Molecular identification and targeting of colorectal cancer stem cells

Tumor initiating or cancer stem cells (CSCs) are suggested to be responsible for tumor initiation and growth. Moreover, therapy resistance and minimal residual disease are thought to result from selective resistance of CSCs. Isolation of CSCs from colon carcinomas can be accomplished by selection of a subpopulation of tumor cells based on expression of one or multiple cell surface markers associated with cancer stemness, like CD133, CD44, CD24, CD29, CD166 and Lgr5. Identification of colon CSCs will lead to a better rational for new therapies that aim to target this fraction specifically. In this review, we analyze known markers used for selection of colon CSCs and their potential function in CSC biology. Moreover, we discuss potential targeting strategies for eradicating CSCs specifically in order to develop more effective therapeutic strategies as well as to address more fundamental questions like the actual role of CSCs in tumor growth

Autophagy in Human Embryonic Stem Cells

Autophagy (macroautophagy) is a degradative process that involves the sequestration of cytosolic material including organelles into double membrane vesicles termed autophagosomes for delivery to the lysosome. Autophagy is essential for preimplantation development of mouse embryos and cavitation of embryoid bodies. The precise roles of autophagy during early human embryonic development, remain however largely uncharacterized. Since human embryonic stem cells constitute a unique model system to study early human embryogenesis we investigated the occurrence of autophagy in human embryonic stem cells. We have, using lentiviral transduction, established multiple human embryonic stem cell lines that stably express GFP-LC3, a fluorescent marker for the autophagosome. Each cell line displays both a normal karyotype and pluripotency as indicated by the presence of cell types representative of the three germlayers in derived teratomas. GFP expression and labelling of autophagosomes is retained after differentiation. Baseline levels of autophagy detected in cultured undifferentiated hESC were increased or decreased in the presence of rapamycin and wortmannin, respectively. Interestingly, autophagy was upregulated in hESCs induced to undergo differentiation by treatment with type I TGF-beta receptor inhibitor SB431542 or removal of MEF secreted maintenance factors. In conclusion we have established hESCs capable of reporting macroautophagy and identify a novel link between autophagy and early differentiation events in hESC

Transthyretin is involved in depression-like behaviour and exploratory activity

Transthyretin (TTR), the major transporter of thyroid hormones and vitamin A in cerebrospinal fluid (CSF), binds the Alzheimer β-peptide and thus might confer protection against neurodegeneration. In addition, altered TTR levels have been described in the CSF of patients with psychiatric disorders, yet its function in the CNS is far from understood. To determine the role of TTR in behaviour we evaluated the performance of TTR-null mice in standardized tasks described to assess depression, exploratory activity and anxiety. We show that the absence of TTR is associated with increased exploratory activity and reduced signs of depressive-like behaviour. In order to investigate the mechanism underlying these alterations, we measured the levels of cathecolamines. We found that the levels of noradrenaline were significantly increased in the limbic forebrain of TTR-null mice. This report represents the first clear indication that TTR plays a role in behaviour, probably by modulation of the noradrenergic system.Fundação para a Ciência e a Tecnologia (FCT) - Programa Operacional "Ciência, Tecnologia, Inovação" POCTI/NSE/37315/2001

Targeting colorectal cancer stem cells with inducible caspase-9

Colorectal cancer stem cells (CSCs) drive tumor growth and are suggested to initiate distant metastases. Moreover, colon CSCs are reportedly more resistant to conventional chemotherapy, which is in part due to upregulation of anti-apoptotic Bcl-2 family members. To determine whether we could circumvent this apoptotic blockade, we made use of an inducible active caspase-9 (iCasp9) construct to target CSCs. Dimerization of iCasp9 with AP20187 in HCT116 colorectal cancer cells resulted in massive and rapid induction of apoptosis. In contrast to fluorouracil (5-FU)-induced apoptosis, iCasp9-induced apoptosis was independent of the mitochondrial pathway as evidenced by Bax/Bak double deficient HCT116 cells. Dimerizer treatment of colon CSCs transduced with iCasp9 (CSC-iCasp9) also rapidly induced high levels of apoptosis, while these cells were unresponsive to 5-FU in vitro. More importantly, injection of the dimerizer into mice that developed a colon CSC-iCasp9-induced tumor resulted in a strong decrease in tumor size, an increase in tumor cell apoptosis and a clear loss of CD133+ CSCs. Taken together, our data indicate that dimerization of iCasp9 circumvents the apoptosis block in CSCs, which results in effective tumor regression in vivo

Regulação da apoptose e diferenciação pelo P53 em células estaminais embrionárias humanas

Tese de doutoramento em Biologia (Biologia Molecular) apresentada à Fac. de Ciências e Tecnologia da Univ. de CoimbraHuman embryonic stem cells (hESC) display pluripotency and unlimited self-renewal while retaining a normal karyotype, suggesting they possess mechanisms to minimize the consequences of DNA damage. The molecular mechanisms controlling DNA-damage induced apoptosis of hESC are poorly understood. This study investigates the role of p53 in DNA damaged-induced apoptosis and cell differentiation. hESC are extremely sensitive to spontaneous apoptosis, as well as gamma irradiation-, hydrogen peroxide- and etoposideinduced apoptosis. Indeed, early passage hESC appear to be far more sensitive to etoposideinduced apoptosis than adapted hESC and cancer cell lines. The data further show that undifferentiated hESC that express Oct4 are much more sensitive to etoposide-induced apoptosis than their more differentiated progeny. It is shown that p53 is constitutively expressed at high levels in the cytoplasm of hESC. Etoposide treatment results in rapid and extensive induction of apoptosis and leads to a further increase in p53 and PUMA expression as well as Bax processing. p53 both translocates to the nucleus and associates with the mitochondria, accompanied by colocalisation of Bax with Mcl1. hESC stably transduced with p53 shRNA, display 80 % reduction of endogenous p53 and exhibit an 80 % reduction in etoposide-induced apoptosis accompanied by constitutive downregulation of Bax and an attenuated upregulation of PUMA. Furthermore, gene profile analysis of untreated GFP and p53 shRNA transduced hESC shows that p53 controls a set of genes under normal conditions. Around 70 % of these genes contain p53 responsive elements. No significant differences were observed between cell cycle entry of GFP and p53 shRNA transduced cell lines or distribution of phases of cell cycle and the karyotypes of both cell lines were found to be normal. p53 had a small but reproducible effect in inhibiting spontaneous differentiation. Preliminary studies to address the role of p53 in differentiation of hESC revealed a slight resistance of p53 shRNA transduced cell lines to BMP4-induced differentiation and the opposite was observed in relation to Activin A treatment. Furthermore, these cells formed teratomas (with representative tissues of all three germ layers) when implanted beneath the testis capsule of SCID mice that exhibited a higher proportion of immature neural tissue compared to control cells, suggesting that these cells are more prone to undergo differentiation to the neural lineage, or mature more slowly. This study demonstrates that p53 plays a role in differentiation, is required for etoposide-induced apoptosis of hESC and reveals, at least in part, the molecular mechanism of DNA damage-induced apoptosis in hESC.As células estaminais embrionárias humanas (hESC) possuem características de pluripotência e capacidade ilimitada de auto-renovação, mantendo ao mesmo tempo um cariótipo normal, o que sugere que estas possuem mecanismos que minimizam as consequências de danos no ADN. Os mecanismos moleculares que controlam a apoptose das hESC induzida por danos no ADN são pouco compreendidos. Este estudo investiga o papel do p53 na apoptose induzida por agentes que causam danos no ADN e na diferenciação celular. As hESC são extremamente sensíveis à apoptose espontânea, como também à induzida por radiação gama, peróxido de hidrogénio e etopósido. De facto, as hESC com poucas passagens são muito mais sensíveis à apoptose induzida por etopósido do que hESC adaptadas e linhas celulares cancerígenas. Os dados deste estudo também demostram que as hESC indiferenciadas que expressam Oct4 são mais sensíveis à apoptose induzida pelo etopósido do que a sua progenia diferenciada. O p53 é constitutivamente expresso em níveis elevados no citoplasma das hESC. O tratamento com etopósido leva a uma rápida indução da apoptose e aumento da expressão do p53 e do PUMA, bem como ao processamento do Bax. O p53 transloca para o núcleo e associa-se com a mitocôndria e isto é acompanhado com a co-localização do Bax com o Mcl1. As hESC estavelmente expressam shRNA desenhado para diminuir a expressão do p53, possuem uma reducção em 80 % do p53 endógeno e exibem também uma reducção na apoptose induzida pelo etoposide, acompanhada por uma diminuição nos níveis do Bax e por uma atenuação no aumento dos níveis do PUMA. A análise do perfil da expressão de genes de hESC que expressam shRNA para reduzir a expressão do p53 e GFP mostra que o p53 controla um conjunto de genes em condições normais. Cerca de 70 % destes genes contêm nos seus promotores locais de ligação para o p53. Não foram observadas diferenças significativas na distribuição das fases do ciclo celular das hESC que expressam shRNA para reduzir a expressão do p53 e GFP e os cariótipos destas células são normais. O p53 tem um pequeno, mas reprodutivél efeito na inibição da differenciação espontânea. Estudos preliminares para investigar o papel do p53 na diferenciação das hESC revelaram uma pequena resistência das células que expressam shRNA para diminuir os níveis do p53 à diferenciação induzida pelo BMP4, sendo o oposto observado em relação ao tratamento com Activina A. Estas células formam teratomas (com tecidos representativos das três camadas germinativas) quando implantadas na cápsula de murganhos immunodeprimidos SCID que exibem uma maior proporção de tecido neural imaturo quando comparadas com teratomas formados pelas células controlo, o que sugere que elas preferencialmente se diferenciam numa linhagem neural ou que o seu processo de maturação é mais lento. Este estudo demonstra que nas hESC, o p53 desempenha um papel na diferenciação celular, é necessário para a apoptose induzida pelo etopósido e revela em parte o mecanismo molecular que controla a apoptose induzida por danos no ADN das hESC

Role of the Human ST6GalNAc-I and ST6GalNAc-II in the Synthesis of the Cancer-Associated Sialyl-Tn Antigen

International audienc

STRAIGHT-IN enables high-throughput targeting of large DNA payloads in human pluripotent stem cells

Inserting large DNA payloads (>10 kb) into specific genomic sites of mammalian cells remains challenging. Applications ranging from synthetic biology to evaluating the pathogenicity of disease-associated variants for precision medicine initiatives would greatly benefit from tools that facilitate this process. Here, we merge the strengths of different classes of site-specific recombinases and combine these with CRISPR-Cas9-mediated homologous recombination to develop a strategy for stringent site-specific replacement of genomic fragments at least 50 kb in size in human induced pluripotent stem cells (hiPSCs). We demonstrate the versatility of STRAIGHT-IN (serine and tyrosine recombinase-assisted integration of genes for high-throughput investigation) by (1) inserting various combinations of fluorescent reporters into hiPSCs to assess the excitation-contraction coupling cascade in derivative cardiomyocytes and (2) simultaneously targeting multiple variants associated with inherited cardiac arrhythmic disorders into a pool of hiPSCs. STRAIGHT-IN offers a precise approach to generate genetically matched panels of hiPSC lines efficiently and cost effectively

Isogenic Sets of hiPSC-CMs Harboring Distinct KCNH2 Mutations Differ Functionally and in Susceptibility to Drug-Induced Arrhythmias

Mutations in KCNH2 can lead to long QT syndrome type 2. Variable disease manifestation observed with this channelopathy is associated with the location and type of mutation within the protein, complicating efforts to predict patient risk. Here, we demonstrated phenotypic differences in cardiomyocytes derived from isogenic human induced pluripotent stem cells (hiPSC-CMs) genetically edited to harbor mutations either within the pore or tail region of the ion channel. Electrophysiological analysis confirmed that the mutations prolonged repolarization of the hiPSC-CMs, with differences between the mutations evident in monolayer cultures. Blocking the hERG channel revealed that the pore-loop mutation conferred greater susceptibility to arrhythmic events. These findings showed that subtle phenotypic differences related to KCNH2 mutations could be captured by hiPSC-CMs under genetically matched conditions. Moreover, the results support hiPSC-CMs as strong candidates for evaluating the underlying severity of individual KCNH2 mutations in humans, which could facilitate patient risk stratification

Kvalita veřejných služeb

V první kapitole se zabývám definicí veřejného sektoru, popisuji metodologické přístupy a metody hodnocení kvality veřejným sektorem.V druhé části popisuji město Turnov, jak v samosprávné činnosti, tak i ve výkonu státní správy.Hodnocení jsem provedla formou dotazníku