Pharmacological and genetic characterisation of the canine P2X4 receptor.

BACKGROUND AND PURPOSE: P2X4 receptors are emerging therapeutic targets for treating chronic pain and cardiovascular disease. Dogs are well-recognised natural models of human disease, but information regarding P2X4 receptors in dogs is lacking. To aid the development and validation of P2X4 receptor ligands, we have characterised and compared canine and human P2X4 receptors. EXPERIMENTAL APPROACH: Genomic DNA was extracted from whole blood samples from 101 randomly selected dogs and sequenced across the P2RX4 gene to identify potential missense variants. Recombinant canine and human P2X4 receptors tagged with Emerald GFP were expressed in 1321N1 and HEK293 cells and analysed by immunoblotting and confocal microscopy. In these cells, receptor pharmacology was characterised using nucleotide-induced Fura-2 AM measurements of intracellular Ca2+ and known P2X4 receptor antagonists. P2X4 receptor-mediated inward currents in HEK293 cells were assessed by automated patch clamp. KEY RESULTS: No P2RX4 missense variants were identified in any canine samples. Canine and human P2X4 receptors were localised primarily to lysosomal compartments. ATP was the primary agonist of canine P2X4 receptors with near identical efficacy and potency at human receptors. 2'(3')-O-(4-benzoylbenzoyl)-ATP, but not ADP, was a partial agonist with reduced potency for canine P2X4 receptors compared to the human orthologues. Five antagonists inhibited canine P2X4 receptors, with 1-(2,6-dibromo-4-isopropyl-phenyl)-3-(3-pyridyl)urea displaying reduced sensitivity and potency at canine P2X4 receptors. CONCLUSION AND IMPLICATIONS: P2X4 receptors are highly conserved across dog pedigrees and display expression patterns and pharmacological profiles similar to human receptors, supporting validation and use of therapeutic agents for P2X4 receptor-related disease onset and management in dogs and humans.

Probenecid directly impairs activation of the canine P2X7 receptor.

The current study aimed to determine if probenecid could directly impair the canine P2X7 receptor, a ligand-gated cation channel activated by extracellular adenosine 5'-triphosphate (ATP). Patch clamp measurements demonstrated that probenecid impairs ATP-induced inward currents in HEK-293 cells expressing canine P2X7. Flow cytometric measurements of ethidium+ uptake into HEK-293 cells expressing canine P2X7 showed that probenecid impairs ATP-induced pore formation in a concentration-dependent manner, with a half maximal inhibitory concentration of 158 µM. Finally, ELISA measurements revealed that probenecid impairs ATP-induced interleukin-1ß release in dog blood. In conclusion, this study reveals that probenecid can directly impair canine P2X7 activation.

Tumor-propagating cells and Yap/Taz activity contribute to lung tumor progression and metastasis.

Metastasis is the leading cause of morbidity for lung cancer patients. Here we demonstrate that murine tumor propagating cells (TPCs) with the markers Sca1 and CD24 are enriched for metastatic potential in orthotopic transplantation assays. CD24 knockdown decreased the metastatic potential of lung cancer cell lines resembling TPCs. In lung cancer patient data sets, metastatic spread and patient survival could be stratified with a murine lung TPC gene signature. The TPC signature was enriched for genes in the Hippo signaling pathway. Knockdown of the Hippo mediators Yap1 or Taz decreased in vitro cellular migration and transplantation of metastatic disease. Furthermore, constitutively active Yap was sufficient to drive lung tumor progression in vivo. These results demonstrate functional roles for two different pathways, CD24-dependent and Yap/Taz-dependent pathways, in lung tumor propagation and metastasis. This study demonstrates the utility of TPCs for identifying molecules contributing to metastatic lung cancer, potentially enabling the therapeutic targeting of this devastating disease.

Extracellular adenosine 5'-triphosphate and lipopolysaccharide induce interleukin-1ß release in canine blood.

Binding of extracellular adenosine 5'-triphosphate (ATP) or lipopolysaccharide (LPS) to the damage-associated molecular pattern receptor P2X7 or the pathogen-associated molecular pattern receptor Toll-like receptor (TLR)4, respectively, can induce the release of the pleiotropic cytokine interleukin (IL)-1ß in humans and mice. However, the release of IL-1ß in dogs remains poorly defined. Using a canine IL-1ß enzyme-linked immunosorbent assay, this study investigated whether ATP or LPS could induce IL-1ß release in a canine blood-based assay. Short-term incubations (30 min) with ATP induced IL-1ß release in LPS-primed canine blood, and this process could be near-completely impaired by the P2X7 antagonist, A438079. In contrast, ATP failed to induce IL-1ß release from blood not primed with LPS. ATP-induced IL-1ß release was observed with LPS-primed blood from eight different pedigrees or cross breeds. Long-term incubations (24h) with LPS induced IL-1ß release in canine blood in a concentration-dependent manner. This process was not altered by co-incubation with A438079. LPS-induced IL-1ß release was observed with blood from 10 different pedigrees or cross breeds. These results demonstrate that both extracellular ATP and LPS can induce IL-1ß release in dogs, and that ATP- but not LPS-induced IL-1ß release in blood is dependent on P2X7 activation. These findings support the role of both P2X7 and TLR4 in IL-1ß release in dogs.

Characterization of the cell of origin for small cell lung cancer.

Small cell lung carcinoma (SCLC) is a neuroendocrine subtype of lung cancer that affects more than 200,000 people worldwide every year with a very high mortality rate. Here, we used a mouse genetics approach to characterize the cell of origin for SCLC; in this mouse model, tumors are initiated by the deletion of the Rb and p53 tumor suppressor genes in the lung epithelium of adult mice. We found that mouse SCLCs often arise in the lung epithelium, where neuroendocrine cells are located, and that the majority of early lesions were composed of proliferating neuroendocrine cells. In addition, mice in which Rb and p53 are deleted in a variety of non-neuroendocrine lung epithelial cells did not develop SCLC. These data indicate that SCLC likely arises from neuroendocrine cells in the lung.

Local mesenchymal stem/progenitor cells are a preferential target for initiation of adult soft tissue sarcomas associated with p53 and Rb deficiency.

The cell of origin and pathogenesis of the majority of adult soft tissue sarcomas (STS) remains poorly understood. Because mutations in both the P53 and RB tumor suppressor genes are frequent in STS in humans, we inactivated these genes by Cre-loxP-mediated recombination in mice with floxed p53 and Rb. Ninety-three percent of mice developed spindle cell/pleomorphic sarcomas after a single subcutaneous injection of adenovirus carrying Cre-recombinase. Similar to human STS, these sarcomas overexpress Cxcr4, which contributes to their invasive properties. Using irradiation chimeras generated by transplanting bone marrow cells from mice carrying either the Rosa26StoploxPLacZ or the Z/EG reporter, as well as the floxed p53 and Rb genes, into irradiated p53loxP/loxPRbloxP/loxP mice, it was determined that sarcomas do not originate from bone marrow-derived cells, such as macrophages, but arise from the local resident cells. At the same time, dermal mesenchymal stem cells isolated by strict plastic adherence and low levels of Sca-1 expression (Sca-1low, CD31negCD45neg) have shown enhanced potential for malignant transformation according to soft agar, invasion, and tumorigenicity assays, after the conditional inactivation of both p53 and Rb. Sarcomas formed after transplantation of these cells have features typical for undifferentiated high-grade pleomorphic sarcomas. Taken together, our studies indicate that local Sca-1low dermal mesenchymal stem/progenitor cells are preferential targets for malignant transformation associated with deficiencies in both p53 and Rb.

Primary tumor genotype is an important determinant in identification of lung cancer propagating cells.

Successful cancer therapy requires the elimination or incapacitation of all tumor cells capable of regenerating a tumor. Therapeutic advances therefore necessitate the characterization of the cells that are able to propagate a tumor in vivo. We show an important link between tumor genotype and isolation of tumor-propagating cells (TPCs). Three mouse models of the most common form of human lung cancer each had TPCs with a unique cell-surface phenotype. The cell-surface marker Sca1 did not enrich for TPCs in tumors initiated with oncogenic Kras, and only Sca1-negative cells propagated EGFR mutant tumors. In contrast, Sca1-positive cells were enriched for tumor-propagating activity in Kras tumors with p53 deficiency. Primary tumors that differ in genotype at just one locus can therefore have tumor-propagating cell populations with distinct markers. Our studies show that the genotype of tumor samples must be considered in studies to identify, characterize, and target tumor-propagating cells.