miR-483-5p offsets functional and behavioural effects of stress in male mice through synapse-targeted repression of Pgap2 in the basolateral amygdala.

Severe psychological trauma triggers genetic, biochemical and morphological changes in amygdala neurons, which underpin the development of stress-induced behavioural abnormalities, such as high levels of anxiety. miRNAs are small, non-coding RNA fragments that orchestrate complex neuronal responses by simultaneous transcriptional/translational repression of multiple target genes. Here we show that miR-483-5p in the amygdala of male mice counterbalances the structural, functional and behavioural consequences of stress to promote a reduction in anxiety-like behaviour. Upon stress, miR-483-5p is upregulated in the synaptic compartment of amygdala neurons and directly represses three stress-associated genes: Pgap2, Gpx3 and Macf1. Upregulation of miR-483-5p leads to selective contraction of distal parts of the dendritic arbour and conversion of immature filopodia into mature, mushroom-like dendritic spines. Consistent with its role in reducing the stress response, upregulation of miR-483-5p in the basolateral amygdala produces a reduction in anxiety-like behaviour. Stress-induced neuromorphological and behavioural effects of miR-483-5p can be recapitulated by shRNA mediated suppression of Pgap2 and prevented by simultaneous overexpression of miR-483-5p-resistant Pgap2. Our results demonstrate that miR-483-5p is sufficient to confer a reduction in anxiety-like behaviour and point to miR-483-5p-mediated repression of Pgap2 as a critical cellular event offsetting the functional and behavioural consequences of psychological stress.

TIAR and FMRP shape pro-survival nascent proteome of leukemia cells in the bone marrow microenvironment.

Chronic myeloid leukemia (CML) cells circulate between blood and bone marrow niche, representing different microenvironments. We studied the role of the two RNA-binding proteins, T-cell-restricted intracellular antigen (TIAR), and the fragile X mental retardation protein (FMRP) in the regulation of protein translation in CML cells residing in settings mimicking peripheral blood microenvironment (PBM) and bone marrow microenvironment (BMM). The outcomes showed how conditions shaped the translation process through TIAR and FMRP activity, considering its relevance in therapy resistance. The QuaNCAT mass-spectrometric approach revealed that TIAR and FMRP have a discrete modulatory effect on protein synthesis and thus affect distinct aspects of leukemic cells functioning in the hypoxic niche. In the BMM setup, FMRP impacted metabolic adaptation of cells and TIAR substantially supported the resistance of CML cells to translation inhibition by homoharringtonine. Overall, our results demonstrated that targeting post-transcriptional control should be considered when designing anti-leukemia therapeutic solutions.

Targeting integrated stress response with ISRIB combined with imatinib treatment attenuates RAS/RAF/MAPK and STAT5 signaling and eradicates chronic myeloid leukemia cells.

The integrated stress response (ISR) facilitates cellular adaptation to unfavorable conditions by reprogramming the cellular response. ISR activation was reported in neurological disorders and solid tumors; however, the function of ISR and its role as a possible therapeutic target in hematological malignancies still remain largely unexplored. Previously, we showed that the ISR is activated in chronic myeloid leukemia (CML) cells and correlates with blastic transformation and tyrosine kinase inhibitor (TKI) resistance. Moreover, the ISR was additionally activated in response to imatinib as a type of protective internal signaling. Here, we show that ISR inhibition combined with imatinib treatment sensitized and more effectively eradicated leukemic cells both in vitro and in vivo compared to treatment with single agents. The combined treatment specifically inhibited the STAT5 and RAS/RAF/MEK/ERK pathways, which are recognized as drivers of resistance. Mechanistically, this drug combination attenuated both interacting signaling networks, leading to BCR-ABL1- and ISR-dependent STAT5 activation. Consequently, leukemia engraftment in patient-derived xenograft mice bearing CD34+ TKI-resistant CML blasts carrying PTPN11 mutation responsible for hyperactivation of the RAS/RAF/MAPK and JAK/STAT5 pathways was decreased upon double treatment. This correlated with the downregulation of genes related to the RAS/RAF/MAPK, JAK/STAT5 and stress response pathways and was associated with lower expression of STAT5-target genes regulating proliferation, viability and the stress response. Collectively, these findings highlight the effect of imatinib plus ISRIB in the eradication of leukemic cells resistant to TKIs and suggest potential clinical benefits for leukemia patients with TKI resistance related to RAS/RAF/MAPK or STAT5 signaling. We propose that personalized treatment based on the genetic selection of patients carrying mutations that cause overactivation of the targeted pathways and therefore make their sensitivity to such treatment probable should be considered as a possible future direction in leukemia treatment.

A New Inhibitor of Tubulin Polymerization Kills Multiple Cancer Cell Types and Reveals p21-Mediated Mechanism Determining Cell Death after Mitotic Catastrophe.

Induction of mitotic catastrophe through the disruption of microtubules is an established target in cancer therapy. However, the molecular mechanisms determining the mitotic catastrophe and the following apoptotic or non-apoptotic cell death remain poorly understood. Moreover, many existing drugs targeting tubulin, such as vincristine, have reduced efficacy, resulting from poor solubility in physiological conditions. Here, we introduce a novel small molecule 2-aminoimidazoline derivative-OAT-449, a synthetic water-soluble tubulin inhibitor. OAT-449 in a concentration range from 6 to 30 nM causes cell death of eight different cancer cell lines in vitro, and significantly inhibits tumor development in such xenograft models as HT-29 (colorectal adenocarcinoma) and SK-N-MC (neuroepithelioma) in vivo. Mechanistic studies showed that OAT-449, like vincristine, inhibited tubulin polymerization and induced profound multi-nucleation and mitotic catastrophe in cancer cells. HeLa and HT-29 cells within 24 h of treatment arrested in G2/M cell cycle phase, presenting mitotic catastrophe features, and 24 h later died by non-apoptotic cell death. In HT-29 cells, both agents altered phosphorylation status of Cdk1 and of spindle assembly checkpoint proteins NuMa and Aurora B, while G2/M arrest and apoptosis blocking was consistent with p53-independent accumulation in the nucleus and largely in the cytoplasm of p21/waf1/cip1, a key determinant of cell fate programs. This is the first common mechanism for the two microtubule-dissociating agents, vincristine and OAT-449, determining the cell death pathway following mitotic catastrophe demonstrated in HT-29 cells.

Generation of Transgenic Rats using a Lentiviral Vector Approach.

Transgenic animal models are fundamentally important for modern biomedical research. The incorporation of foreign genes into early mouse or rat embryos is an invaluable tool for gene function analysis in living organisms. The standard transgenesis method is based on microinjecting foreign DNA fragments into a pronucleus of a fertilized oocyte. This technique is widely used in mice but remains relatively inefficient and technically demanding in other animal species. The transgene can also be introduced into one-cell-stage embryos via lentiviral infection, providing an effective alternative to standard pronuclear injections, especially in species or strains with a more challenging embryo structure. In this approach, a suspension that contains lentiviral vectors is injected into the perivitelline space of a fertilized rat embryo, which is technically less demanding and has a higher success rate. Lentiviral vectors were shown to efficiently incorporate the transgene into the genome to determine the generation of stable transgenic lines. Despite some limitations (e.g., Biosafety Level 2 requirements, DNA fragment size limits), lentiviral transgenesis is a rapid and efficient transgenesis method. Additionally, using female rats that are mated with a fertile male strain with a different dominant fur color is presented as an alternative to generate pseudopregnant foster mothers.

IQGAP1-dysfunction leads to induction of senescence in human vascular smooth muscle cells.

Cell senescence - an irreversible proliferation arrest - is one of the possible cellular responses to stress. There is a vast variety of stimuli, extrinsic and intrinsic, known to induce senescence, and several molecular pathways involved in the process; yet much still remains to be explained. Senescent cells can communicate with neighboring cells through secreted factors such as cytokines and chemokines. Several years ago it was shown that cells can also communicate in a more direct manner by an exchange of proteins via cellular bridges (CBs). Recent studies show that in senescent cells the intensity of such transfer increases. The research also revealed that Cdc42 and actin polymerization are indispensable for this process to occur. Here, we evaluate the hypothesis that, apart from actin and Cdc42, also IQGAP1 could be involved in direct intercellular communication. Our results showed that direct transfer occurred preferentially between senescent cells and that IQGAP1 was not essential for this process. Interestingly, cells harboring mutated IQGAP1 had altered morphology and were characterized by decreased proliferation, increased time of division and appearance of some senescence markers (increased activity of senescence-associated ß-galactosidase and induction of senescence-associated secretory phenotype). Our findings suggest that IQGAP1 dysfunction can induce senescence.

Tunneling nanotube-mediated intercellular vesicle and protein transfer in the stroma-provided imatinib resistance in chronic myeloid leukemia cells.

Intercellular communication within the bone marrow niche significantly promotes leukemogenesis and provides protection of leukemic cells from therapy. Secreted factors, intercellular transfer of mitochondria and the receptor-ligand interactions have been shown as mediators of this protection. Here we report that tunneling nanotubes (TNTs)-long, thin membranous structures, which have been identified as a novel mode of intercellular cross-talk-are formed in the presence of stroma and mediate transfer of cellular vesicles from stroma to leukemic cells. Importantly, transmission of vesicles via TNTs from stromal cells increases resistance of leukemic cells to the tyrosine kinase inhibitor, imatinib. Using correlative light-electron microscopy and electron tomography we show that stromal TNTs contain vesicles, provide membrane continuity with the cell bodies and can be open-ended. Moreover, trans-SILAC studies to reveal the non-autonomous proteome showed that specific sets of proteins are transferred together with cellular vesicles from stromal to leukemic cells, with a potential role in survival and adaptation. Altogether, our findings provide evidence for the biological role of the TNT-mediated vesicle exchange between stromal and leukemic cells, implicating the direct vesicle and protein transfer in the stroma-provided protection of leukemic cells.

Distribution of Glutathione-Stabilized Gold Nanoparticles in Feline Fibrosarcomas and Their Role as a Drug Delivery System for Doxorubicin-Preclinical Studies in a Murine Model.

Feline injection site sarcomas (FISS) are malignant skin tumors with high recurrence rates despite the primary treatment of radical surgical resections. Adjunctive radiotherapy or chemotherapy with doxorubicin is mostly ineffective. Cellular and molecular causes of multidrug resistance, specific physio-chemical properties of solid tumors impairing drug transport, and the tumor microenvironment have been indicated for causing standard chemotherapy failure. Gold nanoparticles are promising imaging tools, nanotherapeutics, and drug delivery systems (DDS) for chemotherapeutics, improving drug transport within solid tumors. This study was conducted to assess the distribution of 4-nm glutathione-stabilized gold nanoparticles in FISS and their influence on kidney and liver parameters in nude mice. The role of gold nanoparticles as a doxorubicin DDS in FISS was examined to determine the potential reasons for failure to translate results from in vitro to in vivo studies. Grade III tumors characterized by a large area of necrosis at their core displayed positive immuneexpression of tumor-associated macrophages (TAM) at both the periphery and within the tumor core near the area of necrosis. Gold nanoparticles did not cause necrosis at the injection site and had no negative effect on liver and kidney parameters in nude mice. Gold nanoparticles accumulated in the tumor core and at the periphery and co-internalized with TAM-an important observation and potential therapeutic target warranting further investigation. The large area of necrosis and high immunoexpression of TAM, indicating "pro-tumor macrophages", may be responsible for FISS tumor progression and therapeutic failure. However, further studies are required to test this hypothesis.

Bafilomycin A1 triggers proliferative potential of senescent cancer cells in vitro and in NOD/SCID mice.

Anticancer therapies that induce DNA damage tend to trigger senescence in cancer cells, a process known as therapy-induced senescence (TIS). Such cells may undergo atypical divisions, thus contributing to tumor re-growth. Accumulation of senescent cancer cells reduces survival of patients after chemotherapy. As senescence interplays with autophagy, a dynamic recycling process, we sought to study whether inhibition of autophagy interferes with divisions of TIS cells. We exposed human colon cancer HCT116 cells to repeated cycles of a chemotherapeutic agent - doxorubicin (doxo) and demonstrated induction of hallmarks of TIS (e.g. growth arrest, hypertrophy, poliploidization and secretory phenotype) and certain properties of cancer stem cells (increased NANOG expression, percentages of CD24+ cells and side population). Colonies of small and highly proliferative progeny appeared shortly after drug removal. Treatment with bafilomycin A1 (BAF A1), an autophagy inhibitor, postponed short term in vitro cell re-population. It was associated with reduction in the number of diploid and increase in the number of poliploid cells. In a long term, a pulse of BAF A1 resulted in reactivation of autophagy in a subpopulation of HCT116 cells and increased proliferation. Accordingly, the senescent HCT116 cells treated with BAF A1 when injected into NOD/SCID mice formed tumors, in contrast to the controls.Our results suggest that senescent cancer cells that appear during therapy, can be considered as dormant cells that contribute to cancer re-growth, when chemotherapeutic treatment is stopped. These data unveil new mechanisms of TIS-related cancer maintenance and re-population, triggered by a single pulse of BAF A1 treatment.

Cell-based assay for low- and high-scale screening of the Wnt/ß-catenin signaling modulators.

Deregulation of the Wnt/ß-catenin signaling pathway is associated with many serious disorders, including cancer and Alzheimer's disease. The pivotal player is ß-catenin, which avoids degradation after activation of the pathway and is translocated to the nucleus, where it interacts with TCF/LEF transcription factors and induces expression of genes involved in cell cycle and apoptosis regulation. The identification of small molecules that may affect Wnt/ß-catenin signaling remains an important target during the development of novel therapies. We used the TCF/LEF lentiviral vector and the Wnt-independent H1703 cell line to develop a luciferase reporter-based cell assay for screening of the Wnt/ß-catenin pathway modulators. Following the optimization of cell density, concentration of activator, and stimulation time, the reporter system was validated by demonstrating its specific and dose-dependent response to several established modulators of Wnt/ß-catenin signaling such as Wnt3a, small interfering RNA (siRNA) against ß-catenin, glycogen synthase kinase 3 (GSK-3), and ß-catenin/TCF transcription complex inhibitors. Two pilot screens of inhibitors and activators of Wnt/ß-catenin signaling identified potential novel modulators of this pathway. Our findings suggest that the H1703-7TFP assay constitutes a suitable model of low background and high sensitivity for the low- and high-scale screening of the Wnt/ß-catenin pathway modulators.

Silencing of ICERs (Inducible cAMP Early Repressors) results in partial protection of neurons from programmed cell death.

ICERs proteins (Inducible cAMP Early Repressors) are the most effective endogenous repressors of CREB/CREM/ATF transcription factors family (CREB-cAMP Responsive Element Binding protein, CREM-cAMP Responsive Element Modulator, ATF-Activating Transcription Factor) that have repeatedly been shown to have a prosurvival function. It has been reported previously that neuronal death is accompanied by increased expression of ICERs and, furthermore, their overexpression provokes neuronal cell death in culture. However, it was not explained whether endogenously activated by proapoptotic stimuli ICERs contribute to the neuronal cell death. Herein, we have examined the involvement of endogenous ICERs in the apoptosis by checking whether it is possible to protect neurons from cell death by blocking the ICER gene. We applied two different in vitro models of neuronal death of primary neuronal cultures: excitotoxic death of neurons derived from dentate gyrus, and cortical cell loss provoked by trophic deprivation. Using the lentiviral vector (LV) to deliver shRNA, specifically silencing ICERs, but not other CREM proteins, we have found that silencing of ICERs enhances the CRE-driven transcription and exerts a mild, although significant, neuroprotective effect in both models. Since we demonstrated that silencing of endogenous ICERs have protective effect on neurons exposed to apoptosis-provoking conditions, targeting ICERs might be a novel strategy to prevent neuronal loss during degenerative processes.

Tet system in the brain: transgenic rats and lentiviral vectors approach.

Local and regulated expression of exogenous genes in the central nervous system is one of the major challenges of modern neuroscience. We have approached this issue by applying the inducible tetracycline system to regulate the expression of EGFP reporter gene in double transgenic rats. We have obtained a strong induction of EGFP only in male testes, which correlated with a high level of rtTA expression only in this organ. To overcome the problem of lack of rtTA protein in the transgenic rat brain, we have delivered this Tet system activator with lentiviral vectors into the dentate gyrus of hippocampus of transgenic EGFP rats. As a result, after systemic application of doxycycline we have obtained inducible, stable and restricted to the desired brain region expression of EGFP. An advantage of this strategy is that the transgene is located in the same genetic milieu in every cell of the transgenic organism. This is crucial to obtain uniform expression of the regulated gene within the target brain structure. Combination of rat transgenesis and lentiviral vectors is a novel approach enabling precise spatiotemporal regulation of genes of interest strictly in the brain structure of choice or in other tissues.

Inducible cAMP early repressor (ICER)-evoked delayed neuronal death in the organotypic hippocampal culture.

Programmed cell death involving gene regulation and de novo protein synthesis is a major component of both normal development and a number of disease conditions. Hence, knowledge of its mechanisms, especially transcription factors, that regulate expression of the genes involved in neurodegenerative disorders is of great importance. cAMP-responsive element-binding protein (CREB) has repeatedly been implicated in the neuronal survival. In the present study we showed that inducible cAMP early repressor (ICER), an endogenous CREB antagonist, is expressed during both excitotoxic and spontaneous neuronal cell death in organotypic hippocampal slice cultures in vitro. Furthermore, overexpression of ICER via an adenoviral vector evoked neuronal cell loss in such cultures. The time course of ICER-dependent cell death was hippocampal subdivision specific, with dentate gyrus neurons dying mostly 3-7 days after the adenovector infection, followed by CA3, where neuronal death peaked after 7 days, and then CA1, where most neuronal death occurred after 7-14 days. These results underscore the usefulness of the organotypic cultures for studies of neurodegeneration and point to neuronal loss having a multifaceted nature in a complex cellular environment.

Inducible cAMP early repressor (ICER) isoforms and neuronal apoptosis in cortical in vitro culture.

CREB activation and CREB-dependent signaling pathways are crucial for neuronal survival. The term ICER (inducible cAMP early repressor) refers to four protein isoforms that are all endogenous, inducible antagonists of CREB. Jaworski and others (2003) have previously shown that one of those isoforms, ICER IIgamma, is highly expressed in apoptotic neurons in vitro and its overexpression evokes neuronal death. In this study we investigated the role of all four ICER isoforms in cortical neuronal culture, comparing their expression level in serum-deprived/MK-801-treated neurons and their pro-apoptotic properties towards transfected cortical neurons. We have found that all four isoforms are induced upon pro-apoptotic treatment, and also that each of them separately evokes neuronal cell death following cortical culture transfection with the genes. The most efficiently induced, as well as the most effective in evoking neuronal cell death, were both ICER Igamma and IIgamma isoforms.

Mechanisms of ammonia-induced cell death in rat cortical neurons: roles of NMDA receptors and glutathione.

The occurrence, nature and prevention of ammonia-induced cell death were assayed in cultured primary cortical neurons from newborn rats. Treatment with 1-10 mM ammonium chloride for 24 or 48 h, dose-dependently decreased neuronal survival (MTT assay) and GSH/GSSG ratio in the cultures, whereas total GSH content was significantly reduced only with 10mM ammonia. Treatment with a glutathione synthesis inhibitor, buthionyl sulfoximine (BSO) (10 microM), decreased the GSH content and GSH/GSSG ratio to a degree similar to that of 10 mM ammonia, but it did not decrease cell survival in control cells. This indicates that glutathione depletion per se is not a cause of ammonia-induced neuronal death. However, ammonia-induced decrease of cell viability was attenuated by incubation with glutathione diethyl ester (GEE), which transiently increased the intracellular GSH level in both control and ammonia-treated cells. Neuronal survival in the presence of ammonia was partly improved by the NMDA receptor antagonists MK-801 and APV. Morphological analysis revealed that ammonia treatment causes both apoptotic and non-apoptotic neuronal death, the former not being inhibited by MK-801. Apoptosis was the dominant type of cell death at 10mM ammonia, as concluded both from morphologic examination and the absence of survival improvement in the presence of GABA+nipecotic acid or taurine, model anti-excitotoxic treatments of cortical neurons. The mechanism underlying apoptosis may include inhibition of a survival kinase, Akt, whose activatory phosphorylation at Ser473 is reduced in neurons treated with 10 mM, but not 1 mM ammonia.

The Src family kinase Hck couples BCR/ABL to STAT5 activation in myeloid leukemia cells.

Signal transducer and activator of transcription 5 (STAT5) is constitutively activated by BCR/ABL, the oncogenic tyrosine kinase responsible for chronic myelogenous leukemia. The mechanism of BCR/ABL-mediated STAT5 activation is unknown. We show here that the BCR/ABL SH3 and SH2 domains interact with hematopoietic cell kinase (Hck), leading to the stimulation of Hck catalytic activity. Active Hck phosphorylated STAT5B on Tyr699, which represents an essential step in STAT5B stimulation. Moreover, a kinase-dead Hck mutant and Hck inhibitor PP2 abrogated BCR/ABL-dependent activation of STAT5 and elevation of expression of STAT5 downstream effectors A1 and pim-1. These data identify a novel BCR/ABL-Hck-STAT5 signaling pathway, which plays an important role in BCR/ABL-mediated transformation of myeloid cells.

Phosphatidylinositol-3 kinase inhibitors enhance the anti-leukemia effect of STI571.

BCR/ABL fusion tyrosine kinase is responsible for the initiation and maintenance of the Philadelphia chromosome (Ph(1))-positive chronic myelogenous leukemia (CML) and a cohort of acute lymphocytic leukemias (ALL). STI571 (Gleevec), a novel anti-leukemia drug targeting BCR/ABL kinase can induce remissions of the Ph(1)-positive leukemias. STI571 was recently combined with the standard cytostatic drugs to achieve better therapeutic results and to overcome emerging drug resistance mechanisms. We decided to search for a more specific partner compound for STI571. Our previous studies showed that a signaling protein phosphatidylinositol-3 kinase (PI-3k) is essential for the growth of CML cells, but not of normal hematopoietic cells (Blood, 86:726,1995). Therefore the anti- Ph(1)-leukemia effect of the combination of BCR/ABL kinase inhibitor STI571 and PI-3k inhibitor wortmannin (WT) or LY294002 (LY) was tested. We showed that STI571+WT exerted a synergistic effect against the Ph(1)-positive cell lines, but did not affect the growth of Ph(1)-negative cell line. Moreover, the combinations of STI571+WT or STI571+LY were effective in the inhibition of clonogenic growth of CML-chronic phase and CML-blast crisis patient cells, while sparing normal bone marrow cells. Single colony RT-PCR assay showed that colonies arising from the mixture of CML cells and normal bone marrow cells after treatment with STI571+WT were selectively depleted of BCR/ABL-positive cells. Biochemical analysis of the CML cells after the treatment revealed that combination of STI571+WT caused a more pronounced activation of caspase-3 and induced massive apoptosis, in comparison to STI571 and WT alone. In conclusion, combination of STI571+WT or STI571+LY may represent a novel approach against the Ph(1)-positive leukemias.