Switching the stereochemical outcome of 6-endo-trig cyclizations; Synthesis of 2,6-Cis-6-substituted 4-oxopipecolic acids

A base-mediated 6-endo-trig cyclization of readily accessible enone-derived α-amino acids has been developed for the direct synthesis of novel 2,6-cis-6- substituted-4-oxo-L-pipecolic acids. A range of aliphatic and aryl side chains were tolerated by this mild procedure to give the target compounds in good overall yields. Molecular modeling of the 6-endo-trig cyclization allowed some insight as to how these compounds were formed, with the enolate intermediate generated via an equilibrium process, followed by irreversible tautomerization/neutralization providing the driving force for product formation. Stereoselective reduction and deprotection of the resulting 2,6-cis-6-substituted 4-oxo-L-pipecolic acids to the corresponding 4-hydroxy-L-pipecolic acids was also performed

Relevance of cyclin D1b expression and CCND1 polymorphism in the pathogenesis of multiple myeloma and mantle cell lymphoma

BACKGROUND: The CCND1 gene generates two mRNAs (cyclin D1a and D1b) through an alternative splicing at the site of a common A/G polymorphism. Cyclin D1a and b proteins differ in their C-terminus, a region involved in protein degradation and sub-cellular localization. Recent data have suggested that cyclin D1b could be a nuclear oncogene. The presence of cyclin D1b mRNA and protein has been studied in two hemopathies in which cyclin D1 could be present: multiple myeloma (MM) and mantle cell lymphoma (MCL). The A/G polymorphism of CCND1 has also been verified in a series of patients. METHODS: The expression of cyclin D1 mRNA isoforms has been studied by real-time quantitative PCR; protein isoforms expression, localization and degradation by western blotting. The CCND1 polymorphism was analyzed after sequencing genomic DNA. RESULTS: Cyclin D1 mRNA isoforms a and b were expressed in mantle cell lymphoma (MCL) and multiple myeloma (MM). Cyclin D1b proteins were present in MCL, rarely in MM. Importantly, both protein isoforms localized the nuclear and cytoplasmic compartments. They displayed the same short half-life. Thus, the two properties of cyclin D1b recognized as necessary for its transforming activity are missing in MCL. Moreover, CCND1 polymorphism at the exon/intron boundary had no influence on splicing regulation in MCL cells. CONCLUSION: Our results support the notion that cyclin D1b is not crucial for the pathogenesis of MCL and MM

3′UTR-Mediated Gene Silencing of the Mixed Lineage Leukemia (MLL) Gene

Translocations involving the Mixed Lineage Leukemia (MLL) gene generate in-frame fusions of MLL with more than 50 different partner genes (PGs). Common to all MLL translocations is the exchange not only of coding regions, but also of MLL and PG 3′-untranslated regions (3′UTRs). As a result, the MLL-PG fusion is normally highly expressed and considered the main driver of leukemia development, whereas the function of the PG-MLL fusions in leukemic disease is unclear. As 3′UTRs have been recognized as determinant regions for regulation of gene expression, we hypothesized that loss of the MLL 3′UTR could have a role in generating high MLL-PG levels and leukemia development. Here, we first tested the MLL-PG and PG-MLL mRNA levels in different leukemic cells and tumours and uncovered differential expression that indicates strong repression by the MLL-3′UTR. Reporter assays confirmed that the 3′UTR of MLL, but not of its main PGs, harbours a region that imposes a strong gene silencing effect. Gene suppression by the MLL 3′UTR was largely microRNA independent and did not affect mRNA stability, but inhibited transcription. This effect can at least partially be attributed to a tighter interaction of the MLL 3′UTR with RNA polymerase II than PG 3′UTRs, affecting its phosphorylation state. Altogether, our findings indicate that MLL translocations relieve oncogenic MLL-PG fusions from the repressive MLL 3′UTR, contributing to higher activity of these genes and leukaemia development

c-Abl phosphorylation of ΔNp63α is critical for cell viability

The p53 family member p63 has been shown to be critical for growth, proliferation and chemosensitivity. Here we demonstrate that the c-Abl tyrosine kinase phosphorylates the widely expressed ΔNp63α isoform and identify multiple sites by mass spectrometry in vitro and in vivo. Phopshorylation by c-Abl results in greater protein stability of both ectopically expressed and endogenous ΔNp63α. c-Abl phosphorylation of ΔNp63α induces its binding to Yes-associated protein (YAP) and silencing of YAP by siRNA reduces the c-Abl-induced increase of ΔNp63α levels. We further show that cisplatin induces c-Abl phosphorylation of ΔNp63α and its binding to YAP. Overexpression of ΔNp63α, but not the c-Abl phosphosites mutant, protects cells from cisplatin treatment. Finally, we demonstrate the rescue of p63 siRNA-mediated loss of viability with p63siRNA insensitive construct of ΔNp63α but not the phosphosites mutant. These results demonstrate that c-Abl phosphorylation of ΔNp63α regulates its protein stability, by inducing binding of YAP, and is critical for cell viability

Toxicity Testing in the 21st Century: Defining New Risk Assessment Approaches Based on Perturbation of Intracellular Toxicity Pathways

The approaches to quantitatively assessing the health risks of chemical exposure have not changed appreciably in the past 50 to 80 years, the focus remaining on high-dose studies that measure adverse outcomes in homogeneous animal populations. This expensive, low-throughput approach relies on conservative extrapolations to relate animal studies to much lower-dose human exposures and is of questionable relevance to predicting risks to humans at their typical low exposures. It makes little use of a mechanistic understanding of the mode of action by which chemicals perturb biological processes in human cells and tissues. An alternative vision, proposed by the U.S. National Research Council (NRC) report Toxicity Testing in the 21st Century: A Vision and a Strategy, called for moving away from traditional high-dose animal studies to an approach based on perturbation of cellular responses using well-designed in vitro assays. Central to this vision are (a) “toxicity pathways” (the innate cellular pathways that may be perturbed by chemicals) and (b) the determination of chemical concentration ranges where those perturbations are likely to be excessive, thereby leading to adverse health effects if present for a prolonged duration in an intact organism. In this paper we briefly review the original NRC report and responses to that report over the past 3 years, and discuss how the change in testing might be achieved in the U.S. and in the European Union (EU). EU initiatives in developing alternatives to animal testing of cosmetic ingredients have run very much in parallel with the NRC report. Moving from current practice to the NRC vision would require using prototype toxicity pathways to develop case studies showing the new vision in action. In this vein, we also discuss how the proposed strategy for toxicity testing might be applied to the toxicity pathways associated with DNA damage and repair

c-Abl downregulates the slow phase of double-strand break repair

c-Abl tyrosine kinase is activated by agents that induce double-strand DNA breaks (DSBs) and interacts with key components of the DNA damage response and of the DSB repair machinery. However, the functional significance of c-Abl in these processes, remained unclear. In this study, we demonstrate, using comet assay and pulsed-field gel electrophoresis, that c-Abl inhibited the repair of DSBs induced by ionizing radiation, particularly during the second and slow phase of DSB repair. Pharmacological inhibition of c-Abl and c-Abl depletion by siRNA-mediated knockdown resulted in higher DSB rejoining. c-Abl null MEFs exhibited higher DSB rejoining compared with cells reconstituted for c-Abl expression. Abrogation of c-Abl kinase activation resulted in higher H2AX phosphorylation levels and higher numbers of post-irradiation γH2AX foci, consistent with a role of c-Abl in DSB repair regulation. In conjunction with these findings, transient abrogation of c-Abl activity resulted in increased cellular radioresistance. Our findings suggest a novel function for c-Abl in inhibition of the slow phase of DSB repair

The Anaphase-Promoting Complex or Cyclosome Supports Cell Survival in Response to Endoplasmic Reticulum Stress

The anaphase-promoting complex or cyclosome (APC/C) is a multi-subunit ubiquitin ligase that regulates exit from mitosis and G1 phase of the cell cycle. Although the regulation and function of APC/CCdh1 in the unperturbed cell cycle is well studied, little is known of its role in non-genotoxic stress responses. Here, we demonstrate the role of APC/CCdh1 (APC/C activated by Cdh1 protein) in cellular protection from endoplasmic reticulum (ER) stress. Activation of APC/CCdh1 under ER stress conditions is evidenced by Cdh1-dependent degradation of its substrates. Importantly, the activity of APC/CCdh1 maintains the ER stress checkpoint, as depletion of Cdh1 by RNAi impairs cell cycle arrest and accelerates cell death following ER stress. Our findings identify APC/CCdh1 as a regulator of cell cycle checkpoint and cell survival in response to proteotoxic insults

Allelic imbalance at 1p36 may predict prognosis of chemoradiation therapy for bladder preservation in patients with invasive bladder cancer

Invasive bladder cancers have been treated by irradiation combined with cis- platinum (CDDP) as a bladder preservative option. The aim of this study was to find a marker for predicting patient outcome as well as clinical response after chemoradiation therapy (CRT) by investigating allelic loss of apoptosis-related genes. A total of 67 transitional cell carcinomas of the bladder treated by CRT (median dose: 32.4 Gy of radiation and 232 mg of CDDP) were studied. We investigated allelic imbalances at 14 loci on chromosomes 17p13 and 1p36 including the p53 and p73 gene regions by fluorescent multiplex PCR based on DNA from paraffin-embedded tumour specimens and peripheral blood. The response to CRT was clinical response (CR) in 21 patients (31%), partial response (PR) in 31 (46%), and no change(NC) in 15 (22%). There was no statistical correlation between treatment response and clinical parameters, such as tumour grade, stage, radiation dose, or CDDP dose. The frequencies of allelic imbalance for TP53 and TP73 were 21 and 56%, respectively; neither was correlated with clinical treatment response and tumour stage or grade. There was no statistical correlation between treatment response and allelic imbalance at the other 12 loci. We found a significant correlation between cancer-specific survival and an imbalance of D1S243 (P=0.0482) or TP73 (P=0.0013) using a Log-rank test, although other loci including TP53 did not correlate with survival (P=0.4529 Multivariate analysis showed performance status (P=0.0047), recurrence (P=0.0017), and radiation doses (P=0.0468) were independent predictive factors for cancer-specific survival. However, an allelic imbalance of TP73 was the most remarkable independent predictive factor of poor patient survival (P=0.0002, risk ratio: 3382). Our results suggest that the allelic loss of the p73 gene predicts a clinical outcome of locally advanced bladder cancer when treated by CRT