RESUMO
Some types of cells, if not all, that undergo signal exchanges in culture need to contact other cells for various reasons, such as cell-to-cell contact for growth inhibition. However, signal exchanges by cell-to-cell contact before proliferation have never been reported. Using time-lapse recording, we discovered the emergence of several astonishing cell-to-cell contact modes in bone marrow-derived mesenchymal stem/stromal cells (MSCs) before the cells divided. When the cells contacted with another, a huge temporary synapse-like structure formed for molecule exchanges; a cell-tissue particle was taken in by a recipient cell; two cell membranes formed infusion-like structure for a short time; and even a 20-µm long and 5-µm wide cell tail was grafted to another cell. A total of 87% of cells underwent cell-to-cell contact before dividing. After epidermal growth factor-green fluorescent protein (EGF-GFP) vectors were transfected into MSCs and the cells were cocultured with unmanipulated MSCs, the unmanipulated MSCs took in EGF-GFP particles from EGF-GFP expressed MSCs, immediately increased in mitogen genes, and then divided. These results suggest that cells which may lack signal molecules may need to obtain these molecules from other cells through various types of cell-to-cell contact, as mentioned above. Our study provided valuable information to better understand the behaviors of cell-to-cell contact and communication before mitosis.
Assuntos
Células da Medula Óssea/fisiologia , Comunicação Celular , Células-Tronco Mesenquimais/fisiologia , Transdução de Sinais , Adulto , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Células Cultivadas , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , MitoseRESUMO
Grade IV glioblastoma is characterized by increased kinase activity of epidermal growth factor receptor (EGFR); however, EGFR kinase inhibitors have failed to improve survival in individuals with this cancer because resistance to these drugs often develops. We showed that tumor necrosis factor-α (TNFα) produced in the glioblastoma microenvironment activated atypical protein kinase C (aPKC), thereby producing resistance to EGFR kinase inhibitors. Additionally, we identified that aPKC was required both for paracrine TNFα-dependent activation of the transcription factor nuclear factor κB (NF-κB) and for tumor cell-intrinsic receptor tyrosine kinase signaling. Targeting aPKC decreased tumor growth in mouse models of glioblastoma, including models of EGFR kinase inhibitor-resistant glioblastoma. Furthermore, aPKC abundance and activity were increased in human glioblastoma tumor cells, and high aPKC abundance correlated with poor prognosis. Thus, targeting aPKC might provide an improved molecular approach for glioblastoma therapy.
Assuntos
Carcinogênese/metabolismo , Receptores ErbB/metabolismo , Glioblastoma/enzimologia , Proteína Quinase C/metabolismo , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Carcinogênese/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Ensaio de Imunoadsorção Enzimática , Fator de Crescimento Epidérmico/farmacologia , Cloridrato de Erlotinib , Citometria de Fluxo , Imunofluorescência , Glioblastoma/tratamento farmacológico , Humanos , Immunoblotting , Imuno-Histoquímica , Imunoprecipitação , Estimativa de Kaplan-Meier , Camundongos , NF-kappa B/metabolismo , Comunicação Parácrina/fisiologia , Proteína Quinase C/antagonistas & inibidores , Quinazolinas/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacosRESUMO
Background. Levels of the protein kinase aPKC have been previously correlated with prostate cancer prognosis in a British cohort. However, prostate cancer incidence and progression rates, as well as genetic changes in this disease, show strong ethnic variance, particularly in Asian populations. Objective. The aim of this study was to validate association of aPKC expression with prostatic adenocarcinoma stages in a Japanese cohort. Methods. Tissue microarrays consisting of 142 malignant prostate cancer cases and 21 benign prostate tissues were subject to immunohistological staining for aPKC. aPKC staining intensity was scored by three independent pathologists and categorized as absent (0), dim (1+), intermediate (2+), and bright (3+). aPKC staining intensities were correlated with Gleason score and tumor stage. Results. Increased aPKC staining was observed in malignant prostate cancer, in comparison to benign tissue. Additionally, aPKC staining levels correlated with Gleason score and tumor stage. Our results extend the association of aPKC with prostate cancer to a Japanese population and establish the suitability of aPKC as a universal prostate cancer biomarker that performs consistently across ethnicities.
RESUMO
CONTEXT: Pulmonary large cell carcinoma (LCC) includes tumors not readily diagnosed as adenocarcinoma (ADC) or squamous cell carcinoma on morphologic grounds, without regard to immunophenotype, according to the World Health Organization (WHO). This ambiguous designation may cause confusion over selection of mutation testing and directed therapies. Several groups have proposed the use of immunohistochemistry (IHC) to recategorize LCC as ADC or squamous cell carcinoma; however, it remains unclear if strictly defined LCCs are a clinicopathologically distinct lung tumor subset. OBJECTIVE: To compare the pathologic, molecular, and clinical features of 2 morphologically similar tumors: solid-subtype ADC and LCC. DESIGN: Tumors were included on the basis of solid growth pattern; tumors with squamous or neuroendocrine differentiation were excluded. Solid ADC (n = 42) and LCC (n = 57) were diagnosed by using WHO criteria (5 intracellular mucin droplets in ≥2 high-power fields for solid ADC) and tested for KRAS, EGFR, and ALK alterations. RESULTS: Both solid ADC and LCC groups were dominated by tumors with "undifferentiated"-type morphology and both had a high frequency of thyroid transcription factor 1 expression. KRAS was mutated in 38% of solid ADCs versus 43% of LCCs (P = .62). One ALK-rearranged and 1 EGFR-mutated tumor were detected in the solid ADC and LCC groups, respectively. There were no significant differences in clinical features or outcomes; the prevalence of smoking in both groups was greater than 95%. CONCLUSIONS: Other than a paucity of intracellular mucin, LCC lacking squamous or neuroendocrine differentiation is indistinguishable from solid-subtype ADC. We propose the reclassification of these tumors as mucin-poor solid adenocarcinomas.
Assuntos
Adenocarcinoma/patologia , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Grandes/patologia , Diferenciação Celular , Neoplasias Pulmonares/patologia , Adenocarcinoma/diagnóstico , Adenocarcinoma/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Quinase do Linfoma Anaplásico , Carcinoma de Células Grandes/diagnóstico , Carcinoma de Células Grandes/metabolismo , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Proteínas de Ligação a DNA/metabolismo , Diagnóstico Diferencial , Receptores ErbB/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/metabolismo , Masculino , Pessoa de Meia-Idade , Mucinas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas p21(ras) , Receptores Proteína Tirosina Quinases/metabolismo , Estudos Retrospectivos , Fatores de Transcrição , Organização Mundial da Saúde , Proteínas ras/metabolismoRESUMO
We describe a genome-wide gain-of-function screen for regulators of NF-kappaB, and identify Rap1 (Trf2IP), as an essential modulator of NF-kappaB-mediated pathways. NF-kappaB is induced by ectopic expression of Rap1, whereas its activity is inhibited by Rap1 depletion. In addition to localizing on telomeres, mammalian Rap1 forms a complex with IKKs (IkappaB kinases), and is crucial for the ability of IKKs to be recruited to, and phosphorylate, the p65 subunit of NF-kappaB to make it transcriptionally competent. Rap1-mutant mice display defective NF-kappaB activation and are resistant to endotoxic shock. Furthermore, levels of Rap1 are positively regulated by NF-kappaB, and human breast cancers with NF-kappaB hyperactivity show elevated levels of cytoplasmic Rap1. Similar to inhibiting NF-kappaB, knockdown of Rap1 sensitizes breast cancer cells to apoptosis. These results identify the first cytoplasmic role of Rap1 and provide a mechanism through which it regulates an important signalling cascade in mammals, independent of its ability to regulate telomere function.
Assuntos
Quinase I-kappa B/metabolismo , NF-kappa B/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Cromatografia em Gel , Células HeLa , Humanos , Quinase I-kappa B/genética , Imuno-Histoquímica , Imunoprecipitação , Estimativa de Kaplan-Meier , Camundongos , NF-kappa B/genética , Fosforilação/genética , Fosforilação/fisiologia , Reação em Cadeia da Polimerase , Ligação Proteica/genética , Ligação Proteica/fisiologia , RNA Interferente Pequeno , Complexo Shelterina , Proteínas de Ligação a Telômeros/genética , Análise Serial de TecidosRESUMO
Creutzfeldt-Jakob disease (CJD) is a fatal progressive infectious encephalopathy of humans characterized by spongiform degeneration of the brain. Detection of protease-resistant low molecular weight proteins, referred to as 'prions', in the brain is essential for diagnosis. Protease-based methods for prion detection are problematic due to variable susceptibility of prion proteins to proteinase-K digestion. Since CJD brain samples are infectious at all stages of the prion extraction process, we set out to develop a laboratory safe method for prion purification. We lysed the tissues with guanidine thiocyanate followed by phenol extraction of the proteins. Western blotting using prion-specific MAB 3F4 revealed primarily low molecular weight unglycosylated prion (UGP) bands in all CJD cases (19) while the predominant banding in all normal brains (14) represented glycosylated prion (GP). Density readings of the blots revealed the UGP/GP ratio to be significantly different in CJD versus normal brains, with an inverse UGP/GP ratio in CJD. Using this method, we discovered one previously undiagnosed CJD case when we screened 19 brains from the Louisiana State University Alzheimer disease brain bank. Our method is a safe and reliable way of detecting abnormal prion proteins (p<0.0001) and is adaptable to both diagnostic and research laboratories.
Assuntos
Western Blotting/métodos , Síndrome de Creutzfeldt-Jakob/diagnóstico , Príons/análise , Química Encefálica , Glicosilação , Guanidinas , Humanos , Fenol , Segurança , TiocianatosRESUMO
It is established that human mesenchymal stem cells (hMSCs) from bone marrow are a source of osteoblast progenitors in vivo and under appropriate conditions, differentiate into osteoblasts ex vivo. Because hMSCs are recovered by iliac crest aspirate and enriched by virtue of their adherence to tissue culture plastic, the cells provide a convenient ex vivo model for the study of osteogenic tissue repair in an experimentally accessible system. Recent advances in the field of skeletal development and osteogenesis have demonstrated that signaling through the canonical wingless (Wnt) pathway is critical for the differentiation of progenitor cell lines into osteoblasts. Inhibition of such signals can predispose MSCs to cell cycle entry and inhibit osteogenesis. Here, we report that synthetic peptides derived from the second cysteine-rich domain of the canonical Wnt inhibitor Dickkopf-1 (Dkk-1) have utility in controlling the growth and recovery of hMSCs from bone marrow stroma. Three peptides corresponding to residues 217-269 in Dkk-1 were each found to enhance the proliferation of hMSCs in culture over 2 days. The most active peptide exhibited agonistic characteristics in that it ablated the proliferation lag observed when cultures of hMSCs receive fresh medium. It also reduced the expression of endogenous Dkk-1 (Gregory, C. A., Singh, H., and Prockop, D. J. (2003) J. Biol. Chem. 278, 28067-28078). When the cytosolic level of beta-catenin was elevated by addition of LiCl to cultures of hMSCs, the peptide also accelerated degradation of beta-catenin on withdrawal of lithium. A second peptide, corresponding to residues 184-204 had preferential and high affinity for hMSCs in the log phase of proliferation. Peptide overlay assays on hMSC lysates confirmed that the peptide bound to a 184-kDa protein corresponding to the molecular mass of LRP6. Cells recovered by this peptide had enhanced osteogenic potential but less chondrogenic potential compared with controls. Because Wnt antagonists increase the number of non-committed hMSCs in culture, they may be of use in increasing the rate of osseous wound healing in vivo by increasing the level of systemically migrating hMSCs. Therefore, such molecules could contribute to the development of a novel family of pharmaceutical agents for the improvement of the healing process in humans.
Assuntos
Células da Medula Óssea/citologia , Cloreto de Lítio/farmacologia , Peptídeos/farmacologia , Proteínas/química , Células-Tronco/citologia , Adulto , Sequência de Aminoácidos , Sequência de Bases , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Dados de Sequência Molecular , Mapeamento de PeptídeosRESUMO
Adult human mesenchymal stem cells from bone marrow stroma (hMSCs) differentiate into numerous mesenchymal tissue lineages and are attractive candidates for cell and gene therapy. When early passage hMSCs are plated or replated at low density, the cultures display a lag phase of 3-5 days, a phase of rapid exponential growth, and then enter a stationary phase without the cultures reaching confluence. We found that as the cultures leave the lag phase, they secrete high levels of dickkopf-1 (Dkk-1), an inhibitor of the canonical Wnt signaling pathway. The addition of recombinant Dkk-1 toward the end of the lag period increased proliferation and decreased the cellular concentration of beta-catenin. The addition of antibodies to Dkk-1 in the early log phase decreased proliferation. Also, expression of Dkk-1 in hMSCs decreased during cell cycle arrest induced by serum starvation. The results indicated that high levels of Dkk-1 allow the cells to reenter the cell cycle by inhibiting the canonical Wnt/beta-catenin signaling pathway. Since antibodies to Dkk-1 also increased the lag phase of an osteosarcoma line that expressed the gene, Dkk-1 may have a similar role in some other cell systems.