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Colorectal cancer; Epidermal growth factor receptor (EGFR); MAPK pathway Càncer colorectal; Receptor epidèrmic de factor del creixement (EGFR); Via MAPK Cáncer colorrectal; Receptor epidérmico de factor del crecimiento (EGFR); Vía MAPK BACKGROUND: Previous studies showed that the combination of an anti-Epidermal growth factor (EGFR) and a MEK-inhibitor is able to prevent the onset of resistance to anti-EGFR monoclonal antibodies in KRAS-wild type colorectal cancer (CRC), while the same combination reverts anti-EGFR primary resistance in KRAS mutated CRC cell lines. However, rapid onset of resistance is a limit to combination therapies in KRAS mutated CRC. METHODS: We generated four different KRAS mutated CRC cell lines resistant to a combination of cetuximab (an anti-EGFR antibody) and refametinib (a selective MEK-inhibitor) after continuous exposure to increasing concentration of the drugs. We characterized these resistant cell lines by evaluating the expression and activation status of a panel of receptor tyrosine kinases (RTKs) and intracellular transducers by immunoblot and qRT-PCR. Oncomine comprehensive assay and microarray analysis were carried out to investigate new acquired mutations or transcriptomic adaptation, respectively, in the resistant cell lines. Immunofluorescence assay was used to show the localization of RTKs in resistant and parental clones. RESULTS: We found that PI3K-AKT pathway activation acts as an escape mechanism in cell lines with acquired resistance to combined inhibition of EGFR and MEK. AKT pathway activation is coupled to the activation of multiple RTKs such as HER2, HER3 and IGF1R, though its pharmacological inhibition is not sufficient to revert the resistant phenotype. PI3K pathway activation is mediated by autocrine loops and by heterodimerization of multiple receptors. CONCLUSIONS: PI3K activation plays a central role in the acquired resistance to the combination of anti-EGFR and MEK-inhibitor in KRAS mutated colorectal cancer cell lines. PI3K activation is cooperatively achieved through the activation of multiple RTKs such as HER2, HER3 and IGF1R.

chlorpyrifos (CPF) is an organophosphate insecticide used to control pests on a variety of food and feed crops. In mammals, maternal exposure to CPF has been reported to induce cerebral cortex thinning, alteration of long-term brain cognitive function, and Parkinson-like symptoms, but the mechanisms of these processes are not fully understood. In this study, we aimed to gain a deeper understanding of the alterations induced in the brains of mice chronically exposed to CPF by dietary intake. For our purpose, we analysed F1 offspring (sacrificed at 3 and 8 months) of Mus musculus, treated in utero and postnatally with 3 different doses of CPF (0.1-1-10 mg/kg/day). Using RT² Profiler PCR Arrays, we evaluated the alterations in the expression of 84 genes associated with neurodegenerative diseases. In the brains of exposed mice, we evidenced a clear dose-response relationship for AChE inhibition and alterations of gene expression. Some of the genes that were steadily down-regulated, such as Pink1, Park 2, Sv2b, Gabbr2, Sept5 and Atxn2, were directly related to Parkinson's onset. Our experimental results shed light on the possibility that long-term CPF exposure may exert membrane signalling alterations which make brain cells more susceptible to develop neurodegenerative diseases.

Dyskerin is an essential, conserved, multifunctional protein found in the nucleolus, whose loss of function causes the rare genetic diseases X-linked dyskeratosis congenita and Hoyeraal-Hreidarsson syndrome. To further investigate the wide range of dyskerin's biological roles, we set up stable cell lines able to trigger inducible protein knockdown and allow a detailed analysis of the cascade of events occurring within a short time frame. We report that dyskerin depletion quickly induces cytoskeleton remodeling and significant alterations in endocytic Ras-related protein Rab-5A/Rab11 trafficking. These effects arise in different cell lines well before the onset of telomere shortening, which is widely considered the main cause of dyskerin-related diseases. Given that vesicular trafficking affects many homeostatic and differentiative processes, these findings add novel insights into the molecular mechanisms underlining the pleiotropic manifestation of the dyskerin loss-of-function phenotype.

Nanoparticle (NPs) delivery systems in vivo promises to overcome many obstacles associated with the administration of drugs, vaccines, plasmid DNA and RNA materials, making the study of their cellular uptake a central issue in nanomedicine. The uptake of NPs may be influenced by the cell culture stage and the NPs physical-chemical properties. So far, controversial data on NPs uptake have been derived owing to the heterogeneity of NPs and the general use of immortalized cancer cell lines that often behave differently from each other and from primary mammalian cell cultures. Main aims of the present study were to investigate the uptake, endocytosis pathways, intracellular fate and release of well standardized model particles, i.e. fluorescent 44 nm polystyrene NPs (PS-NPs), on two primary mammalian cell cultures, i.e. bovine oviductal epithelial cells (BOEC) and human colon fibroblasts (HCF) by confocal microscopy and spectrofluorimetric analysis. Different drugs and conditions that inhibit specific internalization routes were used to understand the mechanisms that mediate PS-NP uptake. Our data showed that PS-NPs are rapidly internalized by both cell types 1) with similar saturation kinetics; 2) through ATP-independent processes, and 3) quickly released in the culture medium. Our results suggest that PS-NPs are able to rapidly cross the cell membrane through passive translocation during both uptake and release, and emphasize the need to carefully design NPs for drug delivery, to ensure their selective uptake and to optimize their retainment in the targeted cells.

Human dyskerin is an evolutively conserved protein that participates in diverse nuclear complexes: the H/ACA snoRNPs, that control ribosome biogenesis, RNA pseudouridylation, and stability of H/ACA snoRNAs; the scaRNPs, that control pseudouridylation of snRNAs; and the telomerase active holoenzyme, which safeguards telomere integrity. The biological importance of dyskerin is further outlined by the fact that its deficiency causes the X-linked dyskeratosis congenita disease, while its over-expression characterizes several types of cancers and has been proposed as prognostic marker. The role of dyskerin in telomere maintenance has widely been discussed, while its functions as H/ACA sno/scaRNP component has been so far mostly overlooked and represent the main goal of this review. Here we summarize how increasing evidence indicates that the snoRNA/microRNA pathways can be interlaced, and that dyskerin-dependent RNA pseudouridylation represents a flexible mechanism able to modulate RNA function in different ways, including modulation of splicing, change of mRNA coding properties, and selective regulation of IRES-dependent translation. We also propose a speculative model that suggests that the dynamics of pre-assembly and nuclear import of H/ACA RNPs are crucial regulatory steps that can be finely controlled in the cytoplasm in response to developmental, differentiative and stress stimuli.

Background The "PTEN hamartoma tumor syndrome" (PHTS) includes a group of syndromes caused by germline mutations within the tumor suppressor gene "phosphatase and tensin homolog deleted on chromosome ten" (PTEN), characterized by multiple polyps in the gastrointestinal tract and by a highly increased risk of developing malignant tumours in many tissues. The current work clarifies the molecular basis of PHTS in three unrelated Italian patients, and sheds light on molecular pathway disregulation constitutively associated to PTEN alteration. Methods We performed a combination of RT-PCR, PCR, sequencing of the amplified fragments, Real Time PCR and western blot techniques. Results Our data provide the first evidence of β-catenin accumulation in blood cells of patients with hereditary cancer syndrome caused by germ-line PTEN alteration. In addition, for the first time we show, in all PHTS patients analysed, alterations in the expression of TNFα, its receptors and IL-10. Importantly, the isoform of TNFRI that lacks the DEATH domain (TNFRSF1β) was found to be overexpressed. Conclusion In light of our findings, we suggest that the PTEN pathway disregulation could determine, in non-neoplastic cells of PHTS patients, cell survival and pro-inflammatory stimulation, mediated by the expression of molecules such as β-catenin, TNFα and TNFα receptors, which could predispose these patients to the development of multiple cancers.

Mutations in the highly conserved human DKC1 gene cause the rare genetic disease X-linked recessive dyskeratosis congenita (X-DC). X-DC patients and DKC1 hypomorphic mutant mice show an increased susceptibility to cancer, so that it has been suggested that DKC1 can act as potent tumor suppressor. The nucleolar protein encoded by DKC1, named dyskerin, is one of the core components of the nucleolar ribonucleoprotein particles which include the small nucleolar RNAs belonging to the H/ACA family. Within H/ACA snoRNPs, dyskerin is known to play a crucial role in rRNA processing and pseudouridylation, being able to act as pseudouridine synthase. In addition, dyskerin has been recently identified as an essential component of the catalytically active human telomerase complex, together with the human telomerase reverse trascriptase (TERT) and the RNA component of telomerase. Since TERT has been indicated as a potential biomarker for colorectal cancer, we wished to evaluate whether dyskerin and TERT mRNA levels were similarly variable in colorectal cancer. In this study, matched samples of tumors and adjacent non-tumorous mucosa from 8 colorectal carcinomas were collected, and Real-time polymerase chain reaction was used to quantitate DKC1 and TERT expression levels. We found that in the colorectal adenocarcinomas DKC1 mRNA level was significantly higher than in the corresponding non-tumor mucosa (p=0.0004, one-sided t test). Instead, TERT exhibited a variable mRNA level, although in most cases it was up-regulated respect to the matched controls (p=0.066, one-sided t test). These data support the view that DKC1 expression may be considered a less variable and more sensible marker than TERT for proliferative colon disorders. Although analysis of a more exhaustive series of tumors is needed, we suggest that the evaluation of DKC1 expression can provide a useful clue in the diagnosis and prognosis of colorectal cancer.

Background In remodelling ventricles, the progression of heart failure is associated with structural changes involving the extra-cellular matrix (ECM) and the cytoskeleton of cardiomyocytes, associated with fibrosis, cellular damage and death. The role of some cytokines and haematopoietic growth factors in the mechanism of both damage and regeneration of cardiac tissue during acute myocardial infarction has been demonstrated. Following heart damage, the development of scarred tissue was considered to be the only outcome, since myocytes were considered to be terminally differentiated cells. However, recent studies in animal models and adult human hearts have shown that myocytes can proliferate under the modulation of several factors. Aims To assess Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) receptor expression in healthy and diseased human hearts, and to evaluate the possible role of GM-CSF and its receptor in the regeneration of cardiac tissue in chronic cardiomyopathy. Methods and results GM-CSFR expression in human cardiac tissue from explanted hearts of ten patients with end-stage heart failure and in cardiac biopsies from eight normal human hearts was studied by immunohistochemistry, and cellular and molecular biology assays. Our results demonstrated an increase in GM-CSFR in cardiomyocytes from end-stage heart failure tissues as compared to normal control tissues. Conclusions We hypothesize that GM-CSF plays a role in apoptotic and/or ECM deposition processes as well as in cytoskeleton modification in the myocardium.

3 Diabetes mellitus or carbohydrate intolerance is frequently described. 1 The age of onset is variable. The disease usually comes at puberty but several cases have become symptomatic after 40 years. 4 The mole- cular defect that occurs in Friedreich's ataxia is the trinucleotide GAA expansion in the first intron of the X25 gene. This gene encodes for a 210 aa mitochondrial protein called frataxin. 5 Levels of frataxin mRNA and protein are severely reduced as a result of this expansion. 5 Although the exact physiological function of frataxin is not known, its involvement in iron-sulphur (Fe-S) cluster biogenesis 6 has been suggested. The Friedreich's ataxia phenotype shows a variable expression with respect to the age of onset and the presence of some signs or symptoms. Disease duration has been proposed to influence dysarthria, decreased vibration sense, optic atrophy, and diabetes. 7 Some Friedreich's ataxia phenotype features are strongly correlated with GAA expan- sion size. An inverse relationship has been demonstrated between GAA repeat size and the age of onset. 78 Friedreich's ataxia with retained reflexes or absence of cardiomyopathy was associated with shorter expansions. In a previous report, we demonstrated that GAA size accounts for about 70% of the onset age variance, 8

Friedreich ataxia (FRDA) is the most common recessive ataxia caused by reduced expression of frataxin, a nuclear encoded mitochondrial protein. In this study we examined the effects of 3-nitropropionic acid (3-NP) on frataxin expression in FRDA patient and control lymphoblasts and in rat pheochromocytoma cell line (PC12) overexpressing human frataxin. Our studies showed an up-regulation of frataxin expression in both FRDA and control lymphoblasts following exposure to 3-NP. In addition, in transgenic frataxin overexpressing cells 3-NP caused an increase of frataxin protein.

The most common mutation causing Friedreich ataxia (FRDA), an autosomal recessive neurodegenerative disease, is the hyperexpansion of a polymorphic GAA triplet repeat localized within an Alu sequence (GAA-Alu) in the first intron of the frataxin (X25) gene. GAA-Alu belongs to the AluSx subfamily and contains several polymorphisms in strong linkage disequilibrium either with a subgroup of normal alleles, or with hyperexpanded FRDA-associated alleles. GAA repeat sizes in 300 normal chromosomes (97 from carriers and 203 from controls) were distributed in two separate groups: 83% of them contained between six and 10 triplets (small normal alleles), while the remaining 17% had more than 12 triplets, up to 36 (large normal alleles). Sequence analysis showed that no normal, stable allele contained more than 27 uninterrupted GAA triplets. All longer normal alleles were interrupted by a hexanucleotide repeat (GAGGAA). An allele containing an uninterrupted run of 34 GAA triplets was stably transmitted in four instances, but in one case underwent hyperexpansion to 650 triplets. Overall, our results suggest that the FRDA-associated expanded GAA repeats originate from normal alleles by recurrent expansions of alleles at risk.

The severe reduction in mRNA and protein levels of the mitochondrial protein frataxin, encoded by the X25 gene, causes Friedreich ataxia (FRDA), the most common form of recessive hereditary ataxia. Increasing evidence underlines the pathogenetic role of oxidative stress in this disease. We generated an in vitro cellular model of regulated human frataxin overexpression. We identified, by differential display technique, the mitogen activated protein kinase kinase 4 mRNA down regulation in frataxin overexpressing cells. We studied the stress kinases pathway in this cellular model and in fibroblasts from FRDA patients. Frataxin overexpression reduced c-Jun N-terminal kinase phosphorylation. Furthermore, exposure of FRDA fibroblasts to several forms of environmental stress caused an up regulation of phospho-JNK and phospho-c-Jun. To understand if this susceptibility results in cell death, we have investigated the involvement of caspases. A significantly higher activation of caspase-9 was observed in FRDA versus control fibroblasts after serum-withdrawal. Our findings suggest the presence, in FRDA patient cells, of a 'hyperactive' stress signaling pathway. The role of frataxin in FRDA pathogenesis could be explained, at least in part, by this hyperactivity.