Intrinsic resistance to selumetinib, a selective inhibitor of MEK1/2, by cAMP-dependent protein kinase A activation in human lung and colorectal cancer cells.

BACKGROUND: MEK is activated in ∼40% colorectal cancer (CRC) and 20-30% non-small cell lung cancer (NSCLC). Selumetinib is a selective inhibitor of MEK1/2, which is currently in clinical development. METHODS: We evaluated the effects of selumetinib in vitro and in vivo in CRC and NSCLC cell lines to identify cancer cell characteristics correlating with sensitivity to MEK inhibition. RESULTS: Five NSCLC and six CRC cell lines were treated with selumetinib and classified according to the median inhibitory concentration (IC(50)) values as sensitive (≤1 µM) or resistant (>1 µM). In selumetinib-sensitive cancer cell lines, selumetinib treatment induced G1 cell-cycle arrest and apoptosis and suppression of tumour growth as xenografts in immunodeficient mice. Evaluation of intracellular effector proteins and analysis of gene mutations showed no correlation with selumetinib sensitivity. Microarray gene expression profiles revealed that the activation of cAMP-dependent protein kinase A (PKA) was associated with MEK inhibitor resistance. Combined targeting of both MEK and PKA resulted in cancer cell growth inhibition of MEK inhibitor-resistant cancer cell lines in vitro and in vivo. CONCLUSION: This study provides molecular insights to explain resistance to an MEK inhibitor in human cancer cell lines.

Comparison of conservative management and juvenile pubic symphysiodesis in the early treatment of canine hip dysplasia.

OBJECTIVES: To evaluate the efficacy of juvenile pubic symphysiodesis (JPS) in a clinical setting for the early treatment of canine hip dysplasia (CHD), and to identify its indications and contraindications. METHODS: The final degree of CHD using the FCI (Fédération Cynologique Internationale) CHD classification in 5 Grades (A, B, C, D, E) was assessed at skeletal maturity in two homogeneous groups of dogs assessed at the age of 14 to 22 weeks and selected according to their susceptibility to CHD; one group was treated with JPS and one group was conservatively managed. Two hundred seventeen puppies completed the study; 81 were treated with JPS (group 1) and 76 were conservatively managed (group 2). A third group of 60 puppies with normal hips was followed as a negative control group. RESULTS: In group 1, 43.2% of the puppies had regression or a lack of progression of the disease in the final evaluation (Grade A & B), 25.9% had mild CHD (Grade C) and 30.9% had moderate and severe CHD (Grade D & E). In group 2, 23.6% of the puppies did not show any development of the disease (Grade A & B), 21.1% had mild CHD (Grade C) and 55.3% developed moderate to severe CHD (Grade D & E). Further investigation was done by comparing the severity of early signs of susceptibility to CHD with the final FCI Grades at adulthood in both groups. CLINICAL SIGNIFICANCE: The JPS procedure increased the odds of arresting or limiting the progression of CHD in mild to moderate grades of CHD, while it was less effective or ineffective in more severe forms.

Outcome of reconstruction of cutaneous limb defects in dogs using hygroscopic "self-inflating" tissue expanders.

OBJECTIVES: To describe the placement of self-inflating tissue expanders and clinical outcomes in 12 consecutive cases of reconstruction of distal cutaneous limb defects in dogs. MATERIALS AND METHODS: Cases of distal cutaneous limb defect were divided into three groups based on the location of the placement of the self-inflating tissue expanders: Group A (n=4): on, or proximal to, the elbow and stifle; Group B (n=4): distal to the elbow or stifle and proximal to the carpus or tarsus; and Group C (n=4): distal to the carpus or tarsus. Owner satisfaction and clinical outcome were documented. RESULTS: Thirteen cases were originally included, but one was excluded because of incomplete follow-up. In one case, the self-inflating tissue expanders were removed before expansion started. A mean of five expanders were implanted per dog (range 2 to 9). Devices were removed after a mean of 24 days (range 13 to 42 days). Primary closure was achieved in eight of 11 cases, including all cases from Group A and 75% and 33% of cases from Groups B and C, respectively. All incompletely reconstructed defects or areas of wound dehiscence healed by second intention. Eight of 12 owners were satisfied. CLINICAL SIGNIFICANCE: Self-inflating tissue expanders can be used as an alternative for the reconstruction of limb defects in dogs in which direct primary closure would otherwise not be achievable. Defects below the carpus and tarsus are more challenging to treat with this method.

[Alternating chemotherapy and daily multiple-fraction radiotherapy in the treatment of advanced spinocellular carcinoma of the head and neck]. / Alternanza di chemioterapia e radioterapia a frazioni multiple giornaliere nella cura di carcinomi spinocellulari avanzati del distretto cervico-cefalico.

From January 1987 to May 1988 sixteen pts with advanced squamous cell carcinoma of the head and neck received a combined treatment based on an alternation of chemotherapy and MFD--radiotherapy. The chemotherapy regimen consisted of CDDP 20 mg/m2 and 5-FU 200 mg/m2 i.v. push, from day 1 to day 5 during weeks 1, 5 and 9. Radiotherapy was administered in two courses of 32 Gy each (total dose 64 Gy), during weeks 2-3 and 6-7. Each course was given in 2 fractions per day, five days per week. All sixteen patients could be evaluated for toxicity and fifteen for response. The following were observed: 7 CR, 6 PR, 1 SD and 1 PD. The overall response rate was 86.6%. Toxicity was high; 43.7% of the patients developed grade III-IV mucositis. These results suggest that CDDP and 5-FU alternating with MFD-radiotherapy is feasible, although new, less toxic scheduling must be determined.

Cholesterol depletion perturbs calcium handling by rat submandibular glands.

The sensitivity to cholesterol depletion of calcium handling by rat submandibular glands was investigated. The glands were digested with collagenase. After homogenization, the lysate was extracted at 4 degrees C with 0.5% Triton X-100 and the extract was submitted to an ultracentrifugation in a sucrose discontinuous gradient. A population of detergent-resistant membranes (DRM) was collected at the 5%-35% interface. The DRM had a higher content of cholesterol, saturated and long-chain fatty acids. Caveolin-1 and alpha(q/11) were located in these membranes. They were more ordered than vesicles from total cellular lysate as determined by anisotropy measurement. They disappeared after cholesterol extraction with methyl-beta-cyclodextrin (MCD). Exposure of the cellular suspension with MCD nearly abolished the response to carbachol, epinephrine, and substance P and inhibited the activation of phospholipase C (PLC) by these agonists and by sodium fluoride. MCD did not affect the mobilization of intracellular pools of calcium by thapsigargin. It increased the uptake of extracellular calcium or barium and did not inhibit the uptake of calcium after depletion of the intracellular stores of this ion. From these results, it is concluded that Triton X-100 can extract a fraction of membrane resistant to detergents. Treatment of the cells with MCD disrupts these membranes. The coupling between the heterotrimeric GTP-binding protein G(q/11) and poly-phosphoinositide-specific PLC is affected by disruption of these membrane fractions. At the opposite, the store-operated calcium channel (SOCC) is not affected by DRM-disruption.

Analysis of a sea urchin gene cluster coding for the small nuclear U7 RNA, a rare RNA species implicated in the 3' editing of histone precursor mRNAs.

A genomic 9.3-kilobase DNA fragment of the sea urchin Psammechinus miliaris, containing a cluster of five U7-RNA genes (or pseudogenes), has been isolated and analyzed by partial DNA sequencing. The U7-RNA coding sequences differ from one another by one or two nucleotides, one of the five gene sequences being identical to those of the cDNA U73 clone prepared earlier [Strub, K., Galli, G., Busslinger, M. & Birnstiel, M. L. (1984) EMBO J. 3, 2801-2807]. The spacer sequences separating the genes have, on the whole, a low degree of homology; hence, the five genes must have arisen by an ancient duplication event. The sequences preceding the coding portion contain three highly conserved sequence motifs but no "TATA box." The 3' flanking sequences include a highly conserved AAAGNNAGA sequence that is held in common with other U-RNA genes from both sea urchins and vertebrates. Our findings confirm our classification of the U7 RNA as a genuine, if sparsely represented, member of the U-RNA family.