The P2X 7 receptor regulates cell growth through mediation of apoptosis. P2X 7 levels are lower in cancer epithelial cells than in normal cells, and previous studies showed that expression of P2X 7 was regulated post-transcriptionally. The objective of the study was to understand regulation of P2X 7 mRNA stability. Overexpression of a reporter containing the full-length human P2X 7 3-untranslated region (3-UTR) or reporters containing parts of the 3-UTR-P2X 7 were associated with increased abundance of the construct in normal cells and decreased abundance in cancer epithelial cells. Sequences within the 3-UTR-P2X 7 , which are putative target sites for the microRNAs, miR-186 (middle segment) and miR-150 (distal segment), decreased the abundance of the P2X 7 transcript. Overexpression in cancer cells of mutated miR-186 and miR-150 target sites was associated with lower levels of the reporter genes. In normal cells overexpression of the mutated miR-186 target site was associated with marked increased concentration, but overexpression of the miR-150 target site reporters, wild-type and mutant, did not change over time. Levels of miR-186 and miR-150 were higher in cancer than in normal cells, and treatment with miR-186 and miR-150 inhibitors increased P2X 7 mRNA. In human embryonic kidney-293 cells heterologously expressing the full-length 3-UTR-P2X 7 luciferase reporter, miR-186 and miR-150 inhibitors increased luciferase activity, whereas miR-186 and miR-150 mimics decreased luciferase activity after actinomycin D treatment. These data suggest that increased expression of miR-186 and miR-150 in cancer epithelial cells decreases P2X 7 mRNA by activation of miR-186 and miR-150 instability target sites located at the 3-UTR-P2X 7 .The receptor P2X 7 is a membrane-bound, ligand-operated channel (1-6). ATP is the naturally occurring ligand for the P2X 7 , and extracellular levels of ATP may reach low micromolar levels (7-12), which are sufficient to activate the receptor (13). Activation of the receptor may induce formation of pores in the plasma membrane (14), which in epithelial cells mediate apoptosis via the caspase-9 mitochondrial pathway (15, 16). The P2X 7 apoptosis effects can be regulated by receptor glycosylation (16), trafficking, plasma membrane expression (17-20), oligomerization (7, 21), and by receptor post-activation internalization, recycling, and degradation (14,21).In epithelial tissues the P2X 7 receptor is expressed predominantly by proliferative (germinative) epithelial cells (21-23), and it controls the growth of the epithelial cells. Previous studies in human uterine epithelial cells showed that base-line and P2X 7 -mediated apoptosis are lower in cancer cells than in normal cells (12,22,23). The differences were not the result of ligand availability because steady-state levels of ATP in conditioned media of cancer epithelial cells were similar to those of normal epithelial cells (12). Similarly, there were no significant differences in P2X 7 receptor activation, oligomerization, or cycling betwe...
BackgroundThe study tested the hypothesis that apoptosis can prevent and control growth of neoplastic cells. Previous studies in-vitro have shown that the pro-apoptotic P2X7 receptor regulates growth of epithelial cells. The specific objective of the present study was to understand to what degree the P2X7 system controls development and growth of skin cancer in vivo, and what cellular and molecular mechanisms are involved in the P2X7 action.MethodsSkin neoplasias in mice (papillomas, followed by squamous spindle-cell carcinomas) were induced by local application of DMBA/TPA. Experiments in-vitro utilized cultured epidermal keratinocytes generated from wild-type or from P2X7-null mice. Assays involved protein immunostaining and Western blots; mRNA real-time qPCR; and apoptosis (evaluated in situ by TUNEL and quantified in cultured keratinocytes as solubilized DNA or by ELISA). Changes in cytosolic calcium or in ethidium bromide influx (P2X7 pore formation) were determined by confocal laser microscopy.Results(a) Co-application on the skin of the P2X7 specific agonist BzATP inhibited formation of DMBA/TPA-induced skin papillomas and carcinomas. At the completion of study (week 28) the proportion of living animals with cancers in the DMBA/TPA group was 100% compared to 43% in the DMBA/TPA+BzATP group. (b) In the normal skin BzATP affected mainly P2X7-receptor – expressing proliferating keratinocytes, where it augmented apoptosis without evoking inflammatory changes. (c) In BzATP-treated mice the degree of apoptosis was lesser in cancer than in normal or papilloma keratinocytes. (d) Levels of P2X7 receptor, protein and mRNA were 4–5 fold lower in cancer tissues than in normal mouse tissues. (e) In cultured mouse keratinocytes BzATP induced apoptosis, formation of pores in the plasma membrane, and facilitated prolonged calcium influx. (f) The BzATP-induced apoptosis, pore-formation and augmented calcium influx had similar dose-dependence for BzATP. (g) Pore formation and the augmented calcium influx were depended on the expression of the P2X7 receptor, while the BzATP-induced apoptosis depended on calcium influx. (h) The BzATP-induced apoptosis could be blocked by co-treatment with inhibitors of caspase-9 and caspase-3, but not of caspase-8.Conclusion(a) P2X7-dependent apoptosis is an important mechanism that controls the development and progression of epidermal neoplasia in the mouse. (b) The P2X7-dependent apoptosis is mediated by calcium influx via P2X7 pores, and involves the caspase-9 (mitochondrial) pathway. (c) The diminished pro-apoptotic effect of BzATP in mouse cancer keratinocytes is possibly the result of low expression of the P2X7 receptor. (d) Activation of P2X7-dependent apoptosis, e.g. with BzATP could be a novel chemotherapeutic growth-preventive modality for papillomas and epithelial cancers in vivo.
Objectives-To understand the potential role of P2X 7 as biomarker of endometrial cancer, and the molecular mechanisms by which cancerous epithelial cells maintain low expression of P2X 7 .Methods-Feasibility clinical experimental study. Normal (28), simple or complex hyperplasia (7), complex hyperplasia with atypia (6) and cancer endometrial discarded tissues (40) were obtained from a total of 81 women, ages 25-75. Endpoint for P2X 7 protein was average pixel signal density of tissue immunoreactivity with anti-P2X 7 antibody. Endpoint for P2X 7 mRNA was one-step quantitative Real-Time PCR. Experiments in-vitro included normal (hEVEC) and cancerous cervical epithelial cells (HeLa) transfected with reporter plasmid containing luciferase-3′ untranslated region (3′UTR)-P2X 7 cDNA, using as endpoint steady-state luciferase mRNA levels.Results-Levels of P2X 7 protein and mRNA were significantly lower in vivo, in tissues of complex hyperplasia with atypia or endometrial adenocarcinoma, than in tissues of normal endometrium, simple hyperplasia or complex hyperplasia tissues (sensitivity and specificity of 89-100%, p<0.0001-0.01). Steady-state levels of luciferase mRNA increased over a 6 h incubation period in hEVEC cells transfected with the 3′ UTR-P2X 7 -luciferase vector, but decreased in HeLa cells transfected with the reporter plasmid.Conclusions-Tissue levels of P2X 7 protein and mRNA can differentiate effectively and accurately between normal and benign hyperplastic endometrial tissues from pre-cancerous and cancer tissues. Cancerous epithelial cells degrade P2X 7 mRNA by activation of instability domains located at the 3′UTR of the P2X 7 .
Transport of the essential amino acids arginine and lysine is critical for the survival of mammalian cells. The adaptive response to nutritional stress involves increased translation of the arginine/lysine transporter (cat-1) mRNA via an internal ribosome entry site (IRES) within the mRNA leader. Induction of cat-1 IRES activity requires both translation of a small upstream open reading frame (uORF) within the IRES and phosphorylation of the translation initiation factor eIF2alpha. We show here that translation of the upstream ORF unfolds an inhibitory structure in the mRNA leader, eliciting a conformational change that yields an active IRES. The IRES, whose activity is induced by amino acid starvation, is created by RNA-RNA interactions between the 5' end of the leader and downstream sequences. This study suggests that the structure of the IRES is dynamic and regulation of this RNA structure is a mechanism of translational control.
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