Loss of MYC confers resistance to doxorubicin-induced apoptosis by preventing the activation of multiple serine protease- and caspase-mediated pathways

c-Myc plays an essential role in proliferation, differentiation, and apoptosis. Because of its relevance to cancer, most studies have focused on the cellular consequences of c-Myc overexpression. Here, we address the role of physiological levels of c-Myc in drug-induced apoptosis. By using c-MYC null cells we confirm and extend recent reports showing a c-Myc requirement for the induction of apoptosis by a number of anticancer agents. In particular, we show that c-Myc is required for the induction of apoptosis by doxorubicin and etoposide, whereas it is not required for camptothecin-induced cell death. We have investigated the molecular mechanisms involved in executing doxorubicin-induced apoptosis and show caspase-3 activation by both mitochondria-dependent and -independent pathways. Moreover, serine proteases participate in doxorubicin-induced apoptosis partly by contributing to caspase-3 activation. Finally, a complete rescue from doxorubicin-induced apoptosis is obtained only when serine proteases, caspase-3, and mitochondrial activation are inhibited simultaneously. Interestingly, doxorubicin requires c-Myc for the activation of all of these pathways. Our findings therefore support a model in which doxorubicin simultaneously triggers multiple c-Myc-dependent apoptosis pathways.     

Intracranial aneurysms in Finnish families: confirmation of linkage and refinement of the interval to chromosome 19q13.3

We recently reported a two-stage genomewide screen of 48 sib pairs affected with intracranial aneurysms (IAs) that revealed suggestive linkage to chromosome 19q13, with a LOD score of 2.58. The region supporting linkage spanned ~22 cM. Here, we report a follow-up study of the locus at 19q13, with a sample size expanded to 139 affected sib pairs, along with 83 other affected relative pairs (222 affected relative pairs in total). Suggestive linkage was observed in both independent sample sets, and linkage was significant in the combined set at 70 cM (LOD score 3.50; P=.00006) and at 80 cM (LOD score 3.93; P=.00002). Linkage was highly significant at 70 cM (LOD score 5.70; P=.000001) and at 80 cM (LOD score 3.99; P=.00005) when a covariate measuring the number of affected individuals in the nuclear family was included. To evaluate further the contribution to the linkage signal from families with more than two affected relatives, we performed model-based linkage analysis with a recessive model and a range of penetrances, and we obtained maximum linkage at 70 cM (LOD score 3.16; P=.00007) with a penetrance of 0.3. We then estimated location by using GENEFINDER. The most likely location for a gene predisposing to IAs in the Finnish population is in a region with a 95% confidence interval of 11.6 cM (P=.00007) centered 2.0 cM proximal to D19S246.     

Purification and cDNA cloning of UDP-GlcNAc:GlcNAcbeta1-3Galbeta1-4Glc(NAc)-R [GlcNAc to Gal]beta1,6N-acetylglucosaminyltransferase from rat small intestine: a major carrier of dIGnT activity in rat small intestine

A rat intestinal beta1,6N-acetylglucosaminyltransferase (beta1-6GnT) responsible for the formation of the beta1,6-branched poly-N-acetyllactosamine structure has been purified to apparent homogeneity by successive column chromatographic procedures using an assay wherein pyridylaminated lacto- N-triose II (GlcNAcbeta1-3Galbeta1-4Glc-PA) was used as an acceptor substrate and the reaction product was GlcNAcbeta1-3(GlcNAcbeta1-6)Galbeta1-4Glc-PA. The purified enzyme catalyzed the conversion of the polylactosamine acceptor GlcNAcbeta1-3'LacNAc into GlcNAcbeta1-3'(GlcNAcbeta1-6') LacNAc (dIGnT activity), but it could not transfer GlcNAc to LacNAcbeta1-3'LacNAc (cIGnT activity). This enzyme could also convert mucin core 1 and core 3 analogs, Galbeta1-3GalNAcalpha1-O-paranitrophenyl (pNP) and GlcNAcbeta1-3GalNAcalpha1-O-pNP, into Galbeta1-3(GlcNAcbeta1-6) GalNAcalpha1-O-pNP (C2GnT activity) and GlcNAcbeta1-3(GlcNAcbeta1-6)GalNAcalpha1-O-pNP (C4GnT activity), respectively. Based on the partial amino acid sequences of the purified protein, the cDNA encoding this enzyme was cloned. The COS-1 cells transiently transfected with this cDNA had high dI/C2/C4GnT activities in a ratio of 0.34:1.00:0.90, compared with non- or mock-transfected cells. The primary structure shows a significant homology with human and viral mucin-type core 2 beta1-6GnTs (C2GnT-Ms), indicating that this enzyme is the rat ortholog of human and viral C2GnT-Ms. This is the first identification and purification of this enzyme as a major carrier of dIGnT activity in the small intestine. This rat ortholog should mostly be responsible for making distal I-branch structures on poly-N-acetyllactosamine sequences in this tissue, as well as making mucin core 2 and core 4 structures, given that it also has high C2/C4GnT activities.     

Coronary artery diameter can be assessed reliably with transthoracic echocardiography

We studied whether diameters of coronary arteries can be measured accurately with the use of transthoracic echocardiography (TTE). By knowing the anatomic diameter of the coronary artery together with coronary flow velocity it is possible to measure coronary flow volume more precisely by TTE. However, the suitability of TTE for measurement of diameters of all main epicardial coronary arteries has not been systematically validated. We measured the diameters of the left main (LM), left anterior descending (LAD), left circumflex (LCX), and right coronary arteries (RCA) with the use of TTE [manual two-dimensional (2D), color-Doppler, and automated 2D analysis] in 30 patients who had normal coronary anatomy. We compared these diameters to those measured with quantitative coronary angiography (QCA). We could measure diameters of LM, LAD, LCX, and RCA by TTE in up to 37%, 63%, 7%, and 60% of patients, respectively. The overall correlation coefficients between TTE and QCA measurements were 0.83 (P < 0.01) with manual 2D analysis, 0.82 (P < 0.01) with automated 2D analysis, and 0.94 (P < 0.01) with a color-Doppler-based analysis. Interobserver variability of TTE measurements was low (coefficient of variation 5.4 +/- 4.6-7.5 +/- 8.8%). TTE is an accurate method to evaluate coronary artery diameter in patients with healthy coronary arteries.     

The establishment of a network of European human research tissue banks

This is a report of a workshop held on the establishment of human research tissue banking which was held in Levi, Finland 21–24 March 2002.There were 21 participants from 7 European countries. This meeting was attended by representatives from academia, research tissue banks and from the Biotech and Pharmaceutical Industries. The principal aim of the workshop was to find a way to progress the recommendations from ECVAM workshop 44 (ATLA 29, 125–134,2001) and ECVAM workshop 32 (ATLA 26, 763–777, 1998). The workshop represented the first unofficial meeting of the European Network of Research Tissue Banks (ENRTB) steering group. It is expected that in the period preceding the next workshop the ENRTB steering group will co-ordinate the ethical,legislative and organisational aspects of research tissue banking. Key issues dealt with by the Levi workshop included the practical aspects of sharing expertise and experiences across the different European members. Such collaboration between research tissue banks and end users of such material seeks to ultimately enable shared access to human tissue for medical and pharmaco-toxicological research while maintaining strict adherence to differences in legal and ethical aspects related to the use of human tissue in individual countries. This revised version was published online in July 2006 with corrections to the Cover Date.     

麝,獐,麂和鹿间线粒体DNA的差异及其系统进化研究

用PCR技术和序列测定方法从线粒体DNA上得到367bp的细胞色素b基因片段序列。分析得出,麝与獐、麂和鹿的遗传差异在12．53％～14．44％之间,处在科间变化范围之内,在分子水平上进一步表明麝应作为一独立科;獐、麂和虎间序列的平均差异为10．28％,属于业科间的差异,与形态研究结果一致。麝、獐、麂和鹿的系统进化中,麝约在600万年前与鹿科分歧,而鹿科的三个亚科是在350～500万年开始分歧;麝、獐、麂和鹿共同组成一单系群．在600万年前具有一共同祖先。     

Enzymatic in vitro synthesis of radiolabeled pentasaccharides GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc/Glc and the isomeric Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc/Glc.

Four radiolabeled pentasaccharides, GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc, Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc, GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4Glc, and Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4Glc, were prepared in virtually pure form. They were obtained by partial enzymic beta 1,4-galactosylations of the appropriate tetrasaccharide acceptors or by partial enzymic degalactosylations of the appropriate hexasaccharides, followed by paper chromatographic separations. All four pentasaccharides contain two nonidentical distal branches, making them valuable primers for enzymatic in vitro synthesis of larger oligo(N-acetyllactosaminoglycans).     

A novel glycosulfopeptide binds to P-selectin and inhibits leukocyte adhesion to P-selectin.

P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric membrane mucin on leukocytes that binds selectins. The molecular features of PSGL-1 that determine this high affinity binding are unclear. Here we demonstrate the in vitro synthesis of a novel glycosulfopeptide (GSP-6) modeled after the extreme N terminus of PSGL-1, which has been predicted to be important for P-selectin binding. GSP-6 contains three tyrosine sulfate (TyrSO(3)) residues and a monosialylated, core 2-based O-glycan with a sialyl Lewis x (C2-O-sLe(x)) motif at a specific Thr residue. GSP-6 binds tightly to immobilized P-selectin, whereas glycopeptides lacking either TyrSO(3) or C2-O-sLe(x) do not detectably bind. Remarkably, an isomeric glycosulfopeptide to GSP-6, termed GSP-6', which contains sLe(x) on an extended core 1-based O-glycan, does not bind immobilized P-selectin. Equilibrium gel filtration analysis revealed that GSP-6 binds to soluble P-selectin with a K(d) of approximately 350 nM. GSP-6 (<5 microM) substantially inhibits neutrophil adhesion to P-selectin in vitro, whereas free sLe(x) (5 mM) only slightly inhibits adhesion. In contrast to the inherent heterogeneity of post-translational modifications of recombinant proteins, glycosulfopeptides permit the placement of sulfate groups and glycans of precise structure at defined positions on a polypeptide. This approach should expedite the probing of structure-function relationships in sulfated and glycosylated proteins, and may facilitate development of novel drugs to treat inflammatory diseases involving P-selectin-mediated leukocyte adhesion.     

Biosynthesis of sialylated and fucosylated selectin ligands of HL-60 cells in vitro. Midchain alpha3-fucose units inhibit terminal alpha6-sialylation but not alpha3-sialylation of polylactosamines.

Polylactosamines Neu5Ac alpha2-3'Lex beta1-3'Lex beta1-3'Lex and Neu5Ac alpha2-3'LNbeta1-3'Lex beta1-3'Lex [Lex, Gal beta1-4(Fuc alpha1-3)GlcNAc; LN, Gal beta1-4GlcNAc] decorate selectin counterreceptors in human HL-60 cells. Here, we show that HL-60 cell lysates catalyze distal alpha3-sialylation of LNbeta1-3'LNbeta1-3'LN and LNbeta1-3'Lex beta1-3'Lex efficiently, outlining two potential sets of biosynthetic pathways leading to the selectin ligands. In one set, alpha3-sialylation precedes internal fucosylation of the polylactosamine backbone, whereas in the other one, internal fucosylation is initiated before alpha3-sialylation. In contrast to alpha3-sialylation, LNbeta1-3'Lex beta1-3'Lex was alpha6-sialylated much less efficiently than LNbeta1-3'LNbeta1-3'LN by HL-60 cell lysates. Hence, internal fucosylation may regulate the extent of alpha6-sialylation of polylactosamines in these cells.     

Combination treatment optimization using a pan-cancer pathway model

The design of efficient combination therapies is a difficult key challenge in the treatment of complex diseases such as cancers. The large heterogeneity of cancers and the large number of available drugs renders exhaustive in vivo or even in vitro investigation of possible treatments impractical. In recent years, sophisticated mechanistic, ordinary differential equation-based pathways models that can predict treatment responses at a molecular level have been developed. However, surprisingly little effort has been put into leveraging these models to find novel therapies. In this paper we use for the first time, to our knowledge, a large-scale state-of-the-art pan-cancer signaling pathway model to identify candidates for novel combination therapies to treat individual cancer cell lines from various tissues (e.g., minimizing proliferation while keeping dosage low to avoid adverse side effects) and populations of heterogeneous cancer cell lines (e.g., minimizing the maximum or average proliferation across the cell lines while keeping dosage low). We also show how our method can be used to optimize the drug combinations used in sequential treatment plans—that is, optimized sequences of potentially different drug combinations—providing additional benefits. In order to solve the treatment optimization problems, we combine the Covariance Matrix Adaptation Evolution Strategy (CMA-ES) algorithm with a significantly more scalable sampling scheme for truncated Gaussian distributions, based on a Hamiltonian Monte-Carlo method. These optimization techniques are independent of the signaling pathway model, and can thus be adapted to find treatment candidates for other complex diseases than cancers as well, as long as a suitable predictive model is available.