RESUMO
Embryonic carcinoma cells are widely used models for studying the mechanisms of proliferation and differentiation occurring during early embryogenesis. We have now investigated how down-regulation of P2X2 and P2X7 receptor expression by RNA interference (RNAi) affects neural differentiation and phenotype specification of P19 embryonal carcinoma cells. Wild-type P19 embryonal carcinoma cells or cells stably expressing shRNAs targeting P2X2 or P2X7 receptor expression were induced to differentiate into neurons and glial cells in the presence of retinoic acid. Silencing of P2X2 receptor expression along differentiation promoted cell proliferation and an increase in the percentage of cells expressing glial-specific GFAP, while the presence of beta-3 tubulin-positive cells diminished at the same time. Proliferation induction in the presence of stable anti-P2X2 receptor RNAi points at a mechanism where glial proliferation is favored over growth arrest of progenitor cells which would allow neuronal maturation. Differently from the P2X2 receptor, inhibition of P2X7 receptor expression during neural differentiation of P19 cells resulted in a decrease in cell proliferation and GFAP expression, suggesting the need of functional P2X7 receptors for the progress of gliogenesis. The results obtained in this study indicate the importance of purinergic signaling for cell fate determination during neural differentiation, with P2X2 and P2X7 receptors promoting neurogenesis and gliogenesis, respectively. The shRNAs down-regulating P2X2 or P2X7 receptor gene expression, developed during this work, present useful tools for studying mechanisms of neural differentiation in other stem cell models.
Assuntos
Células-Tronco de Carcinoma Embrionário/citologia , Células-Tronco Neurais/citologia , Neurogênese/fisiologia , Neuroglia/citologia , Neurônios/citologia , Receptores Purinérgicos P2X2/fisiologia , Receptores Purinérgicos P2X7/fisiologia , Tretinoína/fisiologia , Animais , Diferenciação Celular/genética , Células-Tronco de Carcinoma Embrionário/metabolismo , Células-Tronco de Carcinoma Embrionário/fisiologia , Camundongos , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/fisiologia , Neurogênese/genética , Neuroglia/metabolismo , Neuroglia/fisiologia , Neurônios/metabolismo , Neurônios/fisiologia , Interferência de RNA/fisiologia , Receptores Purinérgicos P2X2/genética , Receptores Purinérgicos P2X7/genética , Transdução de Sinais/genéticaRESUMO
Connective tissue growth factor (CTGF/CCN2) is a protein of the CCN family that modulates cell-ECM interactions in a variety of cell types. In this study, we investigated the chemotactic and adhesive properties of CCN2 protein in embryonic teratocarcinoma P19 cells. Initially, P19 cells were attracted to CCN2-coated agarose beads. In Boyden chamber experiments, CCN2-containing medium induced a threefold greater migration of P19 cells. CCN2 adhesion properties were studied by using optical tweezers. The specific adhesion times of P19 cells to polystyrene beads coated with laminin, fibronectin, CCN2 and bovine serum albumin were 1.8 ± 0.5s, 2.7 ± 0.4s, 10 ± 2s and 13 ± 2s, respectively, revealing an unexpectedly low adhesive capacity of CCN2 protein for P19 cells. In conclusion, our findings support the chemoattractive role of CCN2 for P19 cells, but not its adhesive role when compared to laminin or fibronectin.
Assuntos
Quimiotaxia , Fator de Crescimento do Tecido Conjuntivo/fisiologia , Células-Tronco de Carcinoma Embrionário/patologia , Animais , Adesão Celular , Linhagem Celular Tumoral , Proliferação de Células , Fator de Crescimento do Tecido Conjuntivo/farmacologia , Células-Tronco de Carcinoma Embrionário/metabolismo , Camundongos , Sefarose/químicaRESUMO
The prostate stromal mesenchyme controls organ-specific development. In cancer, the stromal compartment shows altered gene expression compared to non-cancer. The lineage relationship between cancer-associated stromal cells and normal tissue stromal cells is not known. Nor is the cause underlying the expression difference. Previously, the embryonal carcinoma (EC) cell line, NCCIT, was used by us to study the stromal induction property. In the current study, stromal cells from non-cancer (NP) and cancer (CP) were isolated from tissue specimens and co-cultured with NCCIT cells in a trans-well format to preclude heterotypic cell contact. After 3 days, the stromal cells were analyzed by gene arrays for microRNA (miRNA) and mRNA expression. In co-culture, NCCIT cells were found to alter the miRNA and mRNA expression of NP stromal cells to one like that of CP stromal cells. In contrast, NCCIT had no significant effect on the gene expression of CP stromal cells. We conclude that the gene expression changes in stromal cells can be induced by diffusible factors synthesized by EC cells, and suggest that cancer-associated stromal cells represent a more primitive or less differentiated stromal cell type.
Assuntos
Células-Tronco de Carcinoma Embrionário/metabolismo , MicroRNAs/genética , Próstata/metabolismo , Próstata/patologia , Comunicação Celular , Linhagem Celular Tumoral , Forma Celular , Técnicas de Cocultura , Meios de Cultura , Citoplasma/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , MicroRNAs/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Estromais/metabolismo , Células Estromais/patologiaRESUMO
P19 cells, a pluripotent cell line derived from a teratocarcinoma induced in C3H/HeHa mice, have been widely used as a model system to study cardiac differentiation. We have used these cells to evaluate the extent to which exposure to DMSO and/or cardiogenol C for 4 days in suspension culture enhanced their differentiation into cardiomyocytes. Cardiac differentiation was assessed by observing beating clusters and further confirmed using immunocytochemical, biochemical, and pharmacological approaches. The presence of functional gap junctions in differentiated P19 cells was identified through calcium wave analyses. Proliferation rate and cell death were analyzed by BrdU incorporation and activated caspase-3 immunodetection, respectively. Beating clusters of differentiated P19 cells were only found in cultures treated with DMSO. In addition, groups treated with DMSO up-regulated cardiac troponin-T expression. However, when DMSO was used together with cardiogenol C the up-regulation was less than that with DMSO alone, approximately 1.5 times. Moreover, P19 cells cultured in DMSO or DMSO plus 0.25 microM cardiogenol C had lower proliferation rates and higher numbers of activated caspase-3-positive cells. In summary, using several methodological approaches we have demonstrated that DMSO can induce cardiac differentiation of P19 cells but that cardiogenol C does not.
Assuntos
Compostos de Anilina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Dimetil Sulfóxido/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Pirimidinas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Sinalização do Cálcio/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Células-Tronco de Carcinoma Embrionário/metabolismo , Células-Tronco de Carcinoma Embrionário/patologia , Ativação Enzimática/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C3H , Microscopia Confocal , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Fator 3 de Transcrição de Octâmero/biossíntese , Troponina T/biossíntese , Regulação para Cima/efeitos dos fármacosRESUMO
Changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) play a central role in neuronal differentiation. However, Ca(2+) signaling in this process remains poorly understood and it is unknown whether embryonic and adult stem cells share the same signaling pathways. To clarify this issue, neuronal differentiation was analyzed in two cell lines: embryonic P19 carcinoma stem cells (CSCs) and adult murine bone-marrow mesenchymal stem cells (MSC). We studied Ca(2+) release from the endoplasmic reticulum via intracellular ryanodine-sensitive (RyR) and IP(3)-sensitive (IP(3)R) receptors. We observed that caffeine, a RyR agonist, induced a [Ca(2+)](i) response that increased throughout neuronal differentiation. We also demonstrated a functional coupling between RyRs and L- but not with N-, P-, or Q-type Ca(v)1 Ca(2+) channels, both in embryonal CSC and adult MSC. We also found that agonists of L-type channels and of RyRs increase neurogenesis and neuronal differentiation, while antagonists of these channels have the opposite effect. Thus, our data demonstrate that in both cell lines RyRs control internal Ca(2+) release following voltage-dependent Ca(2+) entry via L-type Ca(2+) channels. This study shows that both in embryonal CSC and adult MSC [Ca(2+)](i) is controlled by a common pathway, indicating that coupling of L-type Ca(2+) channels and RyRs may be a conserved mechanism necessary for neuronal differentiation.