MOUSE ANTI HUMAN CD3 APC

Purpose:  The majority of genomic alterations causing intratumoral heterogeneity (ITH) in colorectal cancer (CRC) are thought to arise during early stages of carcinogenesis as a burst but only after truncal mutations in APC have expanded a single founder clone. We have investigated if the initial source of ITH is consequent to multiple independent lineages derived from different crypts harboring distinct truncal APC and driver KRAS mutations, thus challenging the prevailing monoclonal monocryptal model.

Experimental design: High-depth next-generation sequencing and SNP arrays were performed in whole lesion extracts of 37 FAP colorectal adenomas. Also, ultra-sensitive genotyping of hotspot mutations of APC and KRAS was performed using nanofluidic PCRs in matched bulk biopsies (n=59) and crypts (n=591) from 18 adenomas and 7 carcinomas and adjacent normal tissues.

Results: Multiple co-occurring truncal APC and driver KRAS alterations were uncovered in whole lesion extracts from adenomas and subsequently confirmed to belong to multiple clones. Ultra-sensitive genotyping of bulk biopsies and crypts revealed novel undetected APC mutations that were prominent among carcinomas, whereas abundant wild-type APC crypts were detected in adenomas. KRAS mutational heterogeneity within crypts was evident in both adenomas and carcinomas with a higher degree of concordance between biopsy and crypt genotyping in carcinomas. Non-random heterogeneity among crypts was also observed.

Type 1 diabetes (T1D) results from T-cell mediated autoimmune destruction of pancreatic β cells. Effector T-cell responses emerge early in disease development and expand as disease progresses. Following β cell destruction, a long-lived T-cell memory is generated that represents a barrier to islet transplantation and other cellular insulin-replacement therapies. Development of effective immunotherapies that control or ablate β cell destructive effector and memory T cell responses has the potential to prevent disease progression and recurrence. Targeting antigen expression to antigen-presenting cells inactivates cognate CD8(+) effector and memory T-cell responses and has therapeutic potential. Here we investigated this in the context of insulin-specific responses in the non-obese diabetic mouse where genetic immune tolerance defects could impact on therapeutic tolerance induction. Insulin-specific CD8(+) memory T cells transferred to mice expressing proinsulin in antigen-presenting cells proliferated in response to transgenically-expressed proinsulin and the majority were rapidly deleted. A small proportion of transferred insulin-specific Tmem remained undeleted and these were antigen-unresponsive, exhibited reduced TCR expression and H-2K(d)/insB15-23 tetramer binding and expressed co-inhibitory molecules. Expression of proinsulin in antigen-presenting cells also abolished the diabetogenic capacity of CD8(+) effector T cells. Therefore, destructive insulin-specific CD8(+) T cells are effectively inactivated by enforced proinsulin expression despite tolerance defects that exist in diabetes-prone NOD mice. These findings have important implications in developing immunotherapeutic approaches to T1D and other T cell-mediated autoimmune diseases.Immunology and Cell Biology accepted article preview online, 14 June 2017. doi:10.1038/icb.2017.48. - Source :PubMed

The APC/C-Cdh1 ubiquitin-ligase complex targets cell cycle regulators for proteosomal degradation and helps prevent tumor development and accumulation of chromosomal aberrations. Replication stress has been proposed to be the main driver of genomic instability in the absence of Cdh1, but the real contribution of APC/C-Cdh1 to efficient replication, especially in normal cells, remains unclear. Here we show that, in primary MEFs, acute depletion or permanent ablation of Cdh1 slowed down replication fork movement and increased origin activity. Partial inhibition of origin firing does not accelerate replication forks, suggesting that fork progression is intrinsically limited in the absence of Cdh1. Moreover, exogenous supply of nucleotide precursors, or ectopic overexpression of RRM2, the regulatory subunit of Ribonucleotide Reductase, restore replication efficiency, indicating that dNTP availability could be impaired upon Cdh1 loss. Indeed, we found reduced dNTP levels in Cdh1-deficient MEFs. Importantly, DNA breakage is also significantly alleviated by increasing intracellular dNTP pools, strongly suggesting that genomic instability is the result of aberrant replication. These observations highlight the relevance of APC/C-Cdh1 activity during G1 to ensure an adequate supply of dNTPs to the replisome, prevent replication stress and the resulting chromosomal breaks and, ultimately, suppress tumorigenesis.Oncogene advance online publication, 12 June 2017; doi:10.1038/onc.2017.186. - Source :PubMed

The role of adenomatous polyposis coli (APC) gene in mitosis might be critical for regulation of genomic stability and chromosome segregation. APC gene mutations have been associated to have a role in colon cancer and since gastric and colon tumors share some common genetic lesions, it is relevant to investigate the role of APC tumor suppressor gene in gastric cancer. - Source :PubMed

As part of a program to design small molecules that bind proteins, we require cyclic peptides (or peptidomimetics) that are severely constrained such that they adopt one predominant conformation in solution. This paper describes syntheses of the 13-membered cyclic tetrapeptides 1 containing aminopyrrolidine carboxyl (APC) residues. A linear precursor was prepared and used to determine optimal conditions for cyclization of that substrate. A special linker was prepared to enable cyclization of similar linear peptidomimetics on a solid phase, and the solution-phase cyclization conditions were shown to be appropriate for this too. Stereochemical variations were then used to determine the ideal APC configuration for cyclization of the linear precursors (on a solid phase, using the conditions identified previously). Consequently, a series of compounds were prepared that are representative of compounds 1. Conformational studies of representative compounds in DMSO solution were performed primarily using (i) NOE studies, (ii) quenched molecular dynamics simulations using no constraints from experiment, and (iii) MacroModel calculations with NMR constraints. All three strategies converged to the same conclusion: the backbone of molecules based on 1 tends to adopt one preferential conformation in solution and that conformation can be predicted from the stereochemistries of the α-amino acids involved. - Source :PubMed