P19 embryonal carcinoma cells as in vitro model for studying purinergic receptor expression and modulation of N-methyl-d-aspartate–glutamate and acetylcholine receptors during neuronal differentiation

Extracellular ATP reduces tumor sphere growth and cancer stem cell population in glioblastoma cells

“…In our study, these receptors were also more expressed in the more differentiated population of glioma cells, i.e., the adherent cells when compared to spheres. The opposite was true for receptors more expressed in spheres, which were found to reduce its expression along differentiation of P19 cells, except for P2Y14 which was not analyzed in this study [16]. This suggests that there are similarities in the expression of purinergic receptors between neural stem cells and glioma CSCs and their more differentiated counterparts.…”

“…Three different antagonists of purinergic P2Y and P2X receptors decreased the differentiation of P19 neural stem cells to NMDA-responsive cells suggesting that activation of these receptors is pro-differentiative for neural stem cells [16]. Here we observed that ATP, which can activate P2Y and P2X receptors, reduces the number and size of spheres, increases the number of cells needed to form a sphere, and decreases the number of CD133, Nanog and Oct-4 positive CSCs.…”

“…Activation of P2Y receptors was shown to be mitogenic for mice neural progenitor cells, but had no effect on cell differentiation. Distinct expression of purinergic receptors in undifferentiated versus differentiated cells was observed in P19 embryonic carcinoma cells, a model of neural differentiation [16]. Several purinergic receptors changed their expression over differentiation, with P2X6 and P2X7 presenting a more clear-cut increase in expression.…”

“…We have previously shown that injection of the glioma cells together with apyrase, an enzyme that degrades ATP and adenosine diphosphate (ADP) to adenosine monophosphate (AMP), significantly reduced tumor growth [14], whereas expression of NTPDase2, an enzyme that degrades ATP to produce mainly ADP, strongly increased tumor size due to the pro-angiogenic effects of ADP [15]. ATP-mediated signaling was shown to be important in the differentiation of the murine embryonal carcinoma cell line P19 [16], but no study has addressed the impact of nucleotides and nucleosides in glioma CSCs. Here we show that ATP reduces the sphere formation in human and rat glioma CSCs and that purinergic receptors are differently expressed in tumor spheres when compared to adherent cells.…”

Cardiomyogenesis of embryonic stem cells upon purinergic receptor activation by ADP and ATP

“…Semiquantitative real-time RT-PCR analysis was done to identify messenger RNA (mRNA) expression of P2Y and P2X receptor subtypes in 5-18-day-old cells by using the QuantiFast SYBR Green kit followed by an initial denaturation step at 95°C for 10 min and 50 cycles at 95°C for 15 s and 62°C for 1 min with following primers [38]: P2X1, sense 5′-GAG AGT CGG GCC AGG ACT TC-3′, antisense 5′-GCG AAT CCC AAA CAC CTT CA-3′; P2X4, sense 5′-CCC ACT GCC TGC CCA GAT AT-3′, antisense 5′-ACA CTC ACC AAG GCA TAT GG-3′; P2X6, sense 5′-CCC AGA GCA TCC TTC TGT TCC-3′, antisense 5′-GGC ACC AGC TCC AGA TCT CA-3′; P2X7, sense 5′-GCA CGA ATT ATG GCA CCG TC-3′, antisense 5′-CCC CAC CCT CTG TGA CAT TCT-3′; P2Y1, sense 5′-CGT GCT GGT GTG CCT CAT T-3′, antisense 5′-GGA CCC CGG TAC CTG AGT AGA-3′; P2Y2, sense 5′-TTC CTG CCA TTC CAC GTC A-3′, antisense 5′-TTG AGG GTG TGG CAG CTG A-3′; P2Y4, sense 5′-TTG AGG GTG TGG CAG CTG A-3′, antisense 5′-TGT CCT TTT CCT CAC CTG CAT-3′; P2Y6, sense 5′-CCT GCC CAC AGC CAT CTT-3′, antisense 5′-GGC TGA GGT CAT AGC AGA CAG TG-3′. Experiments were performed in triplicate for each data point.…”