BACKGROUND AND PURPOSE Splice variants of P2X7 receptor transcripts contribute to the diversity of receptor‐mediated responses. Here, we investigated expression and function of C‐terminal truncated (ΔC) variants of the mP2X7 receptor, which are predicted to escape inactivation in one strain of P2X7−/− mice (Pfizer KO). EXPERIMENTAL APPROACH Expression in wild‐type (WT) and Pfizer KO tissue was investigated by reverse transcription (RT)‐PCR and Western blot analysis. ΔC variants were also cloned and expressed in HEK293 cells to investigate their assembly, trafficking and function. KEY RESULTS RT‐PCR indicates expression of a ΔC splice variant in brain, salivary gland (SG) and spleen from WT and Pfizer KO mice. An additional ΔC hybrid transcript, containing sequences of P2X7 upstream of exon 12, part of exon 13 followed in‐frame by the sequence of the vector used to disrupt the P2X7 gene, was also identified in the KO mice. By blue native (BN) PAGE analysis and the use of cross linking reagents followed by SDS‐PAGE, P2X7 trimers, dimers and monomers were detected in the spleen and SG of Pfizer KO mice. The molecular mass was reduced compared with P2X7 in WT mice tissue, consistent with a ΔC variant. When expressed in HEK293 cells the ΔC variants were inefficiently trafficked to the cell surface and agonist‐evoked whole cell currents were small. Co‐expressed with P2X7A, the ΔC splice variant acted in a dominant negative fashion to inhibit function. CONCLUSIONS AND IMPLICATIONS Pfizer KO mice are not null for P2X7 receptor expression but express ΔC variants with reduced function.
Inhibitor of apoptosis (IAP) proteins are expressed at high levels in many cancers and therefore represent attractive targets for therapeutic intervention. Here, we report for the first time that the second mitochondria-derived activator of caspases (Smac) mimetic BV6 sensitizes glioblastoma cells toward Temozolomide (TMZ), the first-line chemotherapeutic agent in the treatment of glioblastoma. BV6 and TMZ synergistically reduce cell viability and trigger apoptosis in glioblastoma cells (combination index o0.4-0.8), which is accompanied by increased loss of mitochondrial-membrane potential, cytochrome c release, caspase activation and caspase-dependent apoptosis. Analysis of the molecular mechanisms reveals that BV6 causes rapid degradation of cIAP1, leading to stabilization of NF-kB-inducing kinase and NF-kB activation. BV6-stimulated NF-kB activation is critically required for sensitization toward TMZ, as inhibition of NF-kB by overexpression of the mutant IkBa super-repressor profoundly reduces loss of mitochondrial membrane potential, cytochrome c release, caspase activation and apoptosis. Of note, BV6mediated sensitization to TMZ is not associated with increased tumor necrosis factor alpha (TNFa) production. Also, TNFa, CD95 or TRAIL-blocking antibodies or knockdown of TNFR1 have no or little effect on combination treatment-induced apoptosis. Interestingly, BV6 and TMZ cooperate to trigger the formation of a RIP1 (receptor activating protein 1)/caspase-8/FADD complex. Knockdown of RIP1 by small interfering RNA significantly reduces BV6-and TMZ-induced caspase-8 activation and apoptosis, showing that RIP1 is necessary for apoptosis induction. By demonstrating that BV6 primes glioblastoma cells for TMZ in a NF-kB-and RIP1-dependent manner, these findings build the rationale for further (pre)clinical development of Smac mimetics in combination with TMZ.
Evasion of apoptosis contributes to radioresistance of glioblastoma, calling for novel strategies to overcome apoptosis resistance. In this study, we investigated the potential of the small molecule Smac mimetic BV6 to modulate radiosensitivity of glioblastoma cells. Here, we identify a novel proapoptotic function of NF-kB in g-irradiation-induced apoptosis of glioblastoma cells by showing, for the first time, that NF-kB is critically required for Smac mimetic-mediated radiosensitization. BV6 significantly increases g-irradiation-triggered apoptosis in several glioblastoma cell lines in a dose-and time-dependent manner. Calculation of combination index (CI) reveals that the interaction of BV6 and g-irradiation is highly synergistic (CI < 0.3). Molecular studies show that BV6 stimulates NF-kB activation, which is critical for radiosensitization, because genetic inhibition of NF-kB by overexpression of the dominant-negative superrepressor IkBa-SR significantly decreases BV6-and g-irradiation-induced apoptosis. Also, the BV6-mediated enhancement of g-irradiation-triggered caspase activation, drop of mitochondrial membrane potential, and cytochrome c release is abolished in cells overexpressing IkBa-SR. Similarly, NF-kB inhibition by ectopic expression of a kinase dead mutant of IKKb prevents the BV6-mediated sensitization for g-irradiation. The clinical relevance is underscored by experiments with primary tumor samples showing that BV6 sensitizes primary cultured glioma cells as well as glioblastoma-initiating cancer stem cells derived from surgical specimens for g-irradiation. In conclusion, we identify NF-kB as a critical mediator of Smac mimetic-conferred radiosensitization of glioblastoma cells. These results have important implications for the development of Smac mimetic-based combination protocols for radiosensitization of glioblastoma. Mol Cancer Ther; 10(10); 1867-75. Ó2011 AACR.
SummaryP2X7 receptors function as ATP-gated cation channels but also interact with other proteins as part of a larger signalling complex to mediate a variety of downstream responses that are dependent upon the cell type in which they are expressed. Receptor-mediated membrane permeabilization to large molecules precedes the induction of cell death, but remains poorly understood. The mechanisms that underlie differential sensitivity to NAD are also unknown. By studying alternative variants of the mouse P2X7 receptor we show that sensitivity to NAD is mediated through the P2X7k variant, which has a much more restricted distribution than the P2X7a receptor, but is expressed in T lymphocytes. The altered N-terminus and TM1 of the P2X7k receptor enhances the stability of the active state of this variant compared with P2X7a, thereby increasing the efficacy of NAD-dependent ADP ribosylation as measured by ethidium uptake, a rise in intracellular Ca 2+ and the activation of inward currents. Co-expression of P2X7k and P2X7a receptors reduced NAD sensitivity. P2X7k-receptor-mediated ethidium uptake was also triggered by much lower BzATP concentrations and was insensitive to the P451L single nucleotide polymorphism. P2X7k-receptor-mediated ethidium uptake occurred independently of pannexin-1 suggesting a pathway intrinsic to the receptor. Only for the P2X7aL451 receptor could we resolve a component of dye uptake dependent upon pannexin-1. Signalling occurred downstream of the activation of caspases rather than involving direct cross talk between the channels. However, an in situ proximity assay showed close association between P2X7 receptors and pannexin-1, which would facilitate ATP efflux through pannexin-1 acting in an autocrine manner.
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