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Tracheal intubation is common in the care of critically ill adults and is frequently complicated by hypotension, cardiac arrest, or death. We aimed to evaluate administration of an intravenous fluid bolus to prevent cardiovascular collapse during intubation of critically ill adults.We did a pragmatic, multicentre, unblinded, randomised trial in nine sites (eight ICUs and one emergency department) around the USA. Critically ill adults (≥18 years) undergoing tracheal intubation were randomly assigned (1:1, block sizes of 2, 4, and 6, stratified by study site) to either an intravenous infusion of 500 mL of crystalloid solution or no fluid bolus. The primary outcome, assessed in the intention-to-treat population, was cardiovascular collapse, defined as a new systolic blood pressure65 mm Hg; new or increased vasopressor receipt between induction and 2 min after tracheal intubation; or cardiac arrest or death within 1 h of tracheal intubation. Adverse events were assessed in the as-treated population. This trial, which is now complete, is registered with ClinicalTrials.gov, number NCT03026777.Patients were enrolled from Feb 6, 2017, to Jan 9, 2018, when the data and safety monitoring board stopped the trial on the basis of futility. By trial termination, 337 (63%) of 537 screened adults had been randomly assigned. Cardiovascular collapse occurred in 33 (20%) of 168 patients in the fluid bolus group compared with 31 (18%) of 169 patients in the no fluid bolus group (absolute difference 1·3% [95% CI -7·1% to 9·7%]; p=0·76). The individual components of the cardiovascular collapse composite outcome did not differ between groups (new systolic blood pressure65 mm Hg 11 [7%] in the bolus group vs ten [6%] in the no-bolus group, new or increased vasopressor 32 [19%] vs 31 [18%], cardiac arrest within 1 h seven [4%] vs two [1%], death within 1 h of intubation two [1%] vs one [1%]). In-hospital mortality was not significantly different in the fluid bolus group (48 [29%]) compared with no fluid bolus (59 [35%]).Administration of an intravenous fluid bolus did not decrease the overall incidence of cardiovascular collapse during tracheal intubation of critically ill adults compared with no fluid bolus in this trial.US National Institutes of Health.

Cellular resistance to platinum anticancer compounds is governed by no less than two molecular processes; DNA repair and cellular accumulation of drug. Gli1 is an upstream regulator of nucleotide excision repair, effecting this process through c-jun. We, therefore, investigated whether Gli1 plays a role in cellular accumulation of cisplatin. Using a Gli1-specific shRNA, we explored the role of Gli1 in the cellular accumulation and efflux of cisplatin, in cisplatin-resistant A2780-CP70 human ovarian cancer cells. When Gli1 is inhibited, cellular uptake of cisplatin was approximately 33% of the level of uptake under control conditions. When Gli1 is inhibited, cellular efflux of cisplatin was completely abrogated, over a 12-h period of observation. We assayed nuclear lysates from these cells, for the ability to bind the DNA sequence that is the Gli-binding site (GBS) in the 5'UTR for each of five known cisplatin transmembrane transporters. Four of these transporters are active in cisplatin uptake; and, one is active in cisplatin efflux. In each case, nuclear lysate from A2780-CP70 cells binds the GBS of the respective cisplatin transport gene. We conclude that Gli1 plays a strong role in total cellular accumulation of cisplatin in these cells; and, that the combined effects on cellular accumulation of drug and on DNA repair may indicate a role for Gli1 in protecting cellular DNA from lethal types of DNA damage.

The transcription factor Activator Protein 1 (AP1), is an important regulator in cisplatin resistance. AP1 is comprised of the two proteins, c-jun and c-fos. The transcription factor Gli1, a member of the hedgehog signaling pathway, is also an important factor in cisplatin resistance and transcriptionally regulated c-jun. The c-jun interface with Gli1 is mediated by a 130 kDa isoform of Gli1. We have investigated whether Gli1 may regulate c-fos by selectively knocking down Gli1 expression using anti-Gli1 shRNA or by treating cells with a Hedgehog pathway inhibitor, cyclopamine. A2780-CP70 cells were treated with an IC50 dose of anti-Gli1 shRNA and the changes in RNA and protein levels were analyzed over a 72 hour time course. No changes in the expression of c-jun or c-fos RNA transcripts were observed versus control. Western blot analyses showed Gli1 protein expression was reduced at 24 hours and undetectable at 48 and 72 hours. Sonic hedgehog (Shh) protein levels were dramatically reduced by 24 hours, low but measureable at 48 hours, and undetectable at 72 hours. Indian hedgehog (Ihh) expression increased at 24 hours and remained stable for the remainder of the 72 hour time course. Treatment of A2780-CP70 cells with an IC50 dose of cyclopamine resulted in a different intracellular response than treatment with Gli1 shRNA. Cyclopamine specifically targets the cell membrane receptor Smoothened. Experiments were performed analyzing RNA and protein expression over a 72 hour time period after treatment with cyclopamine. C-jun and c-fos RNA transcript levels were higher than control 6 hours after treatment and remained high for the duration of the 72 hour experiment. Western blot analyses after cyclopamine treatment showed Gli1 protein expression gradually decreasing during the 72 hours, but were never fully depleted. Expression of Shh increased over 72 hours, while Ihh levels remained stable. The identical Gli-Binding-Site (GBS) in the c-jun promoter was found in the promoter of c-fos. We investigated whether the Gli1 isoform that binds c-jun, also binds c-fos. Simultaneous Western and Southwestern blots show only one of the known Gli1 isoform binds the promoter of c-fos. Further studies with Chromatin Immunoprecipitation (ChIP) assays confirmed the 130 kDa isoform of Gli1 binds the GBS of the c-fos promoter. We conclude that the 130 kDa Gli1 isoform binds both components of AP1, c-jun and c-fos, and modulates the cellular resistance to cisplatin. Citation Format: Lauren Amable, Kenji Kudo, Elaine Gavin, Jason Fain, Eddie Reed. A specific Isoform of Gli1 binds the promoter region of c-fos in A2780-CP70 human ovarian cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-223. doi:10.1158/1538-7445.AM2013-LB-223

We have demonstrated that a specific anti-Gli1 shRNA can ablate Gli1 in A2780-CP70 cells, with no effect on Gli2. Upon ablation of Gli1, platinum-DNA adduct repair is inhibited, along with inhibition of ERCC1, XPD, XRCC1, and c-jun. We therefore asked the question of whether there were other subcellular pharmacologic effects, with respect to cisplatin, in these cells. We assessed total cellular accumulation of cisplatin under three conditions: pretreatment with the anti-Gli1 shRNA; pretreatment with a scrambled shRNA control; and pretreatment with Fugene which was used as the transfection agent. We also assessed platinum-DNA adduct levels, at the same time points that total cellular accumulation was measured. All platinum measurements were performed by AAS with Zeeman background correction. In each independent experiment (n=4), we plated cells on day 1; transfected with shRNA 16-18 hours later; treated with cisplatin for 1 hour at 30 μM (the IC50 under these conditions); and, harvested cells at different time points. Those time points were: zero hours (immediately after the 1 hour cisplatin treatment); 6 hours later; or 12 hours later. At zero hours, measured levels of pt-DNA damage were equivalent between the three conditions. At zero hours, measured levels of total cellular platinum were reduced by 40-45% in anti-Gli1 treated cells; suggesting inhibition of cellular accumulation. At 6 hours, scrambled controls showed reductions in total cellular platinum by ∼40%, demonstrating excretion of cisplatin. This is consistent with previously published work (JCI 1991). Cells treated with anti-Gli1 shRNA showed no reductions in total cellular platinum at 6 hrs, nor at 12 hrs; suggesting inhibition of the processes by which cells may eliminate this drug. Cells treated with anti-Gli1 shRNA showed statistically significant inhibition in platinum-DNA adduct repair, as compared to cells treated with scrambled shRNA. Control cells repaired 67.5% of pt-DNA adducts over 12 hours; as compared to 23.7% repair in cells treated with anti-Gli1 shRNA; p2=0.015. We conclude that ablation of Gli1 in cisplatin-resistant A2780-CP70 human ovarian cancer cells, has at least three effects on the subcellular pharmacology of cisplatin. There is inhibition of processes that determine: cellular uptake of drug; cellular elimination of drug; and platinum-DNA adduct repair. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 771. doi:1538-7445.AM2012-771