Genome-wide functional screening identifies CDC37 as a crucial HSP90-cofactor for KIT oncogenic expression in gastrointestinal stromal tumors.

Most gastrointestinal stromal tumors (GISTs) contain KIT or PDGFRA kinase gain-of-function mutations, and therefore respond clinically to imatinib and other tyrosine kinase inhibitor (TKI) therapies. However, clinical progression subsequently results from selection of TKI-resistant clones, typically containing secondary mutations in the KIT kinase domain, which can be heterogeneous between and within GIST metastases in a given patient. TKI-resistant KIT oncoproteins require HSP90 chaperoning and are potently inactivated by HSP90 inhibitors, but clinical applications in GIST patients are constrained by the toxicity resulting from concomitant inactivation of various other HSP90 client proteins, beyond KIT and PDGFRA. To identify novel targets responsible for KIT oncoprotein function, we performed parallel genome-scale short hairpin RNA (shRNA)-mediated gene knockdowns in KIT-mutant GIST-T1 and GIST882. GIST cells were infected with a lentiviral shRNA pooled library targeting 11 194 human genes, and allowed to proliferate for 5-7 weeks, at which point assessment of relative hairpin abundance identified the HSP90 cofactor, CDC37, as one of the top six GIST-specific essential genes. Validations in treatment-naive (GIST-T1, GIST882) vs imatinib-resistant GISTs (GIST48, GIST430) demonstrated that: (1) CDC37 interacts with oncogenic KIT; (2) CDC37 regulates expression and activation of KIT and downstream signaling intermediates in GIST; and (3) unlike direct HSP90 inhibition, CDC37 knockdown accomplishes prolonged KIT inhibition (>20 days) in GIST. These studies highlight CDC37 as a key biologic vulnerability in both imatinib-sensitive and imatinib-resistant GIST. CDC37 targeting is expected to be selective for KIT/PDGFRA and a subset of other HSP90 clients, and thereby represents a promising strategy for inactivating the myriad KIT/PDGFRA oncoproteins in TKI-resistant GIST patients.

Deletion of the p27Kip1 gene restores normal development in cyclin D1-deficient mice.

D-type cyclins (cyclins D1, D2, and D3) are key components of cell cycle machinery in mammalian cells. These proteins are believed to drive cell cycle progression by associating with their kinase partners, cyclin-dependent kinases, and by directing phosphorylation of critical cellular substrates. In addition, D-cyclins play a kinase-independent role by sequestering cell cycle inhibitors p27(Kip1) and p21(Cip1). In the past, we and others generated cyclin D1-deficient mice and have shown that these mice display developmental abnormalities, hypoplastic retinas, and pregnancy-insensitive mammary glands. To test the significance of cyclin D1-p27(Kip1) interaction within a living mouse, we crossed cyclin D1-deficient mice with mice lacking p27(Kip1), and we generated double-mutant cyclin D1(-/-)p27(-/-) animals. Here we report that ablation of p27(Kip1) restores essentially normal development in cyclin D1-deficient mice. Our results provide genetic evidence that p27(Kip1) functions downstream of cyclin D1.

Preanalytical variables affecting the quantification of fatty acid ethyl esters in plasma and serum samples.

BACKGROUND: Fatty acid ethyl esters (FAEEs) are cytotoxic nonoxidative ethanol metabolites produced by esterification of fatty acids and ethanol. FAEEs are detectable in blood up to 24 h after ethanol consumption. The objective of this study was to assess the impact of gender, serum or plasma triglyceride concentration, time and temperature of specimen storage, type of alcoholic beverage ingested, and the rate of ethanol consumption on FAEE concentrations in plasma or serum. METHODS: For some studies, subject were recruited volunteers; in others, residual blood samples after ethanol quantification were used. FAEEs were isolated by solid-phase extraction and quantified by gas chromatography-mass spectrometry. RESULTS: For weight-adjusted amounts of ethanol intake, FAEE concentrations were twofold greater for men than women (P /=24 h. The type of alcoholic beverage and rate of consumption did not affect FAEE concentrations. CONCLUSION: These studies advance plasma and serum FAEE measurements closer to implementation as a clinical test for ethanol intake.

[Mannitol for the prevention of decompensation symptoms in hydrated patients undergoing long-term hemodialysis]. / Mannitol w zapobieganiu objawom zespolu niewyrównania u chorych przewodnionych, dlugotrwale dializowanych.

Symptoms of decompensation syndrome were frequently noted during sequential ultrafiltration/hemodialysis. In 9 adult patients chronically dialysed 101 cases of overhydration were seen. Mannitol was administered in a 20% solution (250 ml) in a continuous intravenous infusion during 49 dialyses. Changes in body weight were measured, arterial blood pressure, pulse rate, hematocrit, total plasma protein levels, urea, creatinine, sodium and potassium were determined as well as plasma osmolality. Mannitol significantly decreased muscular contractions during dialysis, weakness after dialysis, and incidence of various symptoms of decompensation. The values of analysed clinical parameters and laboratory tests did not differ from those determined without mannitol. Plasma creatinine, total plasma protein levels and hematocrit were significantly lower after several hours after the end of ultrafiltration/hemodialysis. We suggest that mannitol decreases the incidence of the symptoms of decompensation syndrome and is safe. Beneficial effect of mannitol is most probably produced by the changes in body fluids distribution.