Induction of adenocarcinoma from hamster pancreatic islet cells treated with N-nitrosobis(2-oxopropyl)amine in vitro

Our previous studies in the hamster pancreatic cancer model have indicated that pancreatic ductal adenocarcinomas derive not only from ductal/ductular cells but also from islets. To verify the presence of carcinogen-responsive cells within islets, we tested the effect of the pancreatic carcinogen N-nitrosobis(2-oxopropyl)amine (BOP) on recently established continuous hamster pancreatic islet culture. Isolated pure pancreatic islets of hamsters were treated in vitro with BOP at a concentration of 0.25 mM three times a week for 19 weeks. Each treatment week was designed as a stage. The growth of these cells, designated KL5B, was compared with untreated cultured islets, designated KL5N. As in our previous study, between 14 and 21 days of culture, exocrine and intermediary cells developed within both KL5N and KL5B islets, which were then replaced by undifferentiated cells. No differences were found in the growth patterns of KL5N and KL5B until stage 4, when KL5B cells showed accelerated cell growth and cell pleomorphism, which increased gradually at later stages of treatment. Anchorage-independent and in vivo growth did not appear until stage 19. Mutation of c-Ki-ras at codon 12 (GGT→GAT) was detected in KL5B cells but not in KL5N cells. In vivo KL5B cells formed anaplastic invasive cancer with areas of glandular formation, overexpressed TGF-α and EGFR, expressed cytokeratin, vimentin, laminin and α-1 antitrypsin and reacted strongly with L-phytohemagglutinin and tomato lectin. Some cells within islets are responsive to the carcinogenic effects of BOP. Whether these cells represent islet cell precursors (stem cells) or malignant transdifferentiated islet cells remains to be see

De novo design of proteins housing excitonically coupled chlorophyll special pairs

Natural photosystems couple light harvesting to charge separation using a ‘special pair’ of chlorophyll molecules that accepts excitation energy from the antenna and initiates an electron-transfer cascade. To investigate the photophysics of special pairs independently of the complexities of native photosynthetic proteins, and as a first step toward creating synthetic photosystems for new energy conversion technologies, we designed C2-symmetric proteins that hold two chlorophyll molecules in closely juxtaposed arrangements. X-ray crystallography confirmed that one designed protein binds two chlorophylls in the same orientation as native special pairs, whereas a second designed protein positions them in a previously unseen geometry. Spectroscopy revealed that the chlorophylls are excitonically coupled, and fluorescence lifetime imaging demonstrated energy transfer. The cryo-electron microscopy structure of a designed 24-chlorophyll octahedral nanocage with a special pair on each edge closely matched the design model. The results suggest that the de novo design of artificial photosynthetic systems is within reach of current computational methods

Choline kinase inhibition induces exacerbated endoplasmic reticulum stress and triggers apoptosis via CHOP in cancer cells

11 p.-6 fig.Endoplasmic reticulum (ER) is a central organelle in eukaryotic cells that regulates protein synthesis and maturation.Perturbation of ER functions leads to ER stress, which has been previously associated with a broad variety of diseases. ER stress is generally regarded as compensatory, but prolonged ER stress has been involved in apoptosis induced by several cytotoxic agents. Choline kinase a (ChoKa), the first enzyme in the Kennedy pathway, is responsible for the generation of phosphorylcholine (PCho) that ultimately renders phosphatidylcholine. ChoKa overexpression and high PCho levels have been detected in several cancer types. Inhibition of ChoKa has demonstrated antiproliferative and antitumor properties; however, the mechanisms underlying these activities remain poorly understood. Here, we demonstrate that ChoKa inhibitors (ChoKIs), MN58b and RSM932A, induce cell death in cancer cells (T47D,MCF7, MDA-MB231, SW620 and H460), through the prolonged activation of ER stress response. Evidence of ChoKIs-induced ER stress includes enhanced production of glucose-regulated protein, 78 kDa (GRP78), protein disulfide isomerase, IRE1a, CHOP, CCAAT/enhancer-binding protein beta (C/EBPb) and TRB3. Although partial reduction of ChoKa levels by small interfering RNA was not sufficient to increase the production of ER stress proteins, silencing of ChoKa levels also show a decrease in CHOP overproduction induced by ChoKIs, which suggests that ER stress induction is due to a change in ChoKa protein folding after binding to ChoKIs. Silencing of CHOP expression leads to a reduction in C/EBPb, ATF3 and GRP78 protein levels and abrogates apoptosis in tumor cells after treatment with ChoKIs, suggesting that CHOP maintains ER stress responses and triggers the pro-apoptotic signal. Consistent with the differential effect of ChoKIs in cancer and primary cells previously described, ChoKIs only promoted a transient and moderated ER stress response in the non-tumorogenic cells MCF10A. In conclusion, pharmacological inhibition of ChoKa induces cancer cell death through a mechanism that involves the activation of exaggerated and persistent ER stress supported by CHOP overproduction.This work has been funded by the following grants:Comunidad de Madrid (S2010/BMD-2326), Ministerio de Economía y Competitividad (SAF2011-29699, RD06-0020-0016 and RD12/0036/0019) and EU no.259737. ESL is a Sara Borell fellow under the program ISCIII/MICINN.Peer reviewe

Phosphorylation of SRSF1 by SRPK1 regulates alternative splicing of tumor-related Rac1b

The pre-messenger RNA of the majority of human genes can generate various transcripts through alternative splicing, and different tissues or disease states show specific patterns of splicing variants. These patterns depend on the relative concentrations of the splicing factors present in the cell nucleus, either as a consequence of their expression levels or of post-translational modifications such as protein phosphorylation, which are determined by signal transduction pathways. Here we analyzed the contribution of protein kinases to the regulation of alternative splicing variant Rac1b that is overexpressed in certain tumor types. In colorectal cells we found that depletion of AKT2, AKT3, GSK3β and SRPK1 significantly decreased endogenous Rac1b levels. Whereas knockdown of AKT2 and AKT3 affected only Rac1b protein levels suggesting a post-splicing effect, the depletion of GSK3β or SRPK1 decreased Rac1b alternative splicing, an effect mediated through changes in splicing factor SRSF1. In particular, the knockdown of SRPK1 or inhibition of its catalytic activity reduced phosphorylation and subsequent translocation of SRSF1 to the nucleus, limiting its availability to promote the inclusion of alternative exon 3b into the Rac1 pre-mRNA. Altogether, the data identify SRSF1 as a prime regulator of Rac1b expression in colorectal cells and provide further mechanistic insights into how the regulation of alternative splicing events by protein kinases can contribute to sustain tumor cell survival

Effect of island overlap on edge localized mode suppression by resonant magnetic perturbations in DIII-D

Recent DIII-D [J. L. Luxon , Nucl. Fusion 43, 1813 (2003)] experiments show a correlation between the extent of overlap of magnetic islands induced in the edge plasma by perturbation coils and complete suppression of Type-I edge localized modes (ELMs) in plasmas with ITER-like electron pedestal collisionality nu(e)*similar to 0.1, flux surface shape and low edge safety factor (q(95)approximate to 3.6). With fixed amplitude n=3 resonant magnetic perturbation (RMP), ELM suppression is obtained only in a finite window in the edge safety factor (q(95)) consistent with maximizing the resonant component of the applied helical field. ELM suppression is obtained over an increasing range of q(95) by either increasing the n=3 RMP strength, or by adding n=1 perturbations to "fill in" gaps between islands across the edge plasma. The suppression of Type-I ELMs correlates with a minimum width of the edge region having magnetic islands with Chirikov parameter >1.0, based on vacuum calculations of RMP mode components excluding the plasma response or rotational shielding. The fraction of vacuum magnetic field lines that are lost from the plasma, with connection length to the divertor targets comparable to an electron-ion collisional mean free path, increases throughout the island overlap region in the ELM suppressed case compared with the ELMing case. (C) 2008 American Institute of Physics