No abstract

Rationale: Alveolar liquid clearance is regulated by Na 1 uptake through the apically expressed epithelial sodium channel (ENaC) and basolaterally localized Na 1 -K 1 -ATPase in type II alveolar epithelial cells. Dysfunction of these Na 1 transporters during pulmonary inflammation can contribute to pulmonary edema.Objectives: In this study, we sought to determine the precise mechanism by which the TIP peptide, mimicking the lectin-like domain of tumor necrosis factor (TNF), stimulates Na 1 uptake in a homologous cell system in the presence or absence of the bacterial toxin pneumolysin (PLY).Methods: We used a combined biochemical, electrophysiological, and molecular biological in vitro approach and assessed the physiological relevance of the lectin-like domain of TNF in alveolar liquid clearance in vivo by generating triple-mutant TNF knock-in mice that express a mutant TNF with deficient Na 1 uptake stimulatory activity.Measurements and Main Results: TIP peptide directly activates ENaC, but not the Na 1 -K 1 -ATPase, upon binding to the carboxyterminal domain of the a subunit of the channel. In the presence of PLY, a mediator of pneumococcal-induced pulmonary edema, this binding stabilizes the ENaC-PIP2-MARCKS complex, which is necessary for the open probability conformation of the channel and preserves ENaC-a protein expression, by means of blunting the protein kinase C-a pathway. Triple-mutant TNF knock-in mice are more prone than wild-type mice to develop edema with low-dose intratracheal PLY, correlating with reduced pulmonary ENaC-a subunit expression.Conclusions: These results demonstrate a novel TNF-mediated mechanism of direct ENaC activation and indicate a physiological role for the lectin-like domain of TNF in the resolution of alveolar edema during inflammation.

Physical and chemical DNA-damaging agents are used widely in the treatment of cancer. Double-strand break (DSB) lesions in DNA are the most deleterious form of damage and, if left unrepaired, can effectively kill cancer cells. DNA-dependent protein kinase (DNA-PK) is a critical component of nonhomologous end joining (NHEJ), one of the two major pathways for DSB repair. Although DNA-PK has been considered an attractive target for cancer therapy, the development of pharmacologic DNA-PK inhibitors for clinical use has been lagging. Here, we report the discovery and characterization of a potent, selective, and orally bioavailable DNA-PK inhibitor, M3814 (peposertib), and provide in vivo proof of principle for DNA-PK inhibition as a novel approach to combination radiotherapy. M3814 potently inhibits DNA-PK catalytic activity and sensitizes multiple cancer cell lines to ionizing radiation (IR) and DSB-inducing agents. Inhibition of DNA-PK autophosphorylation in cancer cells or xenograft tumors led to an increased number of persistent DSBs. Oral administration of M3814 to two xenograft models of human cancer, using a clinically established 6-week fractionated radiation schedule, strongly potentiated the antitumor activity of IR and led to complete tumor regression at nontoxic doses. Our results strongly support DNA-PK inhibition as a novel approach for the combination radiotherapy of cancer. M3814 is currently under investigation in combination with radiotherapy in clinical trials.

Summary Exposure to oxygen deprivation in vitro has been reported to cause drug resistance in CHO cells (Rice et al., 1986; PNAS 83, 5978) and enhancement of experimental metastatic (colonisation) ability of murine tumour cells (Young et al., 1988; PNAS 85, 9533

(Gut 1999;45:766-774)