Predictive value of apoptosis, proliferation, HER-2, and topoisomerase IIalpha for anthracycline chemotherapy in locally advanced breast cancer

Purpose. Laboratory evidence indicates that tumor growth depends on the balance between cell proliferation and cell death, and many anticancer agents may exert their therapeutic effect by decreasing proliferation and increasing apoptosis. Additionally, clinical observations indicate that overexpression of HER-2 or topoisomerase II alpha ( topo II alpha) may be predictors of better response to anthracyclines in breast cancer. The objective of this study was to determine if proliferation ( Ki-67), apoptosis ( TUNEL), and expression of HER-2 and topo II alpha are affected by anthracycline treatment, and if these molecular markers predict anthracycline responsiveness. Experimental design. Thirty-three women with primary breast tumors >= 3 cm received either doxorubicin ( 75 mg/ m(2)) or epirubicin ( 120 mg/ m(2)) for 4 cycles before surgery. Clinical response was evaluated after 4 cycles of treatment. Changes in molecular markers were assessed from core needle biopsy taken before treatment (D0), at 24 - 48 h (Dl) and on day 7 (D7) while on treatment, and from the surgical specimen excised on day 84 (D84) after the fourth cycle of chemotherapy. Results. The overall clinical response rate was 51% (17 of 33 patients), with a 12% complete clinical response rate ( 4 of 33 patients). There were trends for tumors with higher apoptosis and topo IIa at baseline ( D0) to be more responsive to anthracyclines, p = 0.1 and p = 0.08, respectively. Median apoptosis increased from D0 to Dl ( p = 0.06) while median Ki-67 decreased ( p = 0.07). Overall, expression of HER-2 remained stable throughout the chemotherapy administration. By Day 84, topo II alpha had significantly decreased from baseline in responders, while it increased in non-responders, p = 0.03. Conclusions. In human primary breast cancer, anthracycline treatment causes an early increase in apoptosis and a decrease in proliferation. In this pilot study, higher apoptosis and topo II alpha a levels in primary tumors were associated with greater responsiveness to anthracyclines, and topo II alpha levels declined in responsive tumors

Identification of aspartic acid-203 in human thymidine phosphorylase as an important residue for both catalysis and non-competitive inhibition by the small molecule "crystallization chaperone" 5'-O-tritylinosine (KIN59)

Thymidine phosphorylase (TP) is a catabolic enzyme in thymidine metabolism that is frequently upregulated in many solid tumors. Elevated TP levels are associated with tumor angiogenesis, metastasis and poor prognosis. Therefore, the use of TP inhibitors might offer a promising strategy for cancer treatment. The tritylated inosine derivative 5'-O-tritylinosine (previously designated KIN59) is a noncompetitive inhibitor of TP which was previously found to be instrumental for the crystallization of human TP. A combination of computational studies including normal mode analysis, automated ligand docking and molecular dynamics simulations were performed to define a plausible binding site for 5'-O-tritylinosine on human TP. A cavity in which 5'-O-tritylinosine could fit was identified in the vicinity of the Gly405-WI419 loop at a distance of about 11 angstrom from the substrate-binding site. In the X-ray crystal structure, this pocket is characterized by an intricate hydrogen-bonding network in which Asp203 was found to play an important role to afford the loop stabilization that is required for efficient enzyme catalysis. Site-directed mutagenesis of this amino acid residue afforded a mutant enzyme with a severely compromised catalytic efficiency (V-max /K-m of mutant enzyme similar to 50-fold lower than for wild-type TP) and pronounced resistance to the inhibitory effect of 5'-O-tritylinosine. In contrast, the D203A mutant enzyme kept full sensitivity to the competitive inhibitors 6-aminothymine and 6-amino-5-bromouracil, which is in line with the kinetic properties of these inhibitors. Our findings reveal the existence of a previously unrecognized site in TP that can be targeted by small molecules to inhibit the catalytic activity of TP. (C) 2009 Elsevier Inc. All rights reserved

X-chromosome tiling path array detection of copy number variants in patients with chromosome X-linked mental retardation

Contiene 3 ficheros adicionales con información suplementaria.-- et al.[Background] Aproximately 5–10% of cases of mental retardation in males are due to copy number variations (CNV) on the X chromosome. Novel technologies, such as array comparative genomic hybridization (aCGH), may help to uncover cryptic rearrangements in X-linked mental retardation (XLMR) patients. We have constructed an X-chromosome tiling path array using bacterial artificial chromosomes (BACs) and validated it using samples with cytogenetically defined copy number changes. We have studied 54 patients with idiopathic mental retardation and 20 controls subjects.[Results] Known genomic aberrations were reliably detected on the array and eight novel submicroscopic imbalances, likely causative for the mental retardation (MR) phenotype, were detected. Putatively pathogenic rearrangements included three deletions and five duplications (ranging between 82 kb to one Mb), all but two affecting genes previously known to be responsible for XLMR. Additionally, we describe different CNV regions with significant different frequencies in XLMR and control subjects (44% vs. 20%).[Conclusion] This tiling path array of the human X chromosome has proven successful for the detection and characterization of known rearrangements and novel CNVs in XLMR patients.The authors thank the "Genoma España" and Genome Canada joint R+D+I projects in human health, plants and aquiculture; the former "Departament d'Universitats i Societat de la Informació" (DURSI) and the "Departament de Salut", from the Catalan Autonomous Government (2005SGR00008 - Generalitat de Catalunya); the Instituto de Salud Carlos III (PI041126, CIBER-ESP), the EU's Sixth Framework Programme [FP6-2005-LIFESCIHEALTH-7; ANEUPLOIDY No. 037627] and Fundación Areces (U-2006-FARECES-O).Peer reviewe

Association of MC1R Variants and host phenotypes with melanoma risk in CDKN2A mutation carriers: a GenoMEL study

<p><b>Background</b> Carrying the cyclin-dependent kinase inhibitor 2A (CDKN2A) germline mutations is associated with a high risk for melanoma. Penetrance of CDKN2A mutations is modified by pigmentation characteristics, nevus phenotypes, and some variants of the melanocortin-1 receptor gene (MC1R), which is known to have a role in the pigmentation process. However, investigation of the associations of both MC1R variants and host phenotypes with melanoma risk has been limited.</p> <p><b>Methods</b> We included 815 CDKN2A mutation carriers (473 affected, and 342 unaffected, with melanoma) from 186 families from 15 centers in Europe, North America, and Australia who participated in the Melanoma Genetics Consortium. In this family-based study, we assessed the associations of the four most frequent MC1R variants (V60L, V92M, R151C, and R160W) and the number of variants (1, ≥2 variants), alone or jointly with the host phenotypes (hair color, propensity to sunburn, and number of nevi), with melanoma risk in CDKN2A mutation carriers. These associations were estimated and tested using generalized estimating equations. All statistical tests were two-sided.</p> <p><b>Results</b> Carrying any one of the four most frequent MC1R variants (V60L, V92M, R151C, R160W) in CDKN2A mutation carriers was associated with a statistically significantly increased risk for melanoma across all continents (1.24 × 10−6 ≤ P ≤ .0007). A consistent pattern of increase in melanoma risk was also associated with increase in number of MC1R variants. The risk of melanoma associated with at least two MC1R variants was 2.6-fold higher than the risk associated with only one variant (odds ratio = 5.83 [95% confidence interval = 3.60 to 9.46] vs 2.25 [95% confidence interval = 1.44 to 3.52]; Ptrend = 1.86 × 10−8). The joint analysis of MC1R variants and host phenotypes showed statistically significant associations of melanoma risk, together with MC1R variants (.0001 ≤ P ≤ .04), hair color (.006 ≤ P ≤ .06), and number of nevi (6.9 × 10−6 ≤ P ≤ .02).</p> <p><b>Conclusion</b> Results show that MC1R variants, hair color, and number of nevi were jointly associated with melanoma risk in CDKN2A mutation carriers. This joint association may have important consequences for risk assessments in familial settings.</p&gt

HD 219134 Revisited: Planet d Transit Upper Limit and Planet f Transit Nondetection with ASTERIA and TESS

HD 219134 is a K3V dwarf star with six reported radial-velocity discovered planets. The two innermost planets b and c show transits, raising the possibility of this system to be the nearest (6.53 pc), brightest (V = 5.57) example of a star with a compact multiple transiting planet system. Ground-based searches for transits of planets beyond b and c are not feasible because of the infrequent transits, long transit duration (~5 hr), shallow transit depths (<1%), and large transit time uncertainty (~half a day). We use the space-based telescopes the Arcsecond Space Telescope Enabling Research in Astrophysics (ASTERIA) and the Transiting Exoplanet Survey Satellite (TESS) to search for transits of planets f (P = 22.717 days and M sin i = 7.3 ± 0.04M_⊕) and d (P = 46.859 days and M sin i = 16.7 ± 0.64M_⊕). ASTERIA was a technology demonstration CubeSat with an opportunity for science in an extended program. ASTERIA observations of HD 219134 were designed to cover the 3σ transit windows for planets f and d via repeated visits over many months. While TESS has much higher sensitivity and more continuous time coverage than ASTERIA, only the HD 219134 f transit window fell within the TESS survey's observations. Our TESS photometric results definitively rule out planetary transits for HD 219134 f. We do not detect the Neptune-mass HD 219134 d transits and our ASTERIA data are sensitive to planets as small as 3.6 R_⊕. We provide TESS updated transit times and periods for HD 219134 b and c, which are designated TOI 1469.01 and 1469.02 respectively

Protein Phosphatase-1α Interacts with and Dephosphorylates Polycystin-1

Polycystin signaling is likely to be regulated by phosphorylation. While a number of potential protein kinases and their target phosphorylation sites on polycystin-1 have been identified, the corresponding phosphatases have not been extensively studied. We have now determined that polycystin-1 is a regulatory subunit for protein phosphatase-1α (PP1α). Sequence analysis has revealed the presence of a highly conserved PP1-interaction motif in the cytosolic, C-terminal tail of polycystin-1; and we have shown that transfected PP1α specifically co-immunoprecipitates with a polycystin-1 C-tail construct. To determine whether PP1α dephosphorylates polycystin-1, a PKA-phosphorylated GST-polycystin-1 fusion protein was shown to be dephosphorylated by PP1α but not by PP2B (calcineurin). Mutations within the PP1-binding motif of polycystin-1, including an autosomal dominant polycystic kidney disease (ADPKD)-associated mutation, significantly reduced PP1α-mediated dephosphorylation of polycystin-1. The results suggest that polycystin-1 forms a holoenzyme complex with PP1α via a conserved PP1-binding motif within the polycystin-1 C-tail, and that PKA-phosphorylated polycystin-1 serves as a substrate for the holoenzyme

Biogenesis of mammalian microRNAs by a non-canonical processing pathway

Canonical microRNA biogenesis requires the Microprocessor components, Drosha and DGCR8, to generate precursor-miRNA, and Dicer to form mature miRNA. The Microprocessor is not required for processing of some miRNAs, including mirtrons, in which spliceosome-excised introns are direct Dicer substrates. In this study, we examine the processing of putative human mirtrons and demonstrate that although some are splicing-dependent, as expected, the predicted mirtrons, miR-1225 and miR-1228, are produced in the absence of splicing. Remarkably, knockout cell lines and knockdown experiments demonstrated that biogenesis of these splicing-independent mirtron-like miRNAs, termed ‘simtrons’, does not require the canonical miRNA biogenesis components, DGCR8, Dicer, Exportin-5 or Argonaute 2. However, simtron biogenesis was reduced by expression of a dominant negative form of Drosha. Simtrons are bound by Drosha and processed in vitro in a Drosha-dependent manner. Both simtrons and mirtrons function in silencing of target transcripts and are found in the RISC complex as demonstrated by their interaction with Argonaute proteins. These findings reveal a non-canonical miRNA biogenesis pathway that can produce functional regulatory RNAs

Polycystic kidney diseases: From molecular discoveries to targeted therapeutic strategies

Polycystic kidney diseases (PKDs) represent a large group of progressive renal disorders characterized by the development of renal cysts leading to end-stage renal disease. Enormous strides have been made in understanding the pathogenesis of PKDs and the development of new therapies. Studies of autosomal dominant and recessive polycystic kidney diseases converge on molecular mechanisms of cystogenesis, including ciliary abnormalities and intracellular calcium dysregulation, ultimately leading to increased proliferation, apoptosis and dedifferentiation. Here we review the pathobiology of PKD, highlighting recent progress in elucidating common molecular pathways of cystogenesis. We discuss available models and challenges for therapeutic discovery as well as summarize the results from preclinical experimental treatments targeting key disease-specific pathways

Pathway-Based Analysis of a Melanoma Genome-Wide Association Study: Analysis of Genes Related to Tumour-Immunosuppression

Systemic immunosuppression is a risk factor for melanoma, and sunburn-induced immunosuppression is thought to be causal. Genes in immunosuppression pathways are therefore candidate melanoma-susceptibility genes. If variants within these genes individually have a small effect on disease risk, the association may be undetected in genome-wide association (GWA) studies due to low power to reach a high significance level. Pathway-based approaches have been suggested as a method of incorporating a priori knowledge into the analysis of GWA studies. In this study, the association of 1113 single nucleotide polymorphisms (SNPs) in 43 genes (39 genomic regions) related to immunosuppression have been analysed using a gene-set approach in 1539 melanoma cases and 3917 controls from the GenoMEL consortium GWA study. The association between melanoma susceptibility and the whole set of tumour-immunosuppression genes, and also predefined functional subgroups of genes, was considered. The analysis was based on a measure formed by summing the evidence from the most significant SNP in each gene, and significance was evaluated empirically by case-control label permutation. An association was found between melanoma and the complete set of genes (pemp = 0.002), as well as the subgroups related to the generation of tolerogenic dendritic cells (pemp = 0.006) and secretion of suppressive factors (pemp = 0.0004), thus providing preliminary evidence of involvement of tumour-immunosuppression gene polymorphisms in melanoma susceptibility. The analysis was repeated on a second phase of the GenoMEL study, which showed no evidence of an association. As one of the first attempts to replicate a pathway-level association, our results suggest that low power and heterogeneity may present challenges