The frequency and impact of ROS1 rearrangement on clinical outcomes in never smokers with lung adenocarcinoma.

BACKGROUND: To determine the frequency and predictive impact of ROS1 rearrangements on treatment outcomes in never-smoking patients with lung adenocarcinoma. PATIENTS AND METHODS: We concurrently analyzed ROS1 and ALK rearrangements and mutations in the epidermal growth factor receptor (EGFR), and KRAS in 208 never smokers with lung adenocarcinoma. ROS1 and ALK rearrangements were identified by fluorescent in situ hybridization. RESULTS: Of 208 tumors screened, 7 (3.4%) were ROS1 rearranged, and 15 (7.2%) were ALK-rearranged. CD74-ROS1 fusions were identified in two patients using reverse transcriptase-polymerase chain reaction. The frequency of ROS1 rearrangement was 5.7% (6 of 105) among EGFR/KRAS/ALK-negative patients. Patients with ROS1 rearrangement had a higher objective response rate (ORR; 60.0% versus 8.5%; P = 0.01) and a longer median progression-free survival (PFS; not reached versus 3.3 months; P = 0.008) to pemetrexed than those without ROS1/ALK rearrangement. The PFS to EGFR-tyrosine kinase inhibitors in patients harboring ROS1 rearrangement was shorter than those without ROS1/ALK rearrangement (2.5 versus 7.8 months; P = 0.01). CONCLUSIONS: The frequency of ROS1 rearrangements in clinically selected patients is higher than that reported for unselected patients, suggesting that ROS1 rearrangement is a druggable target in East-Asian never smokers with lung adenocarcinoma. Given the different treatment outcomes to conventional therapies and availability of ROS1 inhibitors, identification of ROS1 rearrangement can lead to successful treatment in ROS1-rearranged lung adenocarcinomas.

The effect of bupivacaine.HCl on the physical properties of neuronal membranes.

Fluorescent probe techniques were used to evaluate the effect of bupivacaine.HCl on the physical properties (transbilayer asymmetric lateral and rotational mobilities, annular lipid fluidity and protein distribution) of synaptosomal plasma membrane vesicles (SPMVs) isolated from bovine cerebral cortex. An experimental procedure was used based on selective quenching of both 1,3-di(1-pyrenyl)propane (Py-3-Py) and 1,6-diphenyl-1,3,5-hexatriene (DPH) by trinitrophenyl groups, and radiationless energy transfer (RET) from the tryptophans of membrane proteins to Py-3-Py. Bupivacaine.HCl increased the bulk lateral and rotational mobilities, and annular lipid fluidity in SPMVs lipid bilayers, and had a greater fluidizing effect on the inner monolayer than that of the outer monolayer. The magnitude of increasing effect on annular lipid fluidity in SPMVs lipid bilayer induced by bupivacaine.HCl was significantly far greater than magnitude of increasing effect of the drug on the lateral and rotational mobilities of bulk SPMVs lipid bilayer. It also caused membrane proteins to cluster. These effects of bupivacaine.HCl on neuronal membranes may be responsible for some, though not all, of the local anesthetic actions of bupivacaine.HCl.

Cloning and characterization of tissue inhibitor of metalloproteinase-3 (TIMP-3) from shark, Scyliorhinus torazame.

We cloned the full-length cDNA encoding TIMP-3 from the cartilage of cloudy dogfish, Scyliorhinus torazame. The entire open reading frame was composed of 645 nucleotides and 214 residues including 12 conserved cysteines and asparagine-184, a putative site for N-linked sugars. It showed about 72% identity to those of other species based on the deduced amino acid sequence. The mRNA of shark TIMP-3 were expressed abundantly in brain and cartilage tissues. To investigate the roles of shark TIMP-3, an expression vector was constructed and transfected into HT1080 human fibrosarcoma cells. Overexpression of shark TIMP-3 reduced the activity of MMP-2 in gelatin zymography. Through human Alu PCR based CAM assay, we also confirmed that shark TIMP-3 transfected HT1080 cells had less intravasation effects.

Human rad21 gene, hHR21(SP), is downregulated by hypoxia in human tumor cells.

To identify genes differentially expressed under normoxic (21% O(2)) or hypoxic (1% O(2)) conditions, we used the technique of mRNA differential display using total RNA extracted from Chang human liver cells. Among downregulated genes by hypoxia, we focused on hHR21(SP) (human homologue of rad21 S. pombe) that is involved in DNA double-strand break repair. Northern blot analysis revealed that mRNA expression of hHR21(SP) was inhibited by hypoxia in various tumor cell lines, such as HepG2, SKHep1, MCF7, and HT1080 cells. We also found that hypoglycemia and heat shock significantly decreased the hHR21(SP) level, indicating that a DNA double-strand break repair gene, hHR21(SP) might be regulated by environmental stresses. In addition, wortmannin, a DNA-dependent protein kinase (DNA-PK) inhibitor, decreased the level of hHR21(SP) mRNA, indicating that DNA-PK might be involved in the regulation of hHR21(SP). These results propose a new understanding of hHR21(SP) regulations in human tumor cells.

Pharmacological effects of novel quinone compounds, 6-(fluorinated-phenyl)amino-5,8-quinolinediones, on inhibition of drug-induced relaxation of rat aorta and their putative action mechanism.

Two 6-(fluorinated-phenyl)amino-5,8-quinolinedione derivatives, OQ21 and OQ1, were newly synthesized as potent inhibitors of endothelial-dependent vasorelaxation. The purpose of the present study was to investigate the effects of OQ21 and OQ1 on different types of vasorelaxation and to pursue their action mechanisms. For acetylcholine both compounds, at a low concentration (0.1 microM), reduced the maximal response with increase of EC(50) values. OQ21 is a novel quinone compound and showed a more potent and efficacious inhibitory effect on acetylcholine-induced relaxation of rat aorta than that of LY83583 (6-anilino-5,8-quinolinedione). Relatively high concentrations (1 microM) of OQ21 and OQ1 inhibited the sodium nitroprusside-induced relaxation of endothelium-denuded ring, producing rightward shifts of the curve for sodium nitroprusside without altering the maximal response. They also prevented acetylcholine and sodium nitroprusside-induced elevations of cyclic GMP. In addition, OQ21 and OQ1 (1 microM) significantly decreased (52-72%) the sensitivity of L-arginine-induced relaxation of precontracted endothelium-denuded aortic rings from lipopolysaccaride-treated (20 mg/kg, i.p.) rats. The inhibitory effect of OQ21 on endothelium-dependent vasodilation was enhanced by N(G)-nitro-L-arginine, which inhibits nitric oxide synthase (NOS) by binding the oxygenase domain of the enzyme, but not by diphenylendiodonium, which inhibits NOS by binding to the reductase domain of the enzyme. Treatment of blood vessels with OQ21 or OQ1 showed a significant increase in chemiluminescence output, which was prevented by adding superoxide dismutase, suggesting that superoxide generation is involved in the action mechanism for OQ21. Present results indicate that a novel naphthoquinone compound, OQ21, potently inhibits endothelial NOS, possibly by interacting with the reductase domain of the enzyme, which leads to induce superoxide formation. The new benzoquinone compounds, OQ21 and OQ1, inhibit not only endothelium-dependent vasorelaxation but also endothelium-independent relaxation induced by exogenous NO generated from a nitrovasodilator via the reduction of cyclic GMP. They also reduced L-arginine-induced vasorelaxation in endotoxin-treated rats, indicating their possession of inhibitory effect on inducible NOS.

Egr-1 mediates transcriptional activation of IGF-II gene in response to hypoxia.

We have previously reported that the exposure of human HepG2 cells to hypoxic conditions results in the overexpression of human insulin-like growth factor II (IGF-II) mRNA whose size is 6.0 kb. This particular size of IGF-II mRNA is transcribed under the control of the IGF-II P3 promoter. In the present study, to delineate the molecular mechanism for the activation of the IGF-II gene, we examined the induction of P3 promoter activity in HepG2 cells by hypoxia in the transient expression system. In this system, hypoxia induced a linear increase within 24 h in the expression of luciferase that was driven by the IGF-II P3 promoter. To further delineate which factors mediate this response, the expression pattern of regulators of the P3 promoter, Egr-1, Sp1, and WT1, were analyzed by reverse transcription-PCR and Northern blot analysis. We found that hypoxia increased the expression of Egr-1 but not of Sp1. In contrast, the level of WT1, a repressor of IGF-II expression, was markedly decreased during hypoxia. The mRNA stability assay revealed that the induction of transcription is the mechanism of underlying Egr-1 mRNA elevation. We then investigated the effects of hypoxia on the DNA binding activity of Egr-1. Both electrophoretic mobility shift assay and supershift assay demonstrated that the DNA binding activity of the Egr-1 protein was increased by hypoxia. In addition, the level of Egr-1 protein was also increased under the hypoxia as determined by Western blot analysis. Cotransfection of HepG2 cells with an Egr-1 expression vector and an IGF-II P3 promoter-luciferase reporter plasmid showed that the transcription of IGF-II was activated by Egr-1 in a dose-dependent manner. Moreover, the elevation of IGF-II P3 promoter activity was induced synergistically by the cotreatment of hypoxia with Egr-1 overexpression. Deletion of sequences in the IGF-II P3 promoter containing Egr-1 binding sites did not respond to hypoxic stress. Taken together, these data strongly indicate that hypoxia-induced IGF-II expression in HepG2 cells is due to the enhanced activity of Egr-1 on the IGF-II P3 promoter and that the Egr-1 binding site in the IGF-II P3 promoter is essential for the transcriptional regulation of IGF-II under hypoxic conditions.

Identification of genes differentially expressed by hypoxia in hepatocellular carcinoma cells.

In order to identify genes differentially expressed under hypoxia (1% O2, 5% CO2, balance N2), we performed mRNA differential display analysis using total RNA extracted from hypoxic and normoxic HepG2, human hepatocellular carcinoma (HCC) cells. Of the differentially expressed genes by hypoxia, some of cDNA fragments were cloned and sequenced. The expression patterns of these clones by hypoxia were confirmed by Northern blot analysis and the quantitative RT-PCR. Down-regulated genes by hypoxia have homology to cDNA sequences encoding cytochrome oxidase subunit II and ADP/ATP translocase, respectively. Up-regulated gene by hypoxia was identified as Homo sapiens oscillin. Moreover, novel genes induced by hypoxia represent partial sequences of cDNAs that have not been reported or functionally identified. Up- or down-regulated expression of these genes in response to hypoxia may contribute to human hepatocarcinogenesis.