Shed HER2 extracellular domain in HER2‐mediated tumor growth and in trastuzumab susceptibility

The question of the serum HER2 extracellular domain (HER2/ECD) measurement for prediction of response to the anti‐HER2 antibody Trastuzumab is still an open and current matter of clinical debate. To elucidate the involvement of shed HER2/ECD in HER2‐driven tumor progression and in guiding therapy of individual patients, we examined biological effects exerted by elevated HER2/ECD in cancer growth and in response to Trastuzumab. To this purpose SKOV3 tumor cells were stably transfected to release a recombinant HER2/ECD molecule (rECD). Transfectants releasing high levels of 110‐kDa rECD, identical in size to native HER2/ECD (nECD), grew significantly slower than did controls, which constitutively released only basal levels of nECD. While transmembrane HER2 and HER1 were expressed at equal levels by both controls and transfected cells, activation of these molecules and of downstream ERK2 and Akt was significantly reduced only in rECD transfectants. Surface plasmon resonance analysis revealed heterodimerization of the rECD with HER1, ‐2, and ‐3. In cell growth bioassays in vitro, shed HER2 significantly blocked HER2‐driven tumor cell proliferation. In mice, high levels of circulating rECD significantly impaired HER2‐driven SKOV3 tumor growth but not that of HER2‐negative tumor cells. In vitro and in mice, Trastuzumab significantly inhibited tumor growth due to the rECD‐facilitated accumulation of the antibody on tumor cells. Globally our findings sustain the biological relevance of elevated HER2/ECD levels in the outcome of HER2‐disease and in the susceptibility to Trastuzumab‐based therapy. J. Cell. Physiol. 225: 256–265, 2010. © 2010 Wiley‐Liss, Inc.     

W7FW14F apomyoglobin amyloid aggregates-mediated apoptosis is due to oxidative stress and AKT inactivation caused by Ras and Rac

We have previously reported that addition of prefibrillar aggregates (PFAs) derived from W7FW14F apomyoglobin mutant to NIH-3T3 cells affects their viability. In this article, we have found that cytotoxicity induced by PFAs in NIH 3T3 and SH-SY5Y human neuroblastoma cells was due to early activation of apoptotic cell death dependent from a caspase-3- and -9-mediated mitochondrial pathway. A time-dependent increase of intracellular ROS and an about twofold decrease of mitochondrial localization of scavenger protein MnSOD was found. The use of the anti-oxidant agent N-acetyl-cysteine (NAC) antagonized both the increase of intracellular ROS and apoptosis induced by PFAs. PFAs caused an about 60% increase of the activity of both Ras and Erk-1/2 at 30 and 45 min while they were restored to basal levels at later time points. This effect was paralleled by a time-dependent decrease of the activity of the survival enzyme Akt. Effects similar to those on Ras activity were also recorded on the activity of the stress involved small GTP binding protein Rac that was about 75% increased after 30 min but resumed to basal levels at later time points. This effect was paralleled by a time-dependent activation of p38 kinase activity and HSP-70 expression. The use of both the ras farnesyltransferase inhibitor tipifarnib and the Rac geranyl-geranyltransferase GGTI-298, but not of the MEK-1 inhibitor U0126 partially antagonized the effects of PFAs on apoptosis occurrence. On the other hand, the PI3K/Akt inhibitor LY 294002 potentiated apoptosis induced by PFAs. Our results indicate a role for Ras and Rac in the induction of both intracellular ROS increased levels and apoptosis mediated by PFAs and disclose a new scenario of intervention in neurodegenerative diseases. J. Cell. Physiol. 221: 412–423, 2009. © 2009 Wiley-Liss, Inc.     

Novel tacrine–benzofuran hybrids as potential multi-target drug candidates for the treatment of Alzheimer’s Disease

Abstract

Pursuing the widespread interest on multi-target drugs to combat Alzheimer´s disease (AD), a new series of hybrids was designed and developed based on the repositioning of the well-known acetylcholinesterase (AChE) inhibitor, tacrine (TAC), by its coupling to benzofuran (BF) derivatives. The BF framework aims to endow the conjugate molecules with ability for inhibition of AChE (bimodal way) and of amyloid-beta peptide aggregation, besides providing metal (Fe, Cu) chelating ability and concomitant extra anti-oxidant activity, for the hybrids with hydroxyl substitution. The new TAC-BF conjugates showed very good activity for AChE inhibition (sub-micromolar range) and good capacity for the inhibition of self- and Cu-mediated Aβ aggregation, with dependence on the linker size and substituent groups of each main moiety. Neuroprotective effects were also found for the compounds through viability assays of neuroblastoma cells, after Aβ_1-42 induced toxicity. Structure-activity relationship analysis provides insights on the best structural parameters, to take in consideration for future studies in view of potential applications in AD therapy.     

Dissecting Pin1 and phospho‐pRb regulation

The activity of the Retinoblastoma protein, the master regulator of the cell cycle, is finely regulated by phosphorylation. CDKs and cyclins are major players in phosphorylation and it has been recently discovered that the prolyl isomerase Pin1 is an essential protein that orchestrates this process. In this article, we report new findings regarding the role of Pin1 in the pRb pathway. Our data suggest that PI3K, CDKs, and the Pin1 axis have a critical role in sustaining the complete phosphorylation of pRb. Furthermore, we analyze the correlation between Pin1 and pRb phosphorylation in vivo. We show that, in human malignant glioma tissue microarrays (TMA) and in Pin1 knockout (KO) mice, there is a positive correlation between Pin1 and pRb phosphorylation. Prospectively, our findings suggest that the synergism between CDKs, Pin1, and PI3K inhibitors hold great promise for targeted pharmacological treatment of cancer patients, with the possibility of reaching high effectiveness at tolerated doses. J. Cell. Physiol. 228: 73–77, 2013. © 2012 Wiley Periodicals, Inc.     

ASB2α, an E3 Ubiquitin Ligase Specificity Subunit,Regulates Cell Spreading and Triggers Proteasomal Degradation of Filamins by Targeting the Filamin Calponin Homology 1 Domain

Filamins are actin-binding and cross-linking proteins that organize the actin cytoskeleton and anchor transmembrane proteins to the cytoskeleton and scaffold signaling pathways. During hematopoietic cell differentiation, transient expression of ASB2α, the specificity subunit of an E3-ubiquitin ligase complex, triggers acute proteasomal degradation of filamins. This led to the proposal that ASB2α regulates hematopoietic cell differentiation by modulating cell adhesion, spreading, and actin remodeling through targeted degradation of filamins. Here, we show that the calponin homology domain 1 (CH1), within the filamin A (FLNa) actin-binding domain, is the minimal fragment sufficient for ASB2α-mediated degradation. Combining an in-depth flow cytometry analysis with mutagenesis of lysine residues within CH1, we find that arginine substitution at each of a cluster of three lysines (Lys-42, Lys-43, and Lys-135) renders FLNa resistant to ASB2α-mediated degradation without altering ASB2α binding. These lysines lie within previously predicted actin-binding sites, and the ASB2α-resistant filamin mutant is defective in targeting to F-actin-rich structures in cells. However, by swapping CH1 with that of α-actinin1, which is resistant to ASB2α-mediated degradation, we generated an ASB2α-resistant chimeric FLNa with normal subcellular localization. Notably, this chimera fully rescues the impaired cell spreading induced by ASB2α expression. Our data therefore reveal ubiquitin acceptor sites in FLNa and establish that ASB2α-mediated effects on cell spreading are due to loss of filamins.     

Assessment of the genetic susceptibility of sheep to scrapie by protein misfolding cyclic amplification and comparison with experimental scrapie transmission studies

The susceptibility of sheep to scrapie is influenced mainly by the prion protein polymorphisms A136V, R154H, and Q171R/H. Here we analyzed the ability of protein misfolding cyclic amplification (PMCA) to model the genetic susceptibility of sheep to scrapie. For this purpose, we studied the efficiency of brain homogenates from sheep with different PrP genotypes to support PrP^Sc amplification by PMCA using an ARQ/ARQ scrapie inoculum. The results were then compared with those obtained in vivo using the same sheep breed, genotypes, and scrapie inoculum. Genotypes associated with susceptibility (ARQ/ARQ, ARQ/AHQ, and AHQ/ARH) were able to sustain PrP^Sc amplification in PMCA reactions, while genotypes associated with resistance to scrapie (ARQ/ARR and ARR/ARR) were unable to support the in vitro conversion. The incubation times of the experimental infection were then compared with the in vitro amplification factors. Linear regression analysis showed that the efficiency of in vitro PrP^Sc amplification of the different genotypes was indeed inversely proportional to their incubation times. Finally, the rare ARQK₁₇₆/ARQK₁₇₆ genotype, for which no in vivo data are available, was studied by PMCA. No amplification was obtained, suggesting ARQK₁₇₆/ARQK₁₇₆ as an additional genotype associated with resistance, at least to the isolate tested. Our results indicate a direct correlation between the ability of different PrP genotypes to undergo PrP^C-to-PrP^Sc conversion by PMCA and their in vivo susceptibility and point to PMCA as an alternative to transmission studies and a potential tool to test the susceptibility of numerous sheep PrP genotypes to a variety of prion sources.     

Rapid identification of Legionella spp. by MALDI-TOF MS based protein mass fingerprinting

A set of reference strains representing 38 different Legionella species were submitted to Whole Cell Mass Spectrometry (WCMS) with MALDI-TOF.The dendrogram computed from strain mass spectral patterns obtained by WCMS was compared to the phylogenetic tree obtained from macrophage infectivity potentiator (mip) sequences. The trees inferred from these two methods revealed significant homologies.Using 453 Legionella isolates previously characterized by genotyping, it was possible to create species-specific SuperSpectra, using appropriate sets of spectral masses, allowing unambiguous differentiation and identification of the most frequently isolated Legionella species. These SuperSpectra were tested for their suitability to identify Legionella strains isolated from water samples, cooling towers, potting soils and patient specimens deposited at the Swiss National Reference Centre for Legionella and previously identified by molecular methods such as mip gene sequencing.99.1% of the tested strains isolated from the environment could be correctly identified by comparison with the new SuperSpectra. The identification of Legionella spp. by MALDI-TOF MS is rapid, easy to perform and has the advantage of being time- and cost-saving, in comparison to sequence-based identification.     

Cloning and developmental expression of zebrafish pdzrn3

Pdzrn3, a member of the PDZRN/SEMCAP/LNX protein family containing a RING finger and two PDZ domains, has been implicated in myoblast and osteoblast differentiation. However, its spatio-temporal expression pattern during embryonic development has not been defined. Here, we describe the cloning and expression pattern of pdzrn3 during zebrafish development. We found that in addition to being expressed in several mesodermal structures, this gene displays specific expression in the central nervous system including rhombomere 1, ventral retina, thalamus and motor neurons, indicating a novel function during neural development. In particular, the absence of expression of pdzrn3 in the ventral retina of noi mutant fish suggests a possible role for this gene in regulating fasciculation and/or navigation of retinal ganglion cell axons.