MEK inhibitor resistance mechanisms and recent developments in combination trials.

The mitogen-activated protein kinase (MAPK) pathway plays a vital role in cellular processes such as gene expression, cell proliferation, cell survival, and apoptosis. Also known as the RAS-RAF-MEK-ERK pathway, the MAPK pathway has been implicated in approximately one-third of all cancers. Mutations in RAS or RAF genes such as KRAS and BRAF are common, and these mutations typically promote malignancies by over-activating MEK and ERK downstream, which drives sustained cell proliferation and uninhibited cell growth. Development of drugs targeting this pathway has been a research area of great interest, especially drugs targeting the inhibition of MEK. In vitro and clinical studies have shown promise for certain MEK inhibitors (MEKi) , and MEKi have become the first treatment option for certain cancers. Despite promising results, not all patients have a response to MEKi, and mechanisms of resistance typically arise in patients who do have a positive initial response. This paper summarizes recent developments regarding MEKi, the mechanisms of adaptive resistance to MEKi, and the potential solutions to the issue of adaptive MEKi resistance.

Measurement system for systematic hydrological characterization of unsaturated fractured welded tuff in a mined underground tunnel.

A field investigation of unsaturated flow through a lithophysal unit of fractured welded tuff containing lithophysal cavities has been initiated. To characterize flow in this spatially heterogeneous medium, a systematic approach has been developed to perform tests in boreholes drilled at regular intervals in an underground tunnel (drift). The purpose of the testing is to quantify the amounts of water seeping into the drift versus the amount of water moving around the drift when released into boreholes at many equidistant locations along the drift. In this paper, we describe the test equipment system that has been built for this purpose. Because the field-scale measurements--of liquid flow in the unsaturated, fractured rocks--require continuous testing for periods of days to weeks, the control of test equipment has been fully automated, allowing operation with no human presence at the field site. Preliminary results from the first set of tests indicate that, while the effects of evaporation on characterization of hydrological properties of the rock can be significant, these effects can be controlled and quantified. These tests give insight into the role of the cavities as potential storage during the initial transient flow prior to the breakthrough of water at the drift crown, as well as the role of connected fractures that provide the subsequent quasi-steady flow. In addition to the stated purpose of realizing the flow partitioning, the results yield values for the effective porosity in the pathways for liquid flow in the regions tested thus far.

Expression of vascular cell adhesion molecule-1 by vascular endothelial cells in immune and nonimmune inflammatory reactions in the skin.

Expression of VCAM-1 was compared with that of E-selectin in cytokine-induced lesions and in delayed-type hypersensitivity reactions to tuberculin purified protein derivative (PPD) in pig skin. Lumenally expressed Ags were quantified by measuring localization in skin of i.v. injected (111)In-mAb 10.2C7 (anti-vascular cell adhesion molecule-1 (anti-VCAM-1), (125)I-mAb 1.2B6 (anti-E-selectin), and (99m)Tc-MOPC21 (control IgG1). Anti-VCAM-1 mAb uptake was greater following intradermal (i.d.) injection of TNF-alpha than following injection of IL-1, while the two cytokines induced similar uptake of anti-E-selectin. In immunologically naive pigs there was no detectable increase in anti-VCAM-1 after i.d. injection of PPD, although anti-E-selectin uptake was increased at 3 and 6 h. In contrast, i.d. injection of PPD in sensitized pigs led to increased uptake of both anti-VCAM-1 and anti-E-selectin at 6, 8, 24, and 48 h, each of which was significantly greater than the uptake of control IgG1 into the same lesions (each p < 0.01). Anti-TNF-alpha mAb abolished the increased uptake of anti-VCAM-1 3 and 8 h following i.d. injection of PPD in sensitized pigs and significantly inhibited uptake at 24 h (p = 0.0025), but did not significantly reduce uptake of anti-E-selectin. We conclude that in this delayed-type hypersensitivity model 1) E-selectin expression by endothelial cells follows sequential Ag nonspecific and immune-specific phases, 2) increased VCAM-1 expression by endothelial cells is only seen in sensitized animals, and 3) expression of VCAM-1 appears to be relatively more dependent on TNF-alpha than E-selectin. Differential expression of E-selectin and VCAM-1 may influence the leukocytic infiltrate during the course of nonspecific and immune-specific inflammatory reactions.

Synergy between L-selectin signaling and chemotactic activation during neutrophil adhesion and transmigration.

L-selectin enables capture and rolling of neutrophils on inflamed endothelium. This may facilitate the binding of agonists such as IL-8 and platelet-activating factor (PAF), which signal CD18-mediated firm adhesion and transmigration. Recent studies demonstrate that L-selectin can mediate transmembrane signaling. However, the functional effects of costimulation through agonist and L-selectin require further study. Here, we quantify cell adhesion, motility, and transmigration in response to co-activation through L-selectin and agonist. The surface expression of CD11b/CD18 increased and L-selectin decreased in proportion to the extent of L-selectin cross-linking. A flow cytometric assay was used to measure CD11b/CD18-dependent adhesion to fluorescent beads adsorbed with albumin. Neutrophil adhesion was detected within seconds of adding PAF (20 pM), IL-8 (50 pM), or cross-linking L-selectin. Costimulation through agonist and L-selectin potentiated by up to threefold the rate and extent of bead capture. Stimulation through L-selectin induced membrane ruffling, whereas PAF or IL-8 induced bipolar shape change. L-selectin cross-linking sustained the transient shape change induced by low concentrations (10-50 pM) of agonist. Chemokinesis stimulated by IL-8 was inhibited in the presence of cross-linking L-selectin. This was attributed to enhanced cell spreading following costimulation. Migration across HUVEC monolayers stimulated with IL-1 was also potentiated in the presence of L-selectin cross-linking. We propose that cross-linking of L-selectin and binding of agonist receptors may act synergistically to amplify neutrophil activation and emigration in the inflamed vasculature.

The role of E-selectin in lymphocyte and polymorphonuclear cell recruitment into cutaneous delayed hypersensitivity reactions in sensitized pigs.

We have studied the role of E-selectin in leukocyte accumulation into Ag-specific cutaneous delayed-type hypersensitivity reactions in pigs sensitized to the topical application of 2,4-dinitro-1-fluorobenzene or to the intradermal injection of bacillus Calmette-Guérin. The delayed-type hypersensitivity reactions were shown to be specific for the sensitizing Ag and characterized by the up-regulation of E-selectin, as demonstrated by the uptake of tracer 99mTc-labeled monoclonal anti-E-selectin mAb and entry of 51Cr-labeled PBL and (111)In-labeled polymorphonuclear cells (PMN). Intravenous injection of 5 mg/kg of a F(ab')2 preparation of a monoclonal anti-E-selectin Ab at peak times of leukocyte entry resulted in a significant inhibition of entry of both PMN and lymphocytes. The anti-E-selectin Ab inhibited PMN recruitment by 70 to 90% and lymphocyte recruitment by 50 to 60%. In comparison, an anti-CD18 treatment reduced PMN recruitment by 70 to 90% and lymphocyte recruitment by 60 to 70% in this model. These data confirm an important role for E-selectin in the recruitment of both PMN and lymphocytes to sites of immune-based dermal inflammation.

Porcine E-selectin: cloning and functional characterization.

E-selectin, a member of the selectin family, is believed to play an important role in mediating the initial adhesive events between leucocytes and the endothelium during inflammation. A monoclonal antibody against human E-selectin was found to cross-react with the porcine equivalent, a glycoprotein of 92,000 MW. Isolation of the cDNA for porcine E-selectin showed that it was 71% homologous with human E-selectin but had two less short consensus repeats. The porcine adhesion molecule could also support the adhesion of both porcine and human neutrophils. Expression of E-selectin on interleukin-1 alpha (IL-1 alpha)- or tumour necrosis factor-alpha (TNF-alpha)-activated porcine aortic endothelial cells in culture was prolonged, persisting for up to 48 hr. Binding studies using a chimeric molecule consisting of the lectin domain of porcine E-selectin and the epidermal growth factor (EGF) domain of human E-selectin fused to the human IgG constant region, further characterized porcine E-selectin as recognizing mainly granulocytic leucocytes and a subpopulation of lymphocytes.

Cloning and expression kinetics of porcine vascular cell adhesion molecule.

Human vascular cell adhesion molecule is believed to play a key role in recruiting leukocytes to sites of injury. Here we report the identification of a homologous molecule in the pig which has five Ig domains and an overall 77% homology with the human protein. The expression of this protein was also characterised on porcine endothelial cells. Like the human adhesion molecule, the porcine protein could be induced by LPS, TNF and IL-1 alpha, although differences were noted in the kinetics of expression.