La phosphatidylinositol-3 kinase (PI3-K) est un complexe comportant une sous-unité catalytique (C) et régulatrice (R). En ce qui concerne la classe I, cinq isoformes R (p85α, p55α, p50α, p85β et p55γ) et quatre isoformes C (p110α, p110β, p110δ et p110γ) sont connues. Plusieurs études ont souligné les rôles cruciaux de la voie PI3-K/Akt dans une panoplie de processus cellulaires, dont la survie. De plus, cette voie est l’une des plus altérées dans plusieurs types de cancers, dont le cancer colorectal (CCR). Par ailleurs, il est bien établi que l’acquisition d’une résistance à l’anoïkose constitue une étape cruciale et limitante dans la progression de plusieurs types de cancers, incluant une fois de plus le CCR. Nous avons précédemment démontré que les complexes isoformes PI3-K sont engagés/recrutés par la signalisation intégrines FAK/Src de suppression d’anoïkose et, conséquemment, exercent des rôles distincts dans la survie cellulaire des entérocytes normaux selon leur état de différenciation. Ainsi, l’hypothèse de recherche de la présente étude est que les complexes isoformes PI3-K contribuent sélectivement à l’acquisition d’une résistance à l’anoïkose, et ce, chez les cellules CCR humaines. Les objectifs principaux sont les suivants : 1) Établir le profil des complexes isoformes PI3-K chez des lignées cancéreuses colorectales humaines exhibant différents degrés de résistance à l'anoïkose (modéré à fortement élevé); et 2) Déterminer fonctionnellement les contributions des complexes isoformes PI3-K identifiés dans le maintien de résistance à l'anoïkose. Nos résultats indiquent que : 1) il existe des distinctions entre les profils de complexes isoformes PI3-K prédominants chez des cellules CCR exhibant différents degrés de résistance à l’anoïkose; 2) Ces complexes PI3-K peuvent jouer ou non des rôles dans la survie cellulaire, et ce, peu importe le degré de résistance à l’anoïkose exhibé; 3) les distinctions de profils de complexes isoformes ne semblent pas corrélées avec la progression de résistance à l’anoïkose; 4) il y aurait un mécanisme anormal de compensation entre les isoformes par rapport aux complexes formés et fonctions spécifiques chez les cellules CCR; et 5) une implication de la voie MEK/Erk compliquerait d’autant plus l’investigation des rôles des complexes PI3-K chez les cellules exhibant un degré élevé de résistance à l’anoïkose. Somme toute, cela laisse entrevoir que la progression dans la résistance à l’anoïkose implique une complexité plus importante qu’anticipée, chez les cellules CCR. / Abstract : The Phosphatidylinositol-3 kinase (PI3-K) is a lipid kinase enzyme complex formed of a catalytic (C) and a regulatory (R) subunit. To date, class I PI3-K members include five R (p85α, p55α, p50α, p85β et p55γ) and four C (p110α, p110β, p110δ et p110γ). Many studies already highlighted the crucial roles of the PI3-K/Akt pathway in many, if not all, cell processes, including cell survival. Moreover, PI3-K/Akt constitutes one of the most altered pathways in cancer, including in colorectal cancer (CRC). Incidentally, it is well established that the acquisition of anoikis resistance constitutes a crucial and limiting step in the progression of many cancers, including once again, in CRC. We have already demonstrated that PI3-K isoform complexes are selectively engaged by the anoikis-suppressing integrins/FAK/Src signaling and that this translates in distinct roles in normal enterocytes survival, according to their state of differentiation. This establishes a basis for the working hypothesis of the present report which is that PI3-K isoform complexes distinctly contributes to the acquisition and maintenance of anoikis resistance in human CRC cells. The main goals were as follows: 1) To establish the expression profiles of PI3-K isoform complexes in human CRC cells displaying differing anoikis resistance degrees; and 2) To define the functional contributions of said PI3-K isoform complexes in the maintenance of anoikis resistance. Our results indicate that 1) the expression profiles of PI3-K isoform complexes are distinct between human CRC cells displaying differing anoikis resistance degrees; 2) these PI3-K isoform complexes can perform, or not, roles in cell survival regardless of the anoikis resistance degree displayed; 3) the observed distinctions of the expression profiles of PI3-K isoform complexes do not correlate with the progression of anoikis resistance; 4) there seems to be an abnormal compensation mechanism between the various PI3-K isoforms linked not only with the nature of the complexes that are formed, but to their specific functions as well; and 5) the MEK/Erk pathway seems implicated in a cross-talk signaling loop, the nature of which further complexifies the investigation of the roles of PI3-K isoform complexes in human CRC cells displaying differing anoikis resistance degrees. Taken together, these observations suggest a more complex network of interactions than previously thought in human CRC cells.

Peripheral T-cell non-Hodgkin lymphoma (PTCL) are heterogeneous, rare, and aggressive diseases mostly incurable with current cell cycle therapies. Aurora kinases (AKs) are key regulators of mitosis that drive PTCL proliferation. Alisertib (AK inhibitor) has a response rate similar to 30% in relapsed and refractory PTCL (SWOG1108). Since PTCL are derived from CD4(+)/CD8(+) cells, we hypothesized that Program Death Ligand-1 (PDL1) expression is essential for uncontrolled proliferation. Combination of alisertib with PI3K alpha (MLN1117) or pan-PI3K inhibition (PF-04691502) or vincristine (VCR) was highly synergistic in PTCL cells. Expression of PD-L1 relative to PD-1 is high in PTCL biopsies (similar to 9-fold higher) and cell lines. Combination of alisertib with pan-PI3K inhibition or VCR significantly reduced PD-L1, NF-kappa B expression and inhibited phosphorylation of AKT, ERK1/2 and AK with enhanced apoptosis. In a SCID PTCL xenograft mouse model, alisertib displayed high synergism with MLN1117. In a syngeneic PTCL mouse xenograft model alisertib demonstrated tumor growth inhibition (TGI) similar to 30%, whilst anti-PD-L1 therapy alone was ineffective. Alisertib + anti-PD-L1 resulted in TGI > 90% indicative of a synthetic lethal interaction. PF-04691502 + alisertib + anti-PD-L1 + VCR resulted in TGI 100%. Overall, mice tolerated the treatments well. Co-targeting AK, PI3K and PD-L1 is a rational and novel therapeutic strategy for PTCL.

Adult skeletal muscles have remarkable regenerative capacity. Muscle regeneration occurs when muscle tissue experiences injury, causing a population of normally quiescent muscle-resident stem cells, called satellite cells, to become activated. The CCAAT/enhancer binding proteins known as C/EBPs are transcription factors belonging to the bZIP family. Previous work from our lab has identified C/EBPβ as an important negative regulator of myogenesis. C/EBPβ expression is localized to muscle satellite cells and is downregulated upon induction to differentiate, mirroring the loss of Pax7 expression in early myogenesis. C/EBPβ expression also negatively regulates MyoD protein expression. Leaky ribosomal scanning of the Cebpb mRNA produces three C/EBPβ isoforms: LAP*, LAP and LIP, though the individual role of each of these isoforms has not been investigated in myoblasts. This thesis focuses on determining the role of each of the C/EBPβ isoforms during skeletal muscle differentiation. Forced expression of the C/EBPβ-LIP isoform in myoblasts led to a decrease in Myf5, MyoD, and myogenin expression under differentiation conditions when compared to empty vector controls. Further, the fusion of cells was greatly reduced following differentiation. C/EBPβ-LIP expressing cells also demonstrated a growth defect, with pronounced G1 arrest and features of senescence. In contrast, myoblasts expressing the C/EBPβ-LAP isoform has impaired differentiation, though this was not as pronounced as in C/EBPβ-LIP expressing cells and proliferated normally. While LIP is not normally expressed in primary myoblasts from healthy muscle, the ratio of LIP:LAP was increased in primary myoblasts isolated from mdx mice, an animal model for Duchenne muscular dystrophy. These findings suggest that the regulation of C/EBPβ isoform expression could regulate stem cell stamina and may contribute to defects in muscle regeneration in disease.

Apart from the p53 protein, the TP53 tumor-suppressor gene is expressed as another eleven protein isoforms with the use of alternative splicing, alternative promotors and alternative translational initiation sites. Abnormal expression of these isoforms has been observed in tumor tissues. The binding properties as well as the biological functions are also modulated, due to sequential and therefore structural differences from the p53 protein. p53 is regulated by these isoforms in both suppressive and supportive manner. Explanation of the p53 isoform regulation mechanism in cells could lead to desired alternative splicing of the chosen isoforms, and modulation of isoform expression could be used in cancer treatment based on p53 therapy. Basic information about p53 protein is summarised in the theoretical part of this master thesis, supplemented with recent advances in the field of p53 isoforms, as well as the Gateway cloning method. The main goal of the experimental part was p53 isoform production in a bacterial expression system. Prior to the protein production, DNA sequences coding twelve p53 isoforms were prepared using PCR and Gateway cloning. In total, twelve entry clones and eight expression clones were prepared by cloning the isoforms’ sequences. After the protein production and purification, the detection using SDS-PAGE and Western Blotting was performed with five p53 protein isoforms: p53, 40p53, 40p53 and 40p53. DNA binding properties of p53 protein isoforms will be tested in subsequent research.

The theoretical part of the thesis summarizes general information about the human TP53 gene and p53 protein, which is encoded by this gene. There is also dealt with the transcriptional variants - p53 isoforms. The experimental part is focused on the detection of isoform p53 and its shorter, yet undescribed form. The starting material is peripheral blood of patients from University Hospital Brno, from which RNA was isolated. Subsequently, RNA was transcribed by reverse transcription into cDNA which was amplified by polymerase chain reaction (PCR). Experimental measurements demonstrate that p53 occurs in all the B-lymphocytes of patients with chronic lymphocytic leukemia. It also demonstrats the existence of a shorter form of p53.

The TCF/LEF transcriptional factors regulate the target genes of the Wnt signalling pathway - one of the key signalling mechanisms involved in development of multicellular organisms. The TCF/LEF genes produce a number of various protein isoforms, which consequently leads to a great functional diversity of the TCF/LEF proteins. In this diploma project we focused on the Caenorhabditis elegans gene pop-1, the ortholog of the TCF/LEF genes, whose isoforms have not been studied yet. Using the Northern blot analysis we tried to identify alternative isoforms of the pop-1 mRNA in C. elegans. Using quantitative RT-PCR we also analyzed the pop-1 mRNA levels during seven developmental stages of C. elegans. Further, we also determined the expression profile of two important partners of pop-1, the bar-1 and sys-1 genes, whose protein products function as transcriptional co-activators. Key words: canonical Wnt signaling pathway, TCF/LEF transcription factors, Caenorhabditis elegans, pop-1

<p>Phospholipase Cs (PLCs) are a family of enzymes that hydrolyze membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2) to generate inositol triphosphate (IP3) and diacylglycerol (DAG). These second messengers activate a variety of intracellular responses, including inflammation, vascular smooth muscle contraction, and cardiac hypertrophy. While much is known about how Gaq-mediated activation of PLCb occurs, the same cannot be said for Gbg-mediated activation. Residues within the PLCb-Gbg binding interface were previously identified in interior regions of the protein, suggesting the PH domain must undergo a conformational change to allow for Gbg-mediated activation. However, the role of PH domain conformational dynamics in Gbg-mediated activation of PLCb has yet to be determined. In this work, I discuss efforts to characterize conformational dynamics of the PLCb PH domain and its role in interactions of the enzyme with liposomes and Gbg. First, I generated a disulfide crosslink between the PH domain and EF hands1/2 of PLCb3, purified under oxidizing or reducing conditions, and conducted biochemical and structural tests to determine any differences in structure and/or function of the protein as compared to wild-type. Results of these studies provided the first direct structural evidence of PLCb PH domain dynamics in solution. Then, I discuss the rationale behind the generation of a surface cysteine-less PLCb for use in solvatochromic fluorescence assays in the presence and absence of liposomes and Gbg. Initial results of these studies suggest the PLCb PH domain favors a buried conformation alone and in the presence of Gbg or liposomes, and likely exists at an equilibrium between open and closed states.</p>

BACKGROUND: α-tocopherol (AT) and γ-tocotrienol (GT3) are vitamin E isoforms considered to have potential chemopreventive properties. AT has been widely studied in vitro and in clinical trials with mixed results. The latest clinical study (SELECT trial) tested AT in prostate cancer patients, determined that AT provided no benefit, and could promote cancer. Conversely, GT3 has shown antineoplastic properties in several in vitro studies, with no clinical studies published to date. GT3 causes apoptosis via upregulation of the JNK pathway; however, inhibition results in a partial block of cell death. We compared side by side the mechanistic differences in these cells in response to AT and GT3. METHODS: The effects of GT3 and AT were studied on androgen sensitive LNCaP and androgen independent PC-3 prostate cancer cells. Their cytotoxic effects were analyzed via MTT and confirmed by metabolic assays measuring ATP. Cellular pathways were studied by immunoblot. Quantitative analysis and the determination of relationships between cell signaling events were analyzed for both agents tested. Non-cancerous prostate RWPE-1 cells were also included as a control. RESULTS: The RAF/RAS/ERK pathway was significantly activated by GT3 in LNCaP and PC-3 cells but not by AT. This activation is essential for the apoptotic affect by GT3 as demonstrated the complete inhibition of apoptosis by MEK1 inhibitor U0126. Phospho-c-JUN was upregulated by GT3 but not AT. No changes were observed on AKT for either agent, and no release of cytochrome c into the cytoplasm was detected. Caspases 9 and 3 were efficiently activated by GT3 on both cell lines irrespective of androgen sensitivity, but not in cells dosed with AT. Cell viability of non-cancerous RWPE-1 cells was affected neither by GT3 nor AT. CONCLUSIONS: c-JUN is a recognized master regulator of apoptosis as shown previously in prostate cancer. However, the mechanism of action of GT3 in these cells also include a significant activation of ERK which is essential for the apoptotic effect of GT3. The activation of both, ERK and c-JUN, is required for apoptosis and may suggest a relevant step in ensuring circumvention of mechanisms of resistance related to the constitutive activation of MEK1.

Protein kinase C (PKC) has been linked to functional and morphological changes in endothelial cells involved in increased microvessel permeability. Bradykinin and TNF are potent inflammatory mediators which translocate PKC from the cytosol to the membrane of various cell types, including endothelial cells. The PKC isoforms α, ε and ζ have been demonstrated as the most prominent in human umbilical vein endothelial cells (HUVEC). We propose that bradykinin and TNF cause increased microvascular permeability via a PKC-dependent endothelial cell signalling pathway. HUVEC were incubated at 37°C and 5% CO2 for 1 min, 15 min and 3 h with either bradykinin (1 μM) or TNF (100 U/ml). PMA incubation served as a positive control (100 nM, 15 min). Cytosolic and membrane-bound extracts were obtained by incubation in digitonin (0.5%) and Triton X100 (1%). PKC isoforms were assayed by Western blot and membrane fractions calculated. These experiments revealed that: HUVEC clearly displayed a non-uniform basal membrane fraction distribution of PKC isoforms, with ζ (35.4%) greater than ε (30.6%) and both much greater than α (8.6%); Bradykinin caused significant translocation of PKC α with 15 min and 3 h of treatment but not 1 min; TNF caused dramatic translocation of PKC α at 1 min treatment which subsided at 15 min and 3 h but remained significantly elevated; and PMA caused dramatic translocation of α and ε but not ζ. Treatments of bradykinin and TNF that translocated PKC also showed cytoskeletal rearrangement of rhodamine-phalloidin stained actin, causing it to become more prevalent near cell membranes and concentrated at focal points between cells. These results suggest that PKC α may contribute to long term low grade increases in microvessel permeability in response to bradykinin, and that PKC α could be involved in both transient and sustained microvessel permeability changes induced by TNF. Also, cytoskeletal actin organization appears to be a downstream pathway in the activation process, possibly leading to alteration in endothelial cell shape and contact points.

Huntington’s disease is a neurodegenerative disorder caused by expansion of the CAG repeat in huntingtin exon 1. Early studies demonstrated the huntingtin gene is transcribed into two 3′UTR isoforms in normal human tissue. Decades later, researchers identified a truncated huntingtin mRNA isoform in disease but not control human brain. We speculated the amount of huntingtin 3′UTR isoforms might also vary between control and Huntington’s disease brains. We provide evidence that the abundance of huntingtin 3′UTR isoforms, including a novel mid-3′UTR isoform, differs between patient and control neural stem cells, fibroblasts, motor cortex, and cerebellum. Both alleles of huntingtin contribute to isoform changes. We show huntingtin 3′UTR isoforms are metabolized differently. The long and mid isoforms have shorter half-lives, shorter polyA tails, and more microRNA and RNA binding protein sites than the short isoform. 3′UTR Isoform changes are not limited to huntingtin. Isoforms from 11% of genes change abundance in Huntington’s motor cortex. Only 17% of genes with isoform alterations are differentially expressed in disease tissue. However, gene ontology analysis suggests they share common pathways with differentially expressed genes. We demonstrate knockdown of the RNA binding protein CNOT6 in control fibroblasts results in huntingtin isoform changes similar to those in disease fibroblasts. This study further characterizes Huntington’s disease molecular pathology and suggests RNA binding protein expression may influence mRNA isoform expression in the Huntington’s disease brain.