Characterization of the Menin-MLL Interaction as Therapeutic Cancer Target.

Inhibiting the interaction of menin with the histone methyltransferase MLL1 (KMT2A) has recently emerged as a novel therapeutic strategy. Beneficial therapeutic effects have been postulated in leukemia, prostate, breast, liver and in synovial sarcoma models. In those indications, MLL1 recruitment by menin was described to critically regulate the expression of disease associated genes. However, most findings so far rely on single study reports. Here we independently evaluated the pathogenic functions of the menin-MLL interaction in a large set of different cancer models with a potent and selective probe inhibitor BAY-155. We characterized the inhibition of the menin-MLL interaction for anti-proliferation, gene transcription effects, and for efficacy in several in vivo xenografted tumor models. We found a specific therapeutic activity of BAY-155 primarily in AML/ALL models. In solid tumors, we observed anti-proliferative effects of BAY-155 in a surprisingly limited fraction of cell line models. These findings were further validated in vivo. Overall, our study using a novel, highly selective and potent inhibitor, shows that the menin-MLL interaction is not essential for the survival of most solid cancer models. We can confirm that disrupting the menin-MLL complex has a selective therapeutic benefit in MLL-fused leukemia. In solid cancers, effects are restricted to single models and more limited than previously claimed.

N-glycosylation of ABC transporters is associated with functional activity in sandwich-cultured rat hepatocytes.

Hepatobiliary elimination via canalicular efflux transport proteins plays a key role in the clearance of endo- and xenobiotics. Correct membrane localization and coordinated action of the transport systems are essential for vectorial transport of drugs from blood into the bile. While basolaterally localized uptake transporters are responsible for the inward transport of substances from the blood into the hepatocyte, apically expressed ATP-dependent transport proteins such as P-glycoprotein (P-gp), multidrug resistance-associated protein (Mrp2) and breast cancer resistance protein (Bcrp) mediate the outward efflux into the bile canaliculus. Using sandwich-cultured rat hepatocytes we have characterized the expression and maturation of P-gp, Mrp2 and Bcrp transport proteins as well as their transport function over several days. The re-differentiation of the hepatocytes, which only occurs in sandwich configuration involves de novo synthesis and subsequent posttranslational N-glycosylation of all three transport proteins. Only fully N-glycosylated isoforms of the transporters were associated with functional activity as visualized by excretion of specific fluorescent substrates into the canalicular network. However, in what way N-glycosylation affects the functional activity of the ABC transporters investigated remains to be determined.

Development of a fluorescence-based assay for drug interactions with human Multidrug Resistance Related Protein (MRP2; ABCC2) in MDCKII-MRP2 membrane vesicles.

PURPOSE: To establish a fluorescence-based assay for drug interactions with the ABC-export-protein MRP2 (ABCC2). METHODS: Apical membrane vesicles were isolated by differential centrifugation from polarized MDCKII cells and MDCKII cells transfected with human MRP2. Vesicle fractions were characterized by electron microscopy, determination of the marker enzyme alkaline phosphatase and Western blot analysis of MRP2. Vesicle orientation was determined by measurement of 5'-nucleotidase activity in the absence and in the presence of detergents. To assess MRP2 activity, the uptake of the fluorescent MRP2-substrate 5-(6)-carboxy-2',7'-dichlorofluorescein (CDF) was determined in the absence and in the presence of other compounds potentially interacting with MRP2. RESULTS: Apical membrane vesicles could be isolated from cells in considerable purity as indicated by electron microscopy, enrichment of alkaline phosphatase and high enrichment of MRP2 in vesicles of MDCKII-MRP2 cells. About half of the vesicles showed "inside-out" orientation. CDF was taken up into the membrane vesicles in a time- and concentration-dependent manner following a Michaelis-Menten type of kinetics with a K(M) of 39 microM and a V(max) of 465.3 fmol/(mgprotein x min). Thereby, uptake into vesicles from transfected cells was significantly higher than uptake into vesicles from control cells. Presence of known MRP2-substrates/inhibitors in the incubation medium decreased CDF uptake into the vesicles in a concentration-dependent manner, whereas nonsubstrates/inhibitors had no effect. CONCLUSIONS: This CDF-based uptake assay can be used as a rapid and sensitive screening system to assess drug interactions with human MRP2 and therefore represents a useful tool in compound profiling.

Characterization of cytochrome P450 protein expression along the entire length of the intestine of male and female rats.

Intestinal cytochrome P450 (P450) proteins play an important role in the biotransformation of drugs and may significantly limit their oral absorption and bioavailability. Therefore, we have investigated the amount of P450 proteins via Western blot analysis along the entire intestine of male and female rats. Despite of the use of an inbred rat strain, controlled housing conditions for the animals, and a timed sample preparation, high interindividual differences in the expression of all P450 proteins was observed. CYP3A (135-243 fmol/mg of protein) and CYP2B1 (107-645 fmol/mg of protein) were the most abundant P450 isoforms in the duodenum and jejunum of rat intestine but were present in neither the ileum nor the colon. Compared with CYP2B1 and CYP3A, CYP2D1 (25-71 fmol/mg of protein) and CYP2C6 (3-10 fmol/mg of protein) were only expressed in minor amounts. CYP2C11 could not be identified in the entire rat intestine. In conclusion, this is the first systematic evaluation and quantification of the expression of P450 proteins along the entire length of the intestine in both male and female rats. These data will provide a basis for a better understanding of the extent of intestinal metabolism along the gastrointestinal tract.

Progress in the identification of stroke-related genes: emerging new possibilities to develop concepts in stroke therapy.

Stroke is a very complex disease influenced by many risk factors: genetic, environmental and comorbidities, such as hypertension, diabetes mellitus, obesity and having had a previous stroke. Neuroprotective therapies that have been found to be successful in laboratory animals have failed to produce the same benefits in clinical trials. Currently, a re-analysis of the clinical trial failures is underway and new therapeutic approaches using the growing knowledge from neurogenesis and neuroinflammation studies, combined with the information from gene expression studies, are taking place. This review focuses on possible ways to identify therapeutic targets using the new discoveries in neuroinflammation and intrinsic regenerative mechanisms of the brain. Molecular events associated with ischaemia trigger an environment for inflammation. Within the ischaemic region and its penumbra, a battery of chemokines and cytokines are released, which have both detrimental and beneficial effects, depending on the specific timepoint after injury and the current activation status of microglia/macrophages. Preventive therapies and treatments for stroke may be established by identifying the genes that are responsible for the induction of those phenotypic changes of microglia/macrophages that switch them to become players in tissue repair and regeneration processes. To aid in the establishment of new target sources for novel therapeutic agents, animal stroke models should closely mimic stroke in humans. To do so, these models should take into account the various risk factors for stroke. For example, hypertensive animals have a more vulnerable blood-brain barrier that in turn may trigger a greater degree of damage after stroke. Furthermore, in aged animals an accelerated astrocytic and microglial reaction has been observed and the regenerative capacity of aged brains is not as high as young brains. Improvements in animal models may also help to ensure better success rates of potential therapies in clinical studies. Inflammation in the brain is a double-edged sword--characterised by the deleterious effect of nerve cell damage and nerve cell death, as well as the beneficial influence on regeneration. The major challenge to develop successful stroke therapies is to broaden the knowledge regarding the underlying pathologic processes and the intrinsic mechanisms of the brain to drive regenerative and plasticity-related changes. On this basis, new concepts can be created leading to better stroke therapy.

CCR1 is an early and specific marker of Alzheimer's disease.

Chemokines are a diverse group of small proteins that effect cell signaling by binding to G-protein-coupled, seven-trans-membrane receptors. Our group had found previously that the chemokine receptor CCR1 was present in neurons and dystrophic processes in a small sample of Alzheimer's disease cases. This expanded immunohistochemical study shows that the number of CCR1-positive plaque-like structures in the hippocampus and entorhinal cortex is highly correlated to dementia state as measured by the clinical dementia rating score. CCR1 immunoreactivity is found in dystrophic, neurofilament-positive, synaptophysin-negative neurites that are associated with senile plaques containing amyloid beta peptides of the 1-42 species (Abeta42). CCR1 was not, however, associated with diffuse deposits of Abeta42. There was limited expression of CCR1 in neurofibrillary tangle-bearing neuritic processes. Astrocytes and microglia were typically negative for CCR1. Human brains from age-matched, nondemented individuals rarely displayed either CCR1 or Abeta42 immunoreactivity. Seven other types of dementing neurodegenerative diseases were examined, and all failed to demonstrate CCR1 immunopositivity unless Abeta42-positive plaques were also present. Thus, neuronal CCR1 is not a generalized marker of neurodegeneration. Rather, it appears to be part of the neuroimmune response to Abeta42-positive neuritic plaques.