR, HER2, EGFR, CK5/6, CK14 and BRCA1 expression. After performing continuous data analysis, mean microcephalin expression decreased with increasing grade (P < 0.006). Mean microcephalin expression was lower in ER/PR negative (P < 0.001) and triple negative cancers (P < 0.004). Conversely, an association with HER2-positive cancers was also identified (P < 0.034). Reduced microcephalin also correlated with reduced nuclear BRCA1 staining (P < 0.001). No association was identified with basal markers. After dichotomising the data into low and high microcephalin expression, reduced expression was identified in 29% (93/319) of breast cancers. An association with low expression was identified in invasive ductal carcinomas with breast cancer-specific survival (BCSS) (P = 0.052). Multivariate analysis of ductal carcinomas showed that microcephalin, together with lymph node involvement and tumour size were independent predictors of BCSS (P = 0.037). Microcephalin expression is reduced in 29% of breast cancers, particularly in higher grade tumours and BRCA1-negative cases. Microcephalin is an independent predictor of BCSS in invasive ductal breast cancer patients and may prove to be a useful biomarker for the identification of aggressive breast cancers.
Assuntos
Proteína BRCA1/biossíntese , Neoplasias da Mama/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Adulto , Idoso , Proteína BRCA1/genética , Biomarcadores Tumorais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patologia , Proteínas de Ciclo Celular , Proteínas do Citoesqueleto , Feminino , Imunofluorescência , Humanos , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/genética , Prognóstico , Receptor ErbB-2/análise , Receptores de Estrogênio/análise , Receptores de Progesterona/análise , Análise Serial de TecidosRESUMO
The Cub Sushi Multiple Domains-1 (CSMD1) protein is a tumour suppressor which has been shown to play a role in regulating human mammary duct development in vitro. CSMD1 knockdown in vitro demonstrated increased cell proliferation, invasion and motility. However, the role of Csmd1 in vivo is poorly characterised when it comes to ductal development and is therefore an area which warrants further exploration. In this study a Csmd1 knockout (KO) mouse model was used to identify the role of Csmd1 in regulating mammary gland development during puberty. Changes in duct development and protein expression patterns were analysed by immunohistochemistry. This study identified increased ductal development during the early stages of puberty in the KO mice, characterised by increased ductal area and terminal end bud number at 6 weeks. Furthermore, increased expression of various proteins (Stat1, Fak, Akt, Slug/Snail and Progesterone receptor) was shown at 4 weeks in the KO mice, followed by lower expression levels from 6 weeks in the KO mice compared to the wild type mice. This study identifies a novel role for Csmd1 in mammary gland development, with Csmd1 KO causing significantly more rapid mammary gland development, suggesting an earlier adult mammary gland formation.
Assuntos
Glândulas Mamárias Animais/crescimento & desenvolvimento , Glândulas Mamárias Animais/metabolismo , Proteínas de Membrana/genética , Organogênese/genética , Proteínas Supressoras de Tumor/genética , Animais , Feminino , Regulação da Expressão Gênica , Imuno-Histoquímica , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Maturidade Sexual/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
BACKGROUND: Mutations in the Abnormal Spindle Microcephaly related gene (ASPM) are the commonest cause of autosomal recessive primary microcephaly (MCPH) a disorder characterised by a small brain and associated mental retardation. ASPM encodes a mitotic spindle pole associated protein. It is suggested that the MCPH phenotype arises from proliferation defects in neural progenitor cells (NPC). RESULTS: We show that ASPM is a microtubule minus end-associated protein that is recruited in a microtubule-dependent manner to the pericentriolar matrix (PCM) at the spindle poles during mitosis. ASPM siRNA reduces ASPM protein at the spindle poles in cultured U2OS cells and severely perturbs a number of aspects of mitosis, including the orientation of the mitotic spindle, the main determinant of developmental asymmetrical cell division. The majority of ASPM depleted mitotic cells fail to complete cytokinesis. In MCPH patient fibroblasts we show that a pathogenic ASPM splice site mutation results in the expression of a novel variant protein lacking a tripeptide motif, a minimal alteration that correlates with a dramatic decrease in ASPM spindle pole localisation. Moreover, expression of dominant-negative ASPM C-terminal fragments cause severe spindle assembly defects and cytokinesis failure in cultured cells. CONCLUSIONS: These observations indicate that ASPM participates in spindle organisation, spindle positioning and cytokinesis in all dividing cells and that the extreme C-terminus of the protein is required for ASPM localisation and function. Our data supports the hypothesis that the MCPH phenotype caused by ASPM mutation is a consequence of mitotic aberrations during neurogenesis. We propose the effects of ASPM mutation are tolerated in somatic cells but have profound consequences for the symmetrical division of NPCs, due to the unusual morphology of these cells. This antagonises the early expansion of the progenitor pool that underpins cortical neurogenesis, causing the MCPH phenotype.
Assuntos
Citocinese , Proteínas do Tecido Nervoso/metabolismo , Fuso Acromático/ultraestrutura , Divisão Celular , Linhagem Celular , Citoesqueleto , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Microtúbulos/metabolismo , Mitose , Mutação , Proteínas do Tecido Nervoso/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Interferência de RNA , Sítios de Splice de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fuso Acromático/metabolismoRESUMO
CUB and SUSHI multiple domain protein 1 (CSMD1) is a candidate tumour suppressor gene that maps to chromosome 8p23, a region deleted in many tumour types including 50% of breast cancers. CSMD1 has homologies to proteins implicated in carcinogenesis. We aimed to study the expression pattern of the CSMD1 protein and evaluate its prognostic importance in invasive ductal carcinoma (IDC). An anti-CSMD1 antibody was developed and validated. The expression pattern of CSMD1 in normal breast and IDC samples was investigated by immunohistochemistry in 275 patients. Univariate and multivariate Cox regression analyses were performed. In normal breast duct epithelial cells, luminal, membranous and cytoplasmic CSMD1 staining was identified. Reduced expression of CSMD1 was detected in 79/275 (28.7%) of IDC cases. Low CSMD1 expression was significantly associated with high tumour grade (P = 0.003). CSMD1 expression was associated with overall survival (OS; HR = 0.607, 95%CI: 0.4-0.91, P = 0.018) but not with disease-free survival (DFS; HR = 0.81, 95%CI: 0.46-1.43, P = 0.48). Multivariate analysis showed that CSMD1, together with Nottingham Prognostic Index, was considered an independent predictor of OS (HR = 0.607, 95%CI: 0.4-0.91, P = 0.018) but not DFS (HR = 0.84, 95%CI: 0.46-1.5, P = 0.573). Reduction of CSMD1 expression was significantly associated with high tumour grade and decreased OS. Therefore, our results support the idea that CSMD1 is a tumour suppressor gene and suggest its possible use as a new prognostic biomarker. The membrane expression pattern of CSMD1 suggests that it may be a receptor or co-receptor involved in the process of signal transduction.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Proteínas de Membrana/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Análise Multivariada , Prognóstico , Modelos de Riscos Proporcionais , Análise de Sobrevida , Proteínas Supressoras de TumorRESUMO
Poor-prognosis breast cancers are treated with cytotoxic chemotherapy, but often without any guidance from therapy predictive markers because universally accepted markers are not currently available. Treatment failure, in the form of recurrences, is relatively common. We aimed to identify chemotherapy predictive markers and resistance pathways in breast cancer. Our hypothesis was that tumor cells remaining after neoadjuvant chemotherapy (NAC) contain somatic variants causing therapy resistance, while variants present pre-NAC but lost post-NAC cause sensitivity. Whole-exome sequencing was performed on matched pre- and post-NAC cancer cells, which were isolated by laser microdissection, from 6 cancer cases, and somatic variants selected for or against by NAC were identified. Somatic variant diversity was significantly reduced after therapy (P < 0.05). MUC17 variants were identified in 3 tumors and were selected against by NAC in each case, while PCNX1 variants were identified in 2 tumors and were selected for in both cases, implicating the function of these genes in defining chemoresponse. In vitro knockdown of MUC17 or PCNX1 was associated with significantly increased or decreased chemotherapy sensitivity, respectively (P < 0.05), further supporting their roles in chemotherapy response. Expression was tested for predictive value in two independent cohorts of chemotherapy-treated breast cancers (n = 53, n = 303). Kaplan-Meier analyses revealed that low MUC17 expression was significantly associated with longer survival after chemotherapy, whereas low PCNX1 was significantly associated with reduced survival. We concluded that therapy-driven selection of somatic variants allows identification of chemotherapy response genes. With respect to MUC17 and PCNX1, therapy-driven selection acting on somatic variants, in vitro knockdown data concerning drug sensitivity, and survival analysis of expression levels in patient cohorts all define the genes as mediators of and predictive markers for chemotherapy response in breast cancer.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/genética , Neoplasias da Mama/patologia , Proteínas de Ciclo Celular/genética , Resistencia a Medicamentos Antineoplásicos/genética , Mucinas/genética , Mutação , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Carcinoma Ductal de Mama/tratamento farmacológico , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/patologia , Carcinoma Lobular/tratamento farmacológico , Carcinoma Lobular/genética , Carcinoma Lobular/patologia , Quimioterapia Adjuvante , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Genômica , Humanos , Terapia Neoadjuvante , Prognóstico , Taxa de SobrevidaRESUMO
Parkinson's disease (PD) is defined by the progressive loss of dopaminergic neurons. Mitochondrial dysfunction and oxidative stress are associated with PD although it is not fully understood how neurons respond to these stresses. How adaptive and apoptotic neuronal stress response pathways are regulated and the thresholds at which they are activated remains ambiguous. Utilising SH-SY5Y neuroblastoma cells, we show that MAPK/AP-1 pathways are critical in regulating the response to mitochondrial uncoupling. Here we found the AP-1 transcription factor c-Jun can act in either a pro- or anti-apoptotic manner, depending on the level of stress. JNK-mediated cell death in differentiated cells only occurred once a threshold of stress was surpassed. We also identified a novel feedback loop between Parkin activity and the c-Jun response, suggesting defective mitophagy may initiate MAPK/c-Jun-mediated neuronal loss observed in PD. Our data supports the hypothesis that blocking cell death pathways upstream of c-Jun as a therapeutic target in PD may not be appropriate due to crossover of the pro- and anti-apoptotic responses. Boosting adaptive responses or targeting specific aspects of the neuronal death response may therefore represent more viable therapeutic strategies.
Assuntos
Apoptose , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Mitocôndrias/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neurônios/citologia , Linhagem Celular Tumoral , Retroalimentação Fisiológica , Regulação da Expressão Gênica , Humanos , Estresse Oxidativo , Ubiquitina-Proteína Ligases/genéticaRESUMO
AIMS: The aim of this study was to explore the correlation of hTERT splice variant expression with MCPH1/BRIT1 and BRCA1 expression in epithelial ovarian cancer (EOC) samples. BACKGROUND: Telomerase activation can contribute to the progression of tumors and the development of cancer. However, the regulation of telomerase activity remains unclear. MCPH1 (also known as BRIT1, BRCT-repeat inhibitor of hTERT expression) and BRCA1 are tumor suppressor genes that have been linked to telomerase expression. METHODS: qPCR was used to investigate telomerase splice variants, MCPH1/BRIT1 and BRCA1 expression in EOC tissue and primary cultures. RESULTS: The wild type α+/ß+ hTERT variant was the most common splice variant in the EOC samples, followed by α+/ß- hTERT, a dominant negative regulator of telomerase activity. EOC samples expressing high total hTERT demonstrated significantly lower MCPH1/BRIT1 expression in both tissue (pâ¯=â¯0.05) and primary cultures (pâ¯=â¯0.03). We identified a negative correlation between MCPH1/BRIT1 and α+/ß+ hTERT (pâ¯=â¯0.04), and a strong positive association between MCPH1/BRIT1 and both α-/ß+ hTERT and α-/ß- hTERT (both pâ¯=â¯0.02). A positive association was observed between BRCA1 and α-/ß+ hTERT and α-/ß- hTERT expression (pâ¯=â¯0.003 and pâ¯=â¯0.04, respectively). CONCLUSIONS: These findings support a regulatory effect of MCPH1/BRIT1 and BRCA1 on telomerase activity, particularly the negative association between MCPH1/BRIT1 and the functional form of hTERT (α+/ß+).
Assuntos
Proteína BRCA1/genética , Neoplasias Epiteliais e Glandulares/genética , Proteínas do Tecido Nervoso/genética , Neoplasias Ovarianas/genética , Telomerase/genética , Proteína BRCA1/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma Epitelial do Ovário , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proteínas do Citoesqueleto , Feminino , Expressão Gênica , Genes Supressores de Tumor , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Estimativa de Kaplan-Meier , Neoplasias Epiteliais e Glandulares/enzimologia , Neoplasias Epiteliais e Glandulares/mortalidade , Proteínas do Tecido Nervoso/metabolismo , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/mortalidade , Telomerase/metabolismo , TranscriptomaRESUMO
The CUB and sushi multiple domains 1 (CSMD1) gene maps to chromosome 8p23, a region deleted in many cancers. Loss of CSMD1 expression is associated with poor prognosis in breast cancer suggesting that it acts as a tumour suppressor in this cancer. However, the function of CSMD1 is largely unknown. Herein, we investigated CSMD1 functions in cell line models. CSMD1 expression was suppressed in MCF10A and LNCaP cells using short hairpin RNA. Functional assays were performed focusing on the 'normal' MCF10A cell line. Suppression of CSMD1 significantly increased the proliferation, cell migration and invasiveness of MCF10A cells compared to shcontrols. shCSMD1 cells also showed significantly reduced adhesion to Matrigel and fibronectin. In a three-dimensional Matrigel model of MCF10A cells, reduced CSMD1 expression resulted in the development of larger and more poorly differentiated breast acini-like structures that displayed impaired lumen formation. Loss of CSMD1 expression disrupts a model of mammary duct formation while enhancing proliferation, migration and invasion. Our data suggest that CSMD1 is involved in the suppression of a transformed phenotype.
Assuntos
Neoplasias da Mama/patologia , Movimento Celular , Proliferação de Células , Glândulas Mamárias Humanas/patologia , Proteínas de Membrana/antagonistas & inibidores , Apoptose , Neoplasias da Mama/metabolismo , Células Cultivadas , Feminino , Humanos , Glândulas Mamárias Humanas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Invasividade Neoplásica , RNA Interferente Pequeno/genética , Proteínas Supressoras de TumorRESUMO
Epigenetic mechanisms such as DNA methylation are now recognized to play an important role in neoplasia. The aim of this study is to relate the pattern of expression of multiple cancer genes known to undergo epigenetic inactivation by promoter hypermethylation in breast cancer with histologic and outcome data. We used immunohistochemistry to study expression of the tumor suppressor gene p16, estrogen receptor (ER) alpha, ERbeta, progesterone receptor (PR), and the DNA repair gene MGMT (O6 -methylguanine-DNA methyltransferase) in a panel of 200 breast cancers. Methylation-specific polymerase chain reaction was used to confirm MGMT promoter methylation. Loss of expression of MGMT, ERalpha, ERbeta, PR, and p16 was observed in 19%, 24%, 13%, 40%, and 50% of cases, respectively. A significant correlation was seen between grade III tumor and loss of expression of ERalpha, ERbeta, PR (all P < .0001), and MGMT (P = .04), whereas loss of expression of p16 was associated with grades I and II tumors (P < .001). Cases that expressed 3 or less of the 5 proteins studied had significantly reduced survival (P = .0016). Methylation-specific polymerase chain reaction in a subset of 20 cancers showed DNA methylation associated with the loss of MGMT expression (P < .001). In conclusion, there is silencing of several key genes in breast cancer affecting molecular pathways involved in cell immortalization, DNA repair, and hormonal regulation, and this correlates significantly with risk of cancer-specific death. This expression profile could be linked to epigenetic events, and if so, these pathways have potential as targets for therapeutic strategies based on reversal of epigenetic silencing.
Assuntos
Adenocarcinoma/genética , Neoplasias da Mama/genética , Epigênese Genética , Regulação Neoplásica da Expressão Gênica/genética , Inativação Gênica , Adenocarcinoma/metabolismo , Adenocarcinoma/mortalidade , Adenocarcinoma/secundário , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Metilação de DNA , DNA de Neoplasias/análise , Feminino , Humanos , Técnicas Imunoenzimáticas , Linfonodos/patologia , Metástase Linfática , Glândulas Mamárias Humanas/metabolismo , Glândulas Mamárias Humanas/patologia , Pessoa de Meia-Idade , Estadiamento de Neoplasias , O(6)-Metilguanina-DNA Metiltransferase/genética , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Reação em Cadeia da Polimerase , Taxa de Sobrevida , Reino Unido/epidemiologiaRESUMO
Defects in primary cilium biogenesis underlie the ciliopathies, a growing group of genetic disorders. We describe a whole-genome siRNA-based reverse genetics screen for defects in biogenesis and/or maintenance of the primary cilium, obtaining a global resource. We identify 112 candidate ciliogenesis and ciliopathy genes, including 44 components of the ubiquitin-proteasome system, 12 G-protein-coupled receptors, and 3 pre-mRNA processing factors (PRPF6, PRPF8 and PRPF31) mutated in autosomal dominant retinitis pigmentosa. The PRPFs localize to the connecting cilium, and PRPF8- and PRPF31-mutated cells have ciliary defects. Combining the screen with exome sequencing data identified recessive mutations in PIBF1, also known as CEP90, and C21orf2, also known as LRRC76, as causes of the ciliopathies Joubert and Jeune syndromes. Biochemical approaches place C21orf2 within key ciliopathy-associated protein modules, offering an explanation for the skeletal and retinal involvement observed in individuals with C21orf2 variants. Our global, unbiased approaches provide insights into ciliogenesis complexity and identify roles for unanticipated pathways in human genetic disease.
Assuntos
Cílios/genética , Transtornos da Motilidade Ciliar/genética , Marcadores Genéticos , Testes Genéticos/métodos , Genômica/métodos , Células Fotorreceptoras , Interferência de RNA , Anormalidades Múltiplas , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/ultraestrutura , Doenças Cerebelares/genética , Cerebelo/anormalidades , Cílios/metabolismo , Cílios/patologia , Transtornos da Motilidade Ciliar/metabolismo , Transtornos da Motilidade Ciliar/patologia , Proteínas do Citoesqueleto , Bases de Dados Genéticas , Síndrome de Ellis-Van Creveld/genética , Anormalidades do Olho/genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Doenças Renais Císticas/genética , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Fenótipo , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/ultraestrutura , Proteínas da Gravidez/genética , Proteínas da Gravidez/metabolismo , Proteínas/genética , Proteínas/metabolismo , Reprodutibilidade dos Testes , Retina/anormalidades , Fatores Supressores Imunológicos/genética , Fatores Supressores Imunológicos/metabolismo , Transfecção , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
BACKGROUND: Real-time PCR is increasingly being adopted for RNA quantification and genetic analysis. At present the most popular real-time PCR assay is based on the hybridisation of a dual-labelled probe to the PCR product, and the development of a signal by loss of fluorescence quenching as PCR degrades the probe. Though this so-called 'TaqMan' approach has proved easy to optimise in practice, the dual-labelled probes are relatively expensive. RESULTS: We have designed a new assay based on SYBR-Green I binding that is quick, reliable, easily optimised and compares well with the published assay. Here we demonstrate its general applicability by measuring copy number in three different genetic contexts; the quantification of a gene rearrangement (T-cell receptor excision circles (TREC) in peripheral blood mononuclear cells); the detection and quantification of GLI, MYC-C and MYC-N gene amplification in cell lines and cancer biopsies; and detection of deletions in the OPA1 gene in dominant optic atrophy. CONCLUSION: Our assay has important clinical applications, providing accurate diagnostic results in less time, from less biopsy material and at less cost than assays currently employed such as FISH or Southern blotting.
Assuntos
DNA/genética , Compostos Orgânicos/química , Reação em Cadeia da Polimerase/métodos , Benzotiazóis , Linhagem Celular Tumoral , DNA/química , Diaminas , Feminino , Fluorescência , GTP Fosfo-Hidrolases/genética , Amplificação de Genes/genética , Deleção de Genes , Rearranjo Gênico/genética , Humanos , Masculino , Proteínas Oncogênicas/genética , Atrofia Óptica/genética , Quinolinas , Receptores de Antígenos de Linfócitos T/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Transativadores , Fatores de Transcrição/genética , Proteína GLI1 em Dedos de ZincoRESUMO
The chromosomal band 1p36 exhibits frequent loss of heterozygosity in a variety of human malignancies, suggesting the presence of an as yet unidentified tumor suppressor gene. The faint terminal subbands often make cytogenetic analysis of 1p36 particularly difficult. Small deletions at this locus may therefore escape detection on analysis by conventional cytogenetics, a hypothesis that we have explored using fluorescence in situ hybridization (FISH) in malignant lymphoma. The study cohort consisted of 20 cases of lymphoma of various subtypes without any 1p abnormality on G-banded karyotyping. FISH was performed using a human chromosome 1 paint and a bacterial artificial chromosome probe RP4-755G5 localizing to 1p36.33, the most telomeric subband of 1p36. Tumors demonstrating 1p36.33 deletions were additionally analyzed by FISH using a second probe from the proximal 1p36.1 subband, to further define the breakpoint. Eight cases of follicular lymphoma (FL), 5 diffuse large B-cell lymphomas (DLBCL), 2 Hodgkin disease, 2 B-cell small lymphocytic lymphomas, 2 T-cell lymphomas, and 1 marginal zone lymphoma were analyzed. FISH identified deletions at 1p36.33 in 5 of the 20 cases: 3 DLBCL and 2 FL. FISH is considerably more sensitive for identifying lymphoma genetic alterations than conventional cytogenetics. Deletion of the distal part of the 1p36 may be a much more common aberration than previously recognized in lymphoma.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 1 , Hibridização in Situ Fluorescente/métodos , Linfoma/genética , Adolescente , Adulto , Idoso , Análise Citogenética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sensibilidade e EspecificidadeRESUMO
Mutations in the MCPH1 (Microcephalin) and ASPM (abnormal spindle-like microcephaly associated) genes cause primary microcephaly. Both are centrosomal associated proteins involved in mitosis. Microcephalin plays an important role in DNA damage response and ASPM is required for correct division of proliferative neuro-epithelial cells of the developing brain. Reduced MCPH1 mRNA expression and ASPM mRNA over-expression have been implicated in the development of human carcinomas. Epithelial ovarian cancer (EOC) is characterised by highly aneuploid tumours. Previously we have reported low Microcephalin and high ASPM protein levels and associations with clinico-pathological parameters in malignant cells from ascitic fluids. To confirm these previous findings on a larger scale Microcephalin and ASPM expression levels and localisations were evaluated by immunohistochemistry in two cohorts; a training set of 25 samples and a validation set of 322 EOC tissue samples. Results were correlated to the associated histopathological data. In normal ovarian tissues the Microcephalin nuclear staining pattern was consistently strong. In the cancer tissues, we identified low nuclear Microcephalin expression in high grade and advanced stage tumours (p<0.0001 and p = 0.0438 respectively). ASPM had moderate to high nuclear and low to moderate cytoplasmic expression in normal tissue. Cytoplasmic ASPM expression decreased with tumour grade and stage in the serous subtype of EOC (p = 0.023 and p = 0.011 respectively). Cytoplasmic ASPM increased with tumour stage in the endometrioid subtype (p = 0.023). Increasing tumour invasiveness (T3) and lymph node involvement (N1) also correlated with a decrease in cytoplasmic ASPM in EOC (p = 0.02 and p = 0.04 respectively). We have validated previous findings of deregulated expression of Microcephalin and ASPM in EOC by confirming associations for low nuclear Microcephalin levels and high cytoplasmic ASPM levels in a larger scale tumour tissue study. Microcephalin and ASPM may prove useful biomarkers in EOC.
Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas do Tecido Nervoso/metabolismo , Neoplasias Ovarianas/metabolismo , Adulto , Idoso , Biomarcadores Tumorais/metabolismo , Proteínas de Ciclo Celular , Estudos de Coortes , Citoplasma/metabolismo , Proteínas do Citoesqueleto , Feminino , Humanos , Imuno-Histoquímica , Metástase Linfática , Pessoa de Meia-Idade , Invasividade Neoplásica , Proteínas do Tecido Nervoso/genética , Neoplasias Ovarianas/genética , RNA Mensageiro/metabolismo , Adulto JovemRESUMO
Premature chromosome condensation (PCC) is a consequence of early mitotic entry, where mitosis begins before completion of DNA replication. Previously we have identified mutations in MCPH1, a DNA damage response and potential tumor suppressor gene, as a cause of primary microcephaly and PCC. Here we describe a high-throughput assay to identify modifiers of PCC. Reverse transfection of control siRNA followed by a forward transfection of MCPH1 small interfering RNA (siRNA) was performed to induce PCC. Condensin II subunits CAPG2 and CAPH2 were validated as PCC modifiers and therefore positive controls. Cell nuclei were detected by DAPI staining using an Operetta imaging system. PCC and nuclei number were determined using Columbus analysis software. Two batches of nine plates were used to determine assay efficacy. Each plate contained four negative (nontargeting) and eight positive control siRNAs. Mean % PCC was 12.35% (n = 72) for negative controls and 4.25% (n = 144) for positive controls. Overall false-positive and false-negative rates were 0% (n = 72) and 2.1% (n = 144), respectively. This assay is currently being used to screen a human druggable genome siRNA library to identify novel therapeutic targets for cancer treatment. The assay can also be used to identify novel compounds and genes that induce PCC.