Parallels between oncogene-driven cardiac hyperplasia and heart regeneration in zebrafish.

The human heart is poorly regenerative and cardiac tumors are extremely rare. Whether the adult zebrafish myocardium is responsive to oncogene overexpression and how this condition affects its intrinsic regenerative capacity remains unknown. Here, we have established a strategy of inducible and reversible expression of HRASG12V in zebrafish cardiomyocytes. This approach stimulated a hyperplastic cardiac enlargement within 16 days. The phenotype was suppressed by rapamycin-mediated inhibition of TOR signaling. As TOR signaling is also required for heart restoration after cryoinjury, we compared transcriptomes of hyperplastic and regenerating ventricles. Both conditions were associated with upregulation of cardiomyocyte dedifferentiation and proliferation factors, as well as with similar microenvironmental responses, such as deposition of nonfibrillar Collagen XII and recruitment of immune cells. Among the differentially expressed genes, many proteasome and cell-cycle regulators were upregulated only in oncogene-expressing hearts. Preconditioning of the heart with short-term oncogene expression accelerated cardiac regeneration after cryoinjury, revealing a beneficial synergism between both programs. Identification of the molecular bases underlying the interplay between detrimental hyperplasia and advantageous regeneration provides new insights into cardiac plasticity in adult zebrafish.

A review and perspective paper: Ras oncogene gets modest, from kingpin to mere henchman.

The concomitant activation of both the YAP1 co-transcription factor and RAS GTPases is a hallmark of several aggressive cancers, though the intricacies of their relationship and implications for oncogenesis are still poorly understood. This review has presented a cooperative model where YAP1 and RAS are not independently acting oncogenes but rather interdependently acting ones, with each fulfilling an essential role within the oncogenic process. YAP1 is responsible for initiating the expression of key proteins that contribute to various cancer traits. However, these proteins must often be transported into the cytoplasm to exert their effects. We suggest that oncogenic RAS actually facilitates this transport, enabling the phosphorylation and subsequent activation of the nuclear transporter XPO1 (aka Exportin1). This mechanism is particularly crucial for anti-apoptotic proteins. Instead of being sequestered within the nucleus in an ineffective state, these proteins are rather shuttled into the cytoplasm. Within the cytoplasm, they can effectively inhibit apoptosis, undermining by these means the efficacy of chemotherapeutic agents designed to induce cell death in cancer cells. Therefore, a clearer understanding of the oncogenic partnership between RAS and YAP1 will likely provide new insights into the molecular underpinnings of cancer and highlight as well potential targets for therapeutic interventions designed to disrupt this pernicious interaction.

Associations of SEMA7A, SEMA4D, ADAMTS10, and ADAM8 with KRAS, NRAS, BRAF, PIK3CA, and AKT Gene Mutations, Microsatellite Instability Status, and Cytokine Expression in Colorectal Cancer Tissue.

Semaphorins (SEMAs), ADAM, and ADAMTS family members are implicated in various cancer progression events within the tumor microenvironment across different cancers. In this study, we aimed to evaluate the expression of SEMA7A, SEMA4D, ADAM8, and ADAMTS10 in colorectal cancer (CRC) in relation to the mutational landscape of KRAS, NRAS, BRAF, PIK3CA, and AKT genes, microsatellite instability (MSI) status, and clinicopathological features. We also examined the associations between the expression of these proteins and selected cytokines, chemokines, and growth factors, assessed using a multiplex assay. Protein concentrations were quantified using ELISA in CRC tumors and tumor-free surgical margin tissue homogenates. Gene mutations were evaluated via RT-PCR, and MSI status was determined using immunohistochemistry (IHC). GSEA and statistical analyses were performed using R Studio. We observed a significantly elevated expression of SEMA7A in BRAF-mutant CRC tumors and an overexpression of ADAM8 in KRAS 12/13-mutant tumors. The expression of ADAMTS10 was decreased in PIK3CA-mutant CRC tumors. No significant differences in the expression of the examined proteins were observed based on MSI status. The SEMA7A and SEMA4D expressions were correlated with the expression of numerous cytokines associated with various immune processes. The potential immunomodulatory functions of these molecules and their suitability as therapeutic targets require further investigation.

K-Ras(V12) differentially affects the three Akt isoforms in lung and pancreatic carcinoma cells and upregulates E-cadherin and NCAM via Akt3.

K-Ras is the most frequently mutated Ras variant in pancreatic, colon and non-small cell lung adenocarcinoma. Activating mutations in K-Ras result in increased amounts of active Ras-GTP and subsequently a hyperactivation of effector proteins and downstream signaling pathways. Here, we demonstrate that oncogenic K-Ras(V12) regulates tumor cell migration by activating the phosphatidylinositol 3-kinases (PI3-K)/Akt pathway and induces the expression of E-cadherin and neural cell adhesion molecule (NCAM) by upregulation of Akt3. In vitro interaction and co-precipitation assays identified PI3-Kα as a bona fide effector of active K-Ras4B but not of H-Ras or N-Ras, resulting in enhanced Akt phosphorylation. Moreover, K-Ras(V12)-induced PI3-K/Akt activation enhanced migration in all analyzed cell lines. Interestingly, Western blot analyses with Akt isoform-specific antibodies as well as qPCR studies revealed, that the amount and the activity of Akt3 was markedly increased whereas the amount of Akt1 and Akt2 was downregulated in EGFP-K-Ras(V12)-expressing cell clones. To investigate the functional role of each Akt isoform and a possible crosstalk of the isoforms in more detail, each isoform was stably depleted in PANC-1 pancreatic and H23 lung carcinoma cells. Akt3, the least expressed Akt isoform in most cell lines, is especially upregulated and active in Akt2-depleted cells. Since expression of EGFP-K-Ras(V12) reduced E-cadherin-mediated cell-cell adhesion by induction of polysialylated NCAM, Akt3 was analyzed as regulator of E-cadherin and NCAM. Western blot analyses revealed pronounced reduction of E-cadherin and NCAM in the Akt3-kd cells, whereas Akt1 and Akt2 depletion upregulated E-cadherin, especially in H23 lung carcinoma cells. In summary, we identified oncogenic K-Ras4B as a key regulator of PI3-Kα-Akt signaling and Akt3 as a crucial regulator of K-Ras4B-induced modulation of E-cadherin and NCAM expression and localization.

Theoretical, in Vitro Antiproliferative, and in Silico Molecular Docking and Pharmacokinetics Studies of Heteroleptic Nickel(II) and Copper(II) Complexes of Thiosemicarbazone-Based Ligands and Pefloxacin.

Twelve new heteroleptic nickel(II) and copper(II) complexes of the type [M(L1-6 )(Pfx)2 ] (1-12), where L1-6 =2-benzylidenehydrazinecarbothioamide (L1 ), 2-benzylidene-N-methylhydrazinecarbothioamide (L2 ), 2-benzylidene-N-phenylhydrazinecarbothioamide (L3 ), 2-(4-methylbenzylidene)hydrazinecarbothioamide (L4 ), 2-(4-methylbenzylidene)-N-methylhydrazinecarbothioamide (L5 ) and 2-(4-methylbenzylidene)-N-phenylhydrazinecarbothioamide (L6 ), Pfx=pefloxacin and M=Ni(II) or Cu(II) have been synthesised, and their structures were confirmed by different spectral techniques. The spectral data and density functional theory (DFT) calculations supported the bonding of pefloxacin drug molecule via one of the carboxylate oxygen atoms and the pyridone oxygen atom, and the thiosemicarbazone ligand via the imine nitrogen and the thione sulfur atoms with the metal(II) ion, forming distorted octahedral geometry. In vitro antiproliferative activity of the synthesized complexes was evaluated against three human breast cancer (T47D, estrogen negative (MDA-MB-231) and estrogen positive (MCF-7)) as well as non-tumorigenic human breast epithelial (MCF-10a) cell lines, which showed the higher activity for the copper(II) complexes. The interaction of the synthesized complexes with an oncogenic protein H-ras (121 p) was explored by in silico molecular docking studies. Further, in silico pharmacokinetics and ADMET parameters were also analysed to predict the drug-likeness as well as non-toxic and non-carcinogenic behavior, and safe oral administration of the complexes.

Patterning of Oncogenic Ras Clustering in Live Cells Using Vertically Aligned Nanostructure Arrays.

As a dominant oncogenic protein, Ras is well-known to segregate into clusters on the plasma membrane for activating downstream signaling. However, current technologies for direct measurements of Ras clustering are limited to sophisticated high-resolution techniques like electron microscopy and fluorescence lifetime imaging. To further promote fundamental investigations and the related drug development, we hereby introduce a nanobar-based platform which effectively guides Ras clusters into quantifiable patterns in live cells that is resolvable under conventional microscopy. Major Ras isoforms, K-Ras, H-Ras, and N-Ras, were differentiated, as well as their highly prevalent oncogenic mutants G12V and G13D. Moreover, the isoform specificity and the sensitivity of a Ras inhibitor were successfully characterized on nanobars. We envision that this nanobar-based platform will serve as an effective tool to read Ras clustering on the plasma membrane, enabling a novel avenue both to decipher Ras regulations and to facilitate anti-Ras drug development.

Engineered variants of the Ras effector protein RASSF5 (NORE1A) promote anticancer activities in lung adenocarcinoma.

Within the superfamily of small GTPases, Ras appears to be the master regulator of such processes as cell cycle progression, cell division, and apoptosis. Several oncogenic Ras mutations at amino acid positions 12, 13, and 61 have been identified that lose their ability to hydrolyze GTP, giving rise to constitutive signaling and eventually development of cancer. While disruption of the Ras/effector interface is an attractive strategy for drug design to prevent this constitutive activity, inhibition of this interaction using small molecules is impractical due to the absence of a cavity to which such molecules could bind. However, proteins and especially natural Ras effectors that bind to the Ras/effector interface with high affinity could disrupt Ras/effector interactions and abolish procancer pathways initiated by Ras oncogene. Using a combination of computational design and in vitro evolution, we engineered high-affinity Ras-binding proteins starting from a natural Ras effector, RASSF5 (NORE1A), which is encoded by a tumor suppressor gene. Unlike previously reported Ras oncogene inhibitors, the proteins we designed not only inhibit Ras-regulated procancer pathways, but also stimulate anticancer pathways initiated by RASSF5. We show that upon introduction into A549 lung carcinoma cells, the engineered RASSF5 mutants decreased cell viability and mobility to a significantly greater extent than WT RASSF5. In addition, these mutant proteins induce cellular senescence by increasing acetylation and decreasing phosphorylation of p53. In conclusion, engineered RASSF5 variants provide an attractive therapeutic strategy able to oppose cancer development by means of inhibiting of procancer pathways and stimulating anticancer processes.

The GTPase Rab27b regulates the release, autophagic clearance, and toxicity of α-synuclein.

α-Synuclein (αsyn) is the primary component of proteinaceous aggregates termed Lewy bodies that pathologically define synucleinopathies including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). αsyn is hypothesized to spread through the brain in a prion-like fashion by misfolded protein forming a template for aggregation of endogenous αsyn. The cell-to-cell release and uptake of αsyn are considered important processes for its prion-like spread. Rab27b is one of several GTPases essential to the endosomal-lysosomal pathway and is implicated in protein secretion and clearance, but its role in αsyn spread has yet to be characterized. In this study, we used a paracrine αsyn in vitro neuronal model to test the impact of Rab27b on αsyn release, clearance, and toxicity. shRNA-mediated knockdown (KD) of Rab27b increased αsyn-mediated paracrine toxicity. Rab27b reduced αsyn release primarily through nonexosomal pathways, but the αsyn released after Rab27b KD was of higher-molecular-weight species, as determined by size-exclusion chromatography. Rab27b KD increased intracellular levels of insoluble αsyn and led to an accumulation of endogenous light chain 3 (LC3)-positive puncta. Rab27b KD also decreased LC3 turnover after treatment with an autophagosome-lysosome fusion inhibitor, chloroquine, indicating that Rab27b KD induces a defect in autophagic flux. Rab27b protein levels were increased in brain lysates obtained from postmortem tissues of individuals with PD and DLB compared with healthy controls. These data indicate a role for Rab27b in the release, clearance, and toxicity of αsyn and, ultimately, in the pathogenesis of synucleinopathies.

Effect of casein kinase 1α inhibition on autophagy flux and the AKT/phospho-ß-catenin (S552) axis in HCT116, a RAS-mutated colorectal cancer cell line.

The Wnt/ß-catenin pathway, which interferes with cell proliferation, differentiation, and autophagy, is commonly dysregulated in colorectal cancer (CRC). Mutation of the RAS oncogene is the most prevalent genetic alteration in CRC and has been linked to activation of protein kinase B (AKT) signaling. Phosphorylation of ß-catenin at Ser 552 by AKT contributes to ß-catenin stability, transcriptional activity, and increase of cell proliferation. Casein kinase 1 alpha (CK1α) is an enzyme that simultaneously regulates Wnt/ß-catenin and AKT. The link of the AKT and Wnt pathway to autophagy in RAS-mutated CRC cells has not well identified. Therefore, we investigated how pharmacological CK1α inhibition (D4476) is involved in regulation of autophagy, Wnt/ß-catenin, and AKT pathways in RAS-mutated CRC cell lines. qRT-PCR and immunoblotting experiments revealed that phospho-AKT (S473) and phospho-ß-catenin (S552) are constitutively increased in RAS-mutated CRC cell lines, in parallel with augmented CK1α expression. The results also showed that D4476 significantly reduced the AKT/phospho-ß-catenin (S552) axis concomitantly with autophagy flux inhibition in RAS-mutated CRC cells. Furthermore, D4476 significantly induced apoptosis in RAS-mutated CRC cells. In conclusion, our results indicate that CK1α inhibition reduces autophagy flux and promotes apoptosis by interfering with the AKT/phospho-ß-catenin (S552) axis in RAS-mutated CRC cells.

Homotypic and heterotypic trans-assembly of human Rab-family small GTPases in reconstituted membrane tethering.

Membrane tethering is a highly regulated event occurring during the initial physical contact between membrane-bounded transport carriers and their target subcellular membrane compartments, thereby ensuring the spatiotemporal specificity of intracellular membrane trafficking. Although Rab-family small GTPases and specific Rab-interacting effectors, such as coiled-coil tethering proteins and multisubunit tethering complexes, are known to be involved in membrane tethering, how these protein components directly act upon the tethering event remains enigmatic. Here, using a chemically defined reconstitution system, we investigated the molecular basis of membrane tethering by comprehensively and quantitatively evaluating the intrinsic capacities of 10 representative human Rab-family proteins (Rab1a, -3a, -4a, -5a, -6a, -7a, -9a, -11a, -27a, and -33b) to physically tether two distinct membranes via homotypic and heterotypic Rab-Rab assembly. All of the Rabs tested, except Rab27a, specifically caused homotypic membrane tethering at physiologically relevant Rab densities on membrane surfaces (e.g. Rab/lipid molar ratios of 1:100-1:3,000). Notably, endosomal Rab5a retained its intrinsic potency to drive efficient homotypic tethering even at concentrations below the Rab/lipid ratio of 1:3,000. Comprehensive reconstitution experiments further uncovered that heterotypic combinations of human Rab-family isoforms, including Rab1a/6a, Rab1a/9a, and Rab1a/33b, can directly and selectively mediate membrane tethering. Rab1a and Rab9a in particular synergistically triggered very rapid and efficient membrane tethering reactions through their heterotypic trans-assembly on two opposing membranes. In conclusion, our findings establish that, in the physiological context, homotypic and heterotypic trans-assemblies of Rab-family small GTPases can provide the essential molecular machinery necessary to drive membrane tethering in eukaryotic endomembrane systems.

Beyond the Genomic Mutation: Rethinking the Molecular Biomarkers of K-RAS Dependency in Pancreatic Cancers.

Oncogenic v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-RAS) plays a key role in the development and maintenance of pancreatic ductal adenocarcinoma (PDAC). The targeting of K-RAS would be beneficial to treat tumors whose growth depends on active K-RAS. The analysis of K-RAS genomic mutations is a clinical routine; however, an emerging question is whether the mutational status is able to identify tumors effectively dependent on K-RAS for tailoring targeted therapies. With the emergence of novel K-RAS inhibitors in clinical settings, this question is relevant. Several studies support the notion that the K-RAS mutation is not a sufficient biomarker deciphering the effective dependency of the tumor. Transcriptomic and metabolomic profiles of tumors, while revealing K-RAS signaling complexity and K-RAS-driven molecular pathways crucial for PDAC growth, are opening the opportunity to specifically identify K-RAS-dependent- or K-RAS-independent tumor subtypes by using novel molecular biomarkers. This would help tumor selection aimed at tailoring therapies against K-RAS. In this review, we will present studies about how the K-RAS mutation can also be interpreted in a state of K-RAS dependency, for which it is possible to identify specific K-RAS-driven molecular biomarkers in certain PDAC subtypes, beyond the genomic K-RAS mutational status.

[Bacillus pumilus Ribonuclease Inhibits Migration of Human Duodenum Adenocarcinoma HuTu 80 Cells].

Migration of cancer cells from the primary tumor site to nearby tissues is the starting point of the metastatic process. The invasive properties of cells are especially important for carcinomas, since tumor cells need to overcome the basement membrane and go beyond its boundaries to the underlying tissues. Substances that reduce the invasive ability of malignant cells are promising as antimetastatic agents. In the present work, the possibility of inhibiting the ability of different cancer cell lines to migrate under the influence of the Bacillus pumilus ribonuclease (binase) was analyzed using the scratch-wound assay. It was established that binase at non-toxic concentrations (10 µg/mL) reliably suppressed the migratory ability of HuTu 80 human duodenum adenocarcinoma cells incubated with RNase for 48-72 h. The antimetastatic potential of binase is confirmed by molecular modeling data demonstrating the ability of binase to inhibit cellular metalloproteinases that determine the migration of tumor cells.

The regulation of oncogenic Ras/ERK signalling by dual-specificity mitogen activated protein kinase phosphatases (MKPs).

Dual-specificity MAP kinase (MAPK) phosphatases (MKPs or DUSPs) are well-established negative regulators of MAPK signalling in mammalian cells and tissues. By virtue of their differential subcellular localisation and ability to specifically recognise, dephosphorylate and inactivate different MAPK isoforms, they are key spatiotemporal regulators of pathway activity. Furthermore, as they are transcriptionally regulated as downstream targets of MAPK signalling they can either act as classical negative feedback regulators or mediate cross talk between distinct MAPK pathways. Because MAPKs and particularly Ras/ERK signalling are implicated in cancer initiation and development, the observation that MKPs are abnormally regulated in human tumours has been interpreted as evidence that these enzymes can either suppress or promote carcinogenesis. However, definitive evidence of such roles has been lacking. Here we review recent work based on the use of mouse models, biochemical studies and clinical data that demonstrate key roles for MKPs in modulating the oncogenic potential of Ras/ERK signalling and also indicate that these enzymes may play a role in the response of tumours to certain anticancer drugs. Overall, this work reinforces the importance of negative regulatory mechanisms in modulating the activity of oncogenic MAPK signalling and indicates that MKPs may provide novel targets for therapeutic intervention in cancer.

Reversal of the Migratory and Invasive Phenotype of Ras-Transfected Mammary Cells by Photodynamic Therapy Treatment.

Photodynamic therapy (PDT) is a non-thermal technique for inducing tumor damage following administration of a light-activated photosensitizing drug (PS). In a previous work we found that PDT induces cytoskeleton changes in HB4a-Ras cells (human mammary breast carcinoma HB4a cells transfected with the RAS oncogene). In the present work we have studied the migratory and invasive features and the expression of proteins related to these processes on HB4a-Ras cells after three successive cycles of PDT using different PSs: 5-aminolevulinic acid (ALA), Verteporfin (Verte), m-tetrahydroxyphenylchlorin (m-THPC), and Merocyanine 540 (MC). A slight (1.25- to 2-fold) degree of resistance was acquired in cell populations subjected to the three successive PDT treatments. However, complete cell killing was achieved after a light dose increase. Regardless of the PS employed, all the PDT-treated populations had shorter stress fibres than the untreated control HB4a-Ras cells, and the number of dorsal stress fibres was decreased in the PDT-treated populations. E-Cadherin distribution, which was already aberrant in HB4a-Ras cells, became even more diffuse in the PDT-treated populations, though its expression was increased in some of them. The strong migratory and invasive ability of HB4a-Ras cells in vitro was impaired in all the PDT-treated populations, with a behavior that was similar to the parental non-tumoral HB4a cells. MMP-2 and -9 metalloproteinase activities were also impaired in the PDT-treated populations. The evidence presented herein suggests that the cells surviving PDT would be less metastatic than the initial population. These findings encourage the use of PDT in combination with other treatments such as intraoperative or post-surgery therapeutic procedures. J. Cell. Biochem. 118: 464-477, 2017. © 2016 Wiley Periodicals, Inc.

B lymphocytes repress hepatic tumorigenesis but not development in Hras12V transgenic mice.

Increasing reports show noninflammation underlying HCC, challenging our understanding of the roles of the immune system in hepatocarcinogenesis. By exploring a mouse model of hepatic tumor induced by hepatocyte-specific expression of the Hras12V oncogene without obvious inflammation, we found that the proportion of B cells, but not T cells, progressively and significantly decreased in 3, 5-month-old transgenic mice (Tg) compared with non-transgenic mice. Notably, the proportions of total and activated B and T cells all significantly decreased in 9-month-old Tg with multiple massive hepatic tumors. Together with the decreased B cell proportion, serum IgG1/2 also significantly decreased in 5, 9-month-old Tg. Interestingly, homozygous Tg showed significantly higher B cell proportion and IgG2 levels, accompanied by significantly lower incidences of liver nodules but not adenomas and carcinomas compared with heterozygous Tg. Treatment of Tg with PCI-32765, a potent Bruton's tyrosine kinase (BTK) inhibitor for suppressing B cell proliferation and activation, significantly decreased the B cell proportion and IgG2 levels, accompanied by a significantly higher incidence of liver nodules, but had no effects on adenoma and carcinoma. Treatment of Tg with insulin-like growth factor 1 (IGF-1) significantly increased the B cell proportion and IgG2 levels, accompanied by a significantly lower incidence of liver nodules and carcinoma, but had no effects on adenoma. Conclusively, B cells and IgG2 may play important roles in suppressing hepatic tumorigenesis, but not development. In addition, hepatocyte-specific expression of the ras oncogene may play roles in suppressing B cells, while developed hepatic tumors suppress both B and T cells.

Isoprenoids responsible for protein prenylation modulate the biological effects of statins on pancreatic cancer cells.

BACKGROUND: Statin treatment of hypercholesterolemia is accompanied also with depletion of the mevalonate intermediates, including farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) necessary for proper function of small GTPases. These include Ras proteins, prevalently mutated in pancreatic cancer. In our study, we evaluated the effect of three key intermediates of the mevalonate pathway on GFP-K-Ras protein localization and the gene expression profile in pancreatic cancer cells after exposure to individual statins. METHODS: These effects were tested on MiaPaCa-2 human pancreatic cancer cells carrying a K-Ras activating mutation (G12C) after exposure to individual statins (20 µM). The effect of statins (atorvastatin, lovastatin, simvastatin, fluvastatin, cerivastatin, rosuvastatin, and pitavastatin) and mevalonate intermediates on GFP-K-Ras protein translocation was analyzed using fluorescence microscopy. The changes in gene expression induced in MiaPaCa-2 cells treated with simvastatin, FPP, GGPP, and their combinations with simvastatin were examined by whole genome DNA microarray analysis. RESULTS: All tested statins efficiently inhibited K-Ras protein trafficking from cytoplasm to the cell membrane of the MiaPaCa-2 cells. The inhibitory effect of statins on GFP-K-Ras protein trafficking was partially prevented by addition of any of the mevalonate pathway's intermediates tested. Expressions of genes involved in metabolic and signaling pathways modulated by simvastatin treatment was normalized by the concurrent addition of FPP or GGPP. K-Ras protein trafficking within the pancreatic cancer cells is effectively inhibited by the majority of statins; the inhibition is eliminated by isoprenoid intermediates of the mevalonate pathway. CONCLUSIONS: Our data indicate that the anticancer effects of statins observed in numerous studies to a large extent are mediated through isoprenoid intermediates of the mevalonate pathway, as they influence expression of genes involved in multiple intracellular pathways.

Dominant negative Ras attenuates pathological ventricular remodeling in pressure overload cardiac hypertrophy.

The importance of the oncogene Ras in cardiac hypertrophy is well appreciated. The hypertrophic effects of the constitutively active mutant Ras-Val12 are revealed by clinical syndromes due to the Ras mutations and experimental studies. We examined the possible anti-hypertrophic effect of Ras inhibition in vitro using rat neonatal cardiomyocytes (NRCM) and in vivo in the setting of pressure-overload left ventricular (LV) hypertrophy (POH) in rats. Ras functions were modulated via adenovirus directed gene transfer of active mutant Ras-Val12 or dominant negative mutant N17-DN-Ras (DN-Ras). Ras-Val12 expression in vitro activates NFAT resulting in pro-hypertrophic and cardio-toxic effects on NRCM beating and Z-line organization. In contrast, the DN-Ras was antihypertrophic on NRCM, inhibited NFAT and exerted cardio-protective effects attested by preserved NRCM beating and Z line structure. Additional experiments with silencing H-Ras gene strategy corroborated the antihypertrophic effects of siRNA-H-Ras on NRCM. In vivo, with the POH model, both Ras mutants were associated with similar hypertrophy two weeks after simultaneous induction of POH and Ras-mutant gene transfer. However, LV diameters were higher and LV fractional shortening lower in the Ras-Val12 group compared to control and DN-Ras. Moreover, DN-Ras reduced the cross-sectional area of cardiomyocytes in vivo, and decreased the expression of markers of pathologic cardiac hypertrophy. In isolated adult cardiomyocytes after 2 weeks of POH and Ras-mutant gene transfer, DN-Ras improved sarcomere shortening and calcium transients compared to Ras-Val12. Overall, DN-Ras promotes a more physiological form of hypertrophy, suggesting an interesting therapeutic target for pathological cardiac hypertrophy.

Pleiotropic effects of coat colour-associated mutations in humans, mice and other mammals.

The characterisation of the pleiotropic effects of coat colour-associated mutations in mammals illustrates that sensory organs and nerves are particularly affected by disorders because of the shared origin of melanocytes and neurocytes in the neural crest; e.g. the eye-colour is a valuable indicator of disorders in pigment production and eye dysfunctions. Disorders related to coat colour-associated alleles also occur in the skin (melanoma), reproductive tract and immune system. Additionally, the coat colour phenotype of an individual influences its general behaviour and fitness. Mutations in the same genes often produce similar coat colours and pleiotropic effects in different species (e.g., KIT [reproductive disorders, lethality], EDNRB [megacolon] and LYST [CHS]). Whereas similar disorders and similar-looking coat colour phenotypes sometimes have a different genetic background (e.g., deafness [EDN3/EDNRB, MITF, PAX and SNAI2] and visual diseases [OCA2, RAB38, SLC24A5, SLC45A2, TRPM1 and TYR]). The human predilection for fancy phenotypes that ignore disorders and genetic defects is a major driving force for the increase of pleiotropic effects in domestic species and laboratory subjects since domestication has commenced approximately 18,000 years ago.

Identification of post-transcriptional regulatory networks during myeloblast-to-monocyte differentiation transition.

Treatment of leukemia cells with 1,25-dihydroxyvitamin D3 may overcome their differentiation block and lead to the transition from myeloblasts to monocytes. To identify microRNA-mRNA networks relevant for myeloid differentiation, we profiled the expression of mRNAs and microRNAs associated to the low- and high-density ribosomal fractions in leukemic cells and in their differentiated monocytic counterpart. Intersection between mRNAs shifted across the fractions after treatment with putative target genes of modulated microRNAs showed a series of molecular networks relevant for the monocyte cell fate determination, as for example the post-transcriptional regulation of the Polo-like kinase 1 (PLK1) by miR-22-3p and let-7e-5p.

Increased susceptibility of H-Ras(G12V)-transformed human urothelial cells to the genotoxic effects of sodium arsenite.

Inorganic arsenite (iAs) is a human carcinogen. Numerous studies have shown that mutation-activated H-Ras is frequently observed in human urothelial carcinomas. The interaction between iAs, an environmental factor, and H-Ras, an oncogene, is not clear. In this study, we explored the genotoxic effects of iAs in human urothelial cells ectopically expressing H-Ras (G12V) an activated H-Ras oncogene. Our results showed that H-Ras(G12V)-transformed human urothelial cells (HUC-RAS) were more susceptible to arsenite-induced cell death, DNA damage, micronuclei formation and anchorage-independent growth than control cells (HUC-neo). Furthermore, iAs treatment induced higher intracellular levels of reactive oxygen species (ROS) in the HUC-RAS cells than in the HUC-neo cells. N-acetyl-L-cysteine could suppress the iAs-induced increases in ROS and genetic damage. We further demonstrated that the intracellular glutathione levels were significantly elevated by the iAs treatment of the HUC-neo cells, but that this effect was not observed in the HUC-RAS cells. The iAs treatment induced higher superoxide dismutase activity in the HUC-neo cells than in the HUC-RAS cells. Alternatively, catalase activity was higher in the HUC-RAS cells than in the HUC-neo cells, but this enzyme was significantly suppressed by iAs. Moreover, iAs activated the ERK and JNK signaling pathways, which are involved in iAs-induced ROS production and genetic damage. Taken together, our present results suggest that elevated catalase activity in H-Ras(G12V)-transformed cells is significantly suppressed by iAs via activation of ERK and JNK signaling pathways and hence attenuate the defense of the neoplastic transformed cells against iAs-induced oxidative injuries.