Endothelial apoptosis in Braf-deficient mice.

Tyrosine kinase growth factor receptors and Ras/Raf/MEK/MAPK signalling have been implicated in the suppression as well as augmentation of programmed cell death. In addition, a Ras-independent role for Raf as a suppressor of programmed cell death has been suggested by the recent finding that Craf1 interacts with members of the Bcl-2 family at mitochondrial membranes. However, genetic studies of C. elegans and Drosophila, as well as the targeted mutagenesis of the murine Araf gene, have failed to support such a role. Here we show that mice with a targeted disruption in the Braf gene die of vascular defects during mid-gestation. Braf -/- embryos, unlike Araf -/- or Craf1 -/- embryos (L.W. et al., unpublished), show an increased number of endothelial precursor cells, dramatically enlarged blood vessels and apoptotic death of differentiated endothelial cells. These results establish Braf as a critical signalling factor in the formation of the vascular system and provide the first genetic evidence for an essential role of Raf gene in the regulation of programmed cell death.

Rhabdomyosarcomas and radiation hypersensitivity in a mouse model of Gorlin syndrome.

Gorlin (or nevoid basal cell carcinoma) syndrome is characterized by a variety of clinical problems including generalized overgrowth of the body, cysts, developmental abnormalities of the skeleton and a predisposition to benign and malignant tumors. The syndrome results from germline mutations of the human homolog of the drosophila segment polarity gene patched (ptc). Here we report that mice heterozygous for ptc develop many of the features characteristic of Gorlin syndrome and that they exhibit a high incidence of rhabdomyosarcomas (RMS), the most common soft-tissue sarcoma in children. The downstream signalling partner of ptc, gli1, was overexpressed in all RMSs analyzed, indicating that abnormal signalling of the ptc-gli1 pathway may be common for the various tumors associated with the syndrome. igf2, implicated in the formation of RMSs, was also overexpressed, suggesting cross-talk between the ptc and igf2 pathways in tumorigenesis. Developmental defects in Gorlin syndrome resemble those induced by ionizing radiation. We show that ptc heterozygous mice exhibit increased incidence of radiation-induced teratogenesis. This suggests a role for ptc in the response to ionizing radiation and provides a model for both the systemic (developmental) and stochastic (cancer) abnormalities observed in Gorlin syndrome.

Oscillating activity of a Ca(2+)-sensitive K+ channel. A prerequisite for migration of transformed Madin-Darby canine kidney focus cells.

Migration plays an important role in the formation of tumor metastases. Nonetheless, little is known about electrophysiological phenomena accompanying or underlying migration. Previously, we had shown that in migrating alkali-transformed Madin-Darby canine kidney focus (MDCK-F) cells a Ca(2+)-sensitive 53-pS K+ channel underlies oscillations of the cell membrane potential. The present study defines the role this channel plays in migration of MDCK-F cells. We monitored migration of individual MDCK-F cells by video imaging techniques. Under control conditions, MDCK-F cells migrated at a rate of 0.90 +/- 0.03 microns/min (n = 201). Application of K+ channel blockers (1 and 5 mmol/liter Ba2+, 5 mmol/liter tetraethylammonium, 100 mumol/liter 4-aminopyridine, 5 nmol/liter charybdotoxin) caused marked inhibition of migration, pointing to the importance of K+ channels in migration. Using patch-clamp techniques, we demonstrated the sensitivity of the Ca(2+)-sensitive 53-pS K+ channel to these blockers. Blockade of this K+ channel and inhibition of migration were closely correlated, indicating the necessity of oscillating K+ channel activity for migration. Migration of MDCK-F cells was also inhibited by furosemide or bumetanide, blockers of the Na+/K+/2Cl- cotransporter. We present a model for migration in which oscillations of cell volume play a central role. Whenever they are impaired, migration is inhibited.

Spontaneously oscillating K+ channel activity in transformed Madin-Darby canine kidney cells.

Intracellular alkalinization is known to be associated with tumorigenic transformation. Besides phenotypical alterations alkali-transformed Madin-Darby canine kidney (MDCK) cells exhibit a spontaneously oscillating cell membrane potential (PD). Using single-channel patch clamp techniques, it was the aim of this study to identify the ion channel underlying the rhythmic hyperpolarizations of the PD. In the cell-attached patch configuration, we found that channel activity was oscillating. The frequency of channel oscillations is 1.1 +/- 0.1 min-1. At the peak of oscillatory channel activity, single-channel current was -2.7 +/- 0.05 pA, and in the resting state it was -1.95 +/- 0.05 pA. Given the single-channel conductance of 53 +/- 3 pS for inward (and of 27 +/- 5 pS for outward) current the difference of single-channel current amplitude corresponded to a hyperpolarization of approximately 14 mV. The channel is selective for K+ over Na+. Channel kinetics are characterized by one open and by three closed time constants. The channel is Ca2+ sensitive. Half maximal activation in the inside-out patch mode is achieved at a Ca2+ concentration of 10 mumol/liter. In addition, we also found a 13-pS K+ channel that shows no oscillatory activity in the cell-attached patch configuration and that was not Ca2+ sensitive. We conclude that the Ca(2+)-sensitive 53-pS K+ channel is underlying spontaneous oscillations of the PD. It has virtually identical biophysical properties as a Ca(2+)-sensitive K+ channel in nontransformed parent MDCK cells. Hence, alkali-induced transformation of MDCK cells did not affect the channel protein itself but its regulators thereby causing spontaneous fluctuations of the PD.

Identification of a genetic contamination in a commercial mouse strain using two panels of polymorphic markers.

Rapid detection of genetic contamination is critical in mouse studies involving inbred strains. During a Quantitative Trait Locus (QTL) study using simple sequence length polymorphism (SSLP) markers, we noticed heterozygosity at some loci of a commercially available inbred C57BL/6N mouse strain, suggesting a contamination by another mouse strain. A panel of 100 single-nucleotide polymorphism (SNP) markers was used to confirm and specify the genetic contamination suspected. Retrospective analyses demonstrated that the contamination took place as early as autumn 2003 and has persisted ever since at a fairly constant level. Contaminating alleles most probably originated from a DBA strain. Our data demonstrate the suitability of SNP markers for rapid detection and identification of the source of genetic contamination. Further, our results show the importance of a state-of-the-art genetic monitoring of the authenticity of murine inbred strains.

Regulation of A-raf expression.

The Raf family proto-oncogenes encode cytoplasmic protein serine/threonine kinases which play a critical role in cell growth and development. A-raf shares several functional properties with Raf-1 including transforming activity, stimulation of the Raf/MAPK pathway and the ability of dominant negative versions to functionally block Ras signalling. A-raf transcripts are predominantly expressed in the mouse urogenital tissues. Interestingly, the human A-raf promoter region contains three potential glucocorticoid response elements GRE-1, GRE-2 and GRE-3, at positions -17, -34 and -168 respectively from the transcriptional start site. DNA sequence analysis of the mouse A-raf promoter region demonstrated that GRE-1 and -2 were conserved evolutionarily. To determine whether the human A-raf GREs represent functional motifs, an expression vector for the glucocorticoid receptor was cotransfected with A-raf promoter/reporter constructs into HeLa cells. A fivefold dexamethasone-dependent induction of A-raf promoter activity was observed using constructs containing all three GRE motifs whereas point mutations in the GREs either diminished or abolished dexamethasone induction. Electrophoretic mobility shift assays (EMSAs) using purified glucocorticoid receptor DNA binding domain (DBD) demonstrated that both GRE-2 and -3 motifs interact with DBD and oligonucleotide competition experiments established that these have different affinities for DBD. Using nuclear extracts from human and rodent cell lines in EMSAs, a specific protein-DNA complex was observed with GRE-1 which displayed binding properties unlike that of glucocorticoid receptor. These results demonstrate that the A-raf promoter is regulated in part by members of the glucocorticoid family of steroid hormone receptors and suggest a model for the regulation of A-raf expression in urogenital tissues.

Overlapping and specific functions of Braf and Craf-1 proto-oncogenes during mouse embryogenesis.

The three mammalian Raf serine/threonine protein kinases mediate the transduction of proliferative and differentiative signals from cell surface receptors to the nucleus. In vertebrates, Raf signaling has been implicated in the progression of mouse embryos through the two-cell stage and in the induction of posterior mesoderm. However, mouse embryos mutant for each of the Raf genes exhibit no developmental defects before mid-gestation. Here we describe the phenotype of mouse mutants with different combinations of mutant Craf-1 and Braf alleles. Our results show that Raf signaling is indeed indispensable for normal development beyond the blastocyst stage. However, due to a significant redundancy between Craf-1 and Braf, either gene is sufficient for normal development until mid-gestation. The molecular and developmental mechanisms for this redundancy were investigated by monitoring the expression of Raf genes throughout embryogenesis and by biochemical studies in mutant cell lines.

Craf-1 protein kinase is essential for mouse development.

The three mammalian Raf serine/threonine protein kinases mediate the transduction of proliferative and differentiative signals from a variety of cell surface receptors to the nucleus. We report here that Craf-1 is essential for mouse development, as its mutation results in embryonic lethality. Developmental defects are found in mutant placentas as well as in the skin and in the lungs of mutant embryos. Craf-1 mutants also display a generalized growth retardation which is consistent with the ubiquitous expression of Craf-1 and which could be due to the reduced proliferation of mutant cells. Interestingly, the time-point of embryonal death varies depending on the genetic background. This suggests that Craf-1-mediated signaling is affected by genetic background-specific alleles of other genes.

The patched signaling pathway in tumorigenesis and development: lessons from animal models.

The identification of mutations in the human homolog of the Drosophila segment polarity gene Patched in basal cell carcinoma has sparked intense interest in the role of this gene in human disorders. The transmembrane protein Patched is a receptor for the morphogene Sonic Hedgehog. Sonic Hedgehog/Patched signaling involves another transmembrane protein, Smoothened, and its intracellular effectors, including the proto-oncogene GLI1. During the past 2 years it has become evident that mutations in Patched or in one of the components of its signaling pathway contribute to the formation of several common human tumors. It is now well established that Patched is a tumor suppressor gene. The Sonic Hedgehog/Patched/Smoothened signaling pathway is thus rapidly emerging as one of the most important regulators of oncogenic transformation. This pathway also plays an important role during mammalian embryonic development. This dual role is especially visible in humans with inherited Patched mutations. Such patients suffer from Gorlin, or nevoid basal cell carcinoma, syndrome and exhibit a variety of developmental defects accompanied by a predisposition to tumor formation. Activating mutations in Sonic Hedgehog and Smoothened lead to similar phenotypes as do loss-of function mutations in Patched. By means of transgenic and gene targeting technologies the respective mutations have been expressed in the mouse. Such mutant mouse strains exhibit many symptoms observed in humans. These strains are useful models to study the pathogenesis of several common human tumors and developmental defects. Furthermore they provide important tools to study the Sonic Hedgehog/Patched/Smoothened signaling at the molecular and biochemical level.

The genetic determinants of the CYP3A5 polymorphism.

CYP3A proteins comprise a significant portion of the hepatic cytochrome P450 (CYP) protein and they metabolize around 50% of drugs currently in use. The dissection of the individual contributions of the four CYP3A genes identified in humans to overall hepatic CYP3A activity has been hampered by sequence and functional similarities. We have investigated the expression of CYP3A5 and its genetic determinants in a panel of 183 Caucasian liver samples. CYP3A5 expression is increased in 10% of livers in this ethnic group. Using a high density map of CYP3A5 variants, we searched for genetic markers of the increased CYP3A5 expression. In agreement with an independent, recent study, we report that a SNP within intron 3 (g.6986G>A) is the primary cause of the CYP3A5 protein polymorphism. The frequencies of the g.6986A variant which allow for normal splicing of CYP3A5 transcripts are 5% in Caucasians, 29% in Japanese, 27% in Chinese, 30% in Koreans and 73% in African-Americans. In the last ethnic group, the expression of CYP3A5 in some individuals who carry the g.6986A variant is affected adversely by a frame shift mutation (CYP3A5*7, D348., q = 0.10). In summary, these results should add to efforts to identify clinically relevant, CYP3A5-specific reactions and to further elucidate traits responsible for variable expression of the entire CYP3A family.

Genomic organization of the human CYP3A locus: identification of a new, inducible CYP3A gene.

Proteins encoded by the human CYP3A genes metabolize every second drug currently in use. The activity of CYP3A gene products in the general population is highly variable and may affect the efficacy and safety of drugs metabolized by these enzymes. The mechanisms underlying this variability are poorly understood, but they include gene induction, protein inhibition and unknown genetic polymorphisms. To better understand the regulation of CYP3A expression and to provide a basis for a screen of genetic polymorphisms, we determined and analysed the sequence of the human CYP3A locus. The 231 kb locus sequence contains the three CYP3A genes described previously (CYP3A4, CYP3A5 and CYP3A7), three pseudogenes as well as a novel CYP3A gene termed CYP3A43. The gene encodes a putative protein with between 71.5% and 75.8% identity to the other CYP3A proteins. The highest expression level of CYP3A43 mRNA is observed in the prostate, an organ with extensive steroid metabolism. CYP3A43 is also expressed in several other tissues including liver, where it can be induced by rifampicin. CYP3A43 transcripts undergo extensive splicing. The identification of a new member of the CYP3A family and the characterization of the full CYP3A locus will aid efforts to identify the genetic variants underlying its variable expression. This, in turn, will lead to a better optimization of therapies involving the numerous substrates of CYP3A proteins.

Identification and functional characterization of eight CYP3A4 protein variants.

The genetic component of the inter-individual variability in CYP3A4 activity has been estimated to be between 60% and 90%, but the underlying genetic factors remain largely unknown. A study of 213 Middle and Western European DNA samples resulted in the identification of 18 new CYP3A4 variants, including eight protein variants. A total of 7.5% of the population studied was found to be heterozygous for one of these variants. In a bacterial heterologous expression system, two mutants, R130Q and P416L, did not result in detectable P450 holoprotein. One mutant, T363M, expressed at significantly lower levels than wild-type CYP3A4. G56D, V170I, D174H and M445T were not significantly different when compared with wild-type CYP3A4 in expression or steroid hydroxylase activity. L373F displayed a significantly altered testosterone metabolite profile and a four-fold increase in the Km value for 1'-OH midazolam formation. The results suggest a limited contribution of CYP3A4 protein variants to the inter-individual variability of CYP3A4 activity in Caucasians. Some variants may, however, play a role in the atypical response to drugs or altered sensitivity to carcinogens.

[The importance of combining ultrasonography of the carotid artery bifurcation and Doppler imaging of cerebral arteries]. / Znaczenie skojarzonego badania USG rozwidlenia tetnic szyjnych i dopplerowskiego tetnic glowy u pacjentów z udarem niedokrwiennym.

The USG of carotid bifurcation and Doppler imaging of cranial arteries were performed in 74 patients with ischaemic stroke. The Doppler image as well as USG were found to be helpful for prognosis. Arteriosclerotic USG changes corresponded more often with the neurological syndrome in severely ill patients (at discharge from hospital). Also reduction of flow in medial cerebral artery was significantly more often in these patients in the first hours of the illness. In thrombosis of the first part of the internal carotid artery only the succeeding image can indicate it (2 cases). Arteriography only can demonstrate artery tortuosity (1 case).

Resveratrol reduces endothelial oxidative stress by modulating the gene expression of superoxide dismutase 1 (SOD1), glutathione peroxidase 1 (GPx1) and NADPH oxidase subunit (Nox4).

Resveratrol, an important antioxidant found in grapes and wine, is likely to contribute to red wine's potential to prevent human cardiovascular disease. In addition to its known (direct) antioxidant effect, we have found that resveratrol also regulates the gene expression of pro-oxidative and anti-oxidative enzymes in human endothelial cells. NADPH oxidases (Nox) are the predominant producers of superoxide in the vasculature, whereas superoxide dismutase (SOD) and glutathione peroxidase 1 (GPx1) are the major enzymes responsible for the inactivation of superoxide and hydrogen peroxide, respectively. Incubation of human umbilical vein endothelial cells (HUVEC) and HUVEC-derived EA.hy 926 cells with resveratrol resulted in a concentration- and time-dependent downregulation of Nox4, the most abundant NADPH oxidase catalytic subunit (quantitative real-time RT-PCR). The same resveratrol regimen upregulated the mRNA expression of SOD1 and GPx1. The addition the protein levels of SOD1 and GPx1 were enhanced by resveratrol in a concentration-dependent manner (Western blot analyses). Pretreatment of EA.hy 926 cells with resveratrol completely abolished DMNQ-induced oxidative stress. Thus, the expressional suppression of pro-oxidative genes (such as NADPH oxidase) and induction of anti-oxidative enzymes (such as SOD1 and GPx1) might be an important component of the vascular protective effect of resveratrol.

Accounting for haplotype phase uncertainty in linkage disequilibrium estimation.

The characterization of linkage disequilibrium (LD) is applied in a variety of studies including the identification of molecular determinants of the local recombination rate, the migration and population history of populations, and the role of positive selection in adaptation. LD suffers from the phase uncertainty of the haplotypes used in its calculation, which reflects limitations of the algorithms used for haplotype estimation. We introduce a LD calculation method, which deals with phase uncertainty by weighting all possible haplotype pairs according to their estimated probabilities as evaluated by PHASE. In contrast to the expectation-maximization (EM) algorithm as implemented in the HAPLOVIEW and GENETICS packages, our method considers haplotypes based on the entire genetic information available for the candidate region. We tested the method using simulated and real genotyping data. The results show that, for all practical purposes, the new method is advantageous in comparison with algorithms that calculate LD using only the most probable haplotype or bilocus haplotypes based on the EM algorithm. The new method deals especially well with low LD regions, which contribute strongly to phase uncertainty. Altogether, the method is an attractive alternative to standard LD calculation procedures, including those based on the EM algorithm. We implemented the method in the software suite R, together with an interface to the popular haplotype calculation package PHASE.

The interleukin-10 gene promoter polymorphism -1087AG does not correlate with clinical outcome in non-Hodgkin's lymphoma.

The Interleukin 10 (IL-10) gene is highly polymorphic, and the IL-10(-1087AG) (rs1800896) gene variation is the only so far studied intensively in association with certain diseases. Conflicting data have been published about an association of IL-10(-1087AG) gene variation with lower rates of complete remission and lower overall survival (OS) in patients with diffuse large B-cell lymphoma. To further investigate this in malignant lymphoma, we established the IL-10 genotypes in patients from the NHL-B1/ B2 studies from the German High-Grade Non-Hodgkin's Lymphoma Study Group. In our study, allele frequencies of lymphoma patients are comparable as in healthy controls. No increase of IL-10(-1087G) alleles was found. In addition we did not find any difference in OS or event-free survival between patients with IL-10(-1087AA) and the other genotypes. Comparable results were obtained for the IL-10 loci at -3538 (A/T), -1354 (A/G), -824 (C/T) and -597 (A/C) (rs1800890, rs1800893, rs1800871 and rs1800872).

Genetic signature consistent with selection against the CYP3A4*1B allele in non-African populations.

Cytochrome P450 3A enzymes (CYP3A) play a major role in the metabolism of steroid hormones, drugs and other chemicals, including many carcinogens. The individually variable CYP3A expression, which remains mostly unexplained, has been suggested to affect clinical phenotypes. We investigated the CYP3A locus in five ethnic groups. The degree of linkage disequilibrium (LD) differed among ethnic groups, but the most common alleles of the conserved LD regions were remarkably similar. Non-African haplotypes are few; for example, only four haplotypes account for 80% of common European Caucasian alleles. Large LD blocks of high frequencies were suggestive of selection. Accordingly, European Caucasian and Asian cohorts each contained a block of single nucleotide polymorphism (SNPs) with very high P excess values. The overlap between these blocks in these two groups contained only two of the investigated 26 SNPs and one of them was the CYP3A4*1B allele. The region centromeric of CYP3A4*1B exhibited high haplotype homozygosity in European Caucasians as opposed to African-Americans. CYP3A4*1B showed a moderate effect on CYP3A4 mRNA and protein expression, as well as on CYP3A activity assessed as Vmax of testosterone 6beta-hydroxylation in a liver bank. Our data are consistent with a functional relevance of CYP3A4*1B and with selection against this allele in non-African populations. The elimination of CYP3A4*1B involved different parts of the CYP3A locus in European Caucasians and Asians. Because CYP3A4 is involved in the vitamin D metabolism, rickets may have been the underlying selecting factor.

Marmoset CYP3A21, a model for human CYP3A4: protein expression and functional characterization of the promoter.

Due to its small size and the relative evolutionary proximity, the marmoset has been proposed as a model for studies of human drug interactions and metabolism. The current study investigated the expression and regulation of marmoset CYP3A using mass spectrometry and reporter gene techniques. Expression levels of hepatic marmoset CYP3A protein range from 51 to 123 pmol mg-1 total protein (mean 85.2 pmol mg-1, n = 10) and CYP3A21 is the dominant hepatic CYP3A protein in marmosets. The sequence similarity between human CYP3A4 and CYP3A21 across the first 7.5 kb of the cloned CYP3A21 promoter is 88% within the xenobiotic-responsive enhancer module (XREM) and the proximal promoter. Both regulatory modules confer transcriptional activation of CYP3A21-luciferase reporter gene constructs cotransfected with hPXR in intestinal LS174T cells. The pronounced response to rifampin and the moderate response to dexamethasone were similar to those observed with CYP3A4. Taken collectively, these data establish substantial similarities in expression and gene regulation between marmoset CYP3A21 and human CYP3A4. CYP3A21 may be an equivalent of CYP3A4 in New World monkeys, consistent with the phylogenetic relationship between these genes. The marmoset, therefore, appears to be a suitable in vivo model to study CYP3A4 function and regulation.

Hypertonicity in fused Madin-Darby canine kidney cells: transient rise in NaHCO3 followed by sustained KCl accumulation.

We investigated mechanisms of regulatory volume increase in fused Madin-Darby canine kidney (MDCK) cells, a cell line originally derived from renal collecting duct. The intracellular ion concentrations as well as the concentration of the volume marker tetramethylammonium+ were measured by means of ion-selective microelectrodes. Application of hypertonic Ringer bicarbonate solution (+150 mmol/l mannitol) resulted in cell shrinkage to 84 +/- 2% of the initial cell volume (shrinkage expected for an ideal osmometer = 66%), indicating a significant regulatory volume increase. During the first 90 s of the hypertonic stress, a transient increase in intracellular Na+ and HCO3- concentrations was observed. It was followed by a sustained increase in intracellular K+ and Cl- concentrations. Ouabain (0.1 mmol/l) as well as amiloride (1 mmol/l) reduced K+ accumulation significantly, whereas the H+/K(+)-ATPase inhibitor SCH 28080 had no effect. Hypertonic stress hyperpolarized the cell membrane potential by 19 +/- 2 mV, owing to the decrease of the ratio of Cl- conductance to K+ conductance of the cell membrane. We conclude: (a) acute hypertonic stress activates Na+/H+ exchange in MDCK cells; (b) transient alteration of intracellular Na+ and pH stimulates Na+/K(+)-ATPase and Cl-/HCO3- exchange, exchange, both leading to the sustained intracellular accumulation of KCl; (c) a high intracellular KCl concentration is maintained by the partial reversion of the Cl-/K+ conductance ratio of the plasma membrane.