ATM is not required in somatic hypermutation of VH,but is involved in the introduction of mutations in the switch mu region

Class switch recombination (CSR) and somatic hypermutation (SHM) are mechanistically related processes that share common key factors such as activation-induced cytidine deaminase. We have previously shown a role for ATM (mutated in ataxia-telangiectasia) in CSR. In this paper we show that the frequency, distribution, and nature of base pair substitutions in the Ig variable (V) heavy chain genes in ataxia-telangiectasia patients are largely similar to those in normal donors, suggesting a normal SHM process. Characterization of the third complementarity-determining region in B cells from ataxia-telangiectasia patients also shows a normal V(D)J recombination process. SHM-like mutations could be identified in the switch (S) mu region (up to several hundred base pairs upstream of the S mu -S(alpha) breakpoints) in normal in vivo switched human B cells. In the absence of ATM, mutations can still be found in this region, but at less than half the frequency of that in normal donors. The latter mutations are mainly due to transitions (86% compared with 58% in controls) and are biased to A or T nucleotides. An ATM-dependent mechanism, different from that generating SHM in V genes, is therefore likely to be involved in introducing SHM-like mutations in the S region. ATM may thus be one of the factors that is not shared by the CSR and SHM processes.     

Vaccination with a Melan-A peptide selects an oligoclonal T cell population with increased functional avidity and tumor reactivity

Both the underlying molecular mechanisms and the kinetics of TCR repertoire selection following vaccination against tumor Ags in humans have remained largely unexplored. To gain insight into these questions, we performed a functional and structural longitudinal analysis of the TCR of circulating CD8(+) T cells specific for the HLA-A2-restricted immunodominant epitope from the melanocyte differentiation Ag Melan-A in a melanoma patient who developed a vigorous and sustained Ag-specific T cell response following vaccination with the corresponding synthetic peptide. We observed an increase in functional avidity of Ag recognition and in tumor reactivity in the postimmune Melan-A-specific populations as compared with the preimmune blood sample. Improved Ag recognition correlated with an increase in the t(1/2) of peptide/MHC interaction with the TCR as assessed by kinetic analysis of A2/Melan-A peptide multimer staining decay. Ex vivo analysis of the clonal composition of Melan-A-specific CD8(+) T cells at different time points during vaccination revealed that the response was the result of asynchronous expansion of several distinct T cell clones. Some of these T cell clones were also identified at a metastatic tumor site. Collectively, these data show that tumor peptide-driven immune stimulation leads to the selection of high-avidity T cell clones of increased tumor reactivity that independently evolve within oligoclonal populations.     

Optimization and evaluation of T7 based RNA linear amplification protocols for cDNA microarray analysis

Background  

T7 based linear amplification of RNA is used to obtain sufficient antisense RNA for microarray expression profiling. We optimized and systematically evaluated the fidelity and reproducibility of different amplification protocols using total RNA obtained from primary human breast carcinomas and high-density cDNA microarrays.     

Canine Mammary Tumours Are Affected by Frequent Copy Number Aberrations,including Amplification of MYC and Loss of PTEN

BackgroundCopy number aberrations frequently occur during the development of many cancers. Such events affect dosage of involved genes and may cause further genomic instability and progression of cancer. In this survey, canine SNP microarrays were used to study 117 canine mammary tumours from 69 dogs.ResultsWe found a high occurrence of copy number aberrations in canine mammary tumours, losses being more frequent than gains. Increased frequency of aberrations and loss of heterozygosity were positively correlated with increased malignancy in terms of histopathological diagnosis. One of the most highly recurrently amplified regions harbored the MYC gene. PTEN was located to a frequently lost region and also homozygously deleted in five tumours. Thus, deregulation of these genes due to copy number aberrations appears to be an important event in canine mammary tumour development. Other potential contributors to canine mammary tumour pathogenesis are COL9A3, INPP5A, CYP2E1 and RB1. The present study also shows that a more detailed analysis of chromosomal aberrations associated with histopathological parameters may aid in identifying specific genes associated with canine mammary tumour progression.ConclusionsThe high frequency of copy number aberrations is a prominent feature of canine mammary tumours as seen in other canine and human cancers. Our findings share several features with corresponding studies in human breast tumours and strengthen the dog as a suitable model organism for this disease.     

The Sub-Cellular Localization of WRAP53 Has Prognostic Impact in Breast Cancer

WRAP53 protein controls intracellular trafficking of DNA repair proteins, the telomerase enzyme, and splicing factors. Functional loss of the protein has been linked to carcinogenesis, premature aging and neurodegeneration. The aim of this study was to investigate the prognostic significance of WRAP53 protein expression in breast cancer. A tissue microarray was constructed from primary breast tumors and immunostained by a polyclonal WRAP53 antibody to assess the protein expression pattern. Two different patient cohorts with long term follow-up were studied; a test- and a validation set of 154 and 668 breast tumor samples respectively. Breast cancer patients with tumor cells lacking the expression of WRAP53 in the nucleus had a significantly poorer outcome compared to patients with tumor cells expressing this protein in the nuclei (HR = 1.95, 95%CI = 1.09–3.51, p = 0.025). Nuclear localization of WRAP53 was further shown to be an independent marker of prognosis in multivariate analysis (HR = 2.57, 95%CI = 1.27–5.19, p = 0.008), and also significantly associated with better outcome in patients with TP53 mutation. Here we show that the sub-cellular localization of the WRAP53 protein has a significant impact on breast cancer survival, and thus has a potential as a clinical marker in diagnostics and treatment.     

Macrocyclic BACE-1 inhibitors acutely reduce Aβ in brain after po application

A series of macrocyclic peptidic BACE-1 inhibitors was designed. While potency on BACE-1 was rather high, the first set of compounds showed poor brain permeation and high efflux in the MDRI–MDCK assay. The replacement of the secondary benzylamino group with a phenylcyclopropylamino group maintained potency on BACE-1, while P-glycoprotein-mediated efflux was significantly reduced and brain permeation improved. Several compounds from this series demonstrated acute reduction of Aβ in human APP-wildtype transgenic (APP51/16) mice after oral administration.     

Synthesis of fluorinated C-mannopeptides as sialyl Lewisx mimics for E- and P-selectin inhibition

The synthesis of fluorinated C-mannopeptides and their evaluation as E- and P-selectin inhibitors is described. These molecules are difluorinated analogues of CH₂-glycopeptides already reported to act as sLe^x mimics. The α and β anomers of these CF₂-glycopeptides have been prepared, as well as their 1-hydroxy analogues which were present in solution as an equilibrium mixture of α- and β-pyranose and α- and β-furanose forms. These molecules showed inhibitory activities comparable to their CH₂ counterparts with a moderate influence of the pseudo-anomeric center configuration.