p53 and DNA damage checkpoint responses in the human prostate

Prostate cancer is the most common noncutaneous malignancy and the second leading cause of cancer mortality in men. In 2004, 5237 new cases were diagnosed and altogether 25 664 men suffered from prostate cancer in Finland (Suomen Syöpärekisteri). Although extensively investigated, we still have a very rudimentary understanding of the molecular mechanisms leading to the frequent transformation of the prostate epithelium. Prostate cancer is characterized by several unique features including the multifocal origin of tumors and extreme resistance to chemotherapy, and new treatment options are therefore urgently needed. The integrity of genomic DNA is constantly challenged by genotoxic insults. Cellular responses to DNA damage involve elegant checkpoint cascades enforcing cell cycle arrest, thus facilitating damage repair, apoptosis or cellular senescence. Cellular DNA damage triggers the activation of tumor suppressor protein p53 and Wee1 kinase which act as executors of the cellular checkpoint responses. These are essential for genomic integrity, and are activated in early stages of tumorigenesis in order to function as barriers against tumor formation. Our work establishes that the primary human prostatic epithelial cells and prostatic epithelium have unexpectedly indulgent checkpoint surveillance. This is evidenced by the absence of inhibitory Tyr15 phosphorylation on Cdk2, lack of p53 response, radioresistant DNA synthesis, lack of G1/S and G2/M phase arrest, and presence of persistent gammaH2AX damage foci. We ascribe the absence of inhibitory Tyr15 phosphorylation to low levels of Wee1A, a tyrosine kinase and negative regulator of cell cycle progression. Ectopic Wee1A kinase restored Cdk2-Tyr15 phosphorylation and efficiently rescued the ionizing radiation-induced checkpoints in the human prostatic epithelial cells. As variability in the DNA damage responses has been shown to underlie susceptibility to cancer, our results imply that a suboptimal checkpoint arrest may greatly increase the accumulation of genetic lesions in the prostate epithelia. We also show that small molecules can restore p53 function in prostatic epithelial cells and may serve as a paradigm for the development of future therapeutic agents for the treatment of prostate cancer We hypothesize that the prostate has evolved to activate the damage surveillance pathways and molecules involved in these pathways only to certain stresses in extreme circumstances. In doing so, this organ inadvertently made itself vulnerable to genotoxic stress, which may have implications in malignant transformation. Recognition of the limited activity of p53 and Wee1 in the prostate could drive mechanism-based discovery of preventative and therapeutic agents.

VEGFR-3 and Tie pathways in vascular network formation

The blood and lymphatic vascular systems are essential for life, but they may become harnessed for sinister purposes in pathological conditions. For example, tumors learn to grow a network of blood vessels (angiogenesis), securing a source of oxygen and nutrients for sustained growth. On the other hand, damage to the lymph nodes and the collecting lymphatic vessels may lead to lymphedema, a debilitating condition characterized by peripheral edema and susceptibility to infections. Promoting the growth of new lymphatic vessels (lymphangiogenesis) is an attractive approach to treat lymphedema patients. Angiopoietin-1 (Ang1), a ligand for the endothelial receptor tyrosine kinases Tie1 and Tie2. The Ang1/Tie2 pathway has previously been implicated in promoting endothelial stability and integrity of EC monolayers. The studies presented here elucidate a novel function for Ang1 as a lymphangiogenic factor. Ang1 is known to decrease the permeability of blood vessels, and could thus act as a more global antagonist of plasma leakage and tissue edema by promoting growth of lymphatic vessels and thereby facilitating removal of excess fluid and other plasma components from the interstitium. These findings reinforce the idea that Ang1 may have therapeutic value in conditions of tissue edema. VEGFR-3 is present on all endothelia during development, but in the adult its expression becomes restricted to the lymphatic endothelium. VEGF-C and VEGF-D are ligands for VEGFR-3, and potently promote lymphangiogenesis in adult tissues, with direct and remarkably specific effects on the lymphatic endothelium in adult tissues. The data presented here show that VEGF-C and VEGF-D therapy can restore collecting lymphatic vessels in a novel orthotopic model of breast cancer-related lymphedema. Furthermore, the study introduces a novel approach to improve VEGF-C/VEGF-D therapy by using engineered heparin-binding forms of VEGF-C, which induced the rapid formation of organized lymphatic vessels. Importantly, VEGF-C therapy also greatly improved the survival and integration of lymph node transplants. The combination of lymph node transplantation and VEGF-C therapy provides a basis for future therapy of lymphedema. In adults, VEGFR-3 expression is restricted to the lymphatic endothelium and the fenestrated endothelia of certain endocrine organs. These results show that VEGFR-3 is induced at the onset of angiogenesis in the tip cells that lead the formation of new vessel sprouts, providing a tumor-specific vascular target. VEGFR-3 acts downstream of VEGF/VEGFR-2 signals, but, once induced, can sustain angiogenesis when VEGFR-2 signaling is inhibited. The data presented here implicate VEGFR-3 as a novel regulator of sprouting angiogenesis along with its role in regulating lymphatic vessel growth. Targeting VEGFR-3 may provide added efficacy to currently available anti-angiogenic therapeutics, which typically target the VEGF/VEGFR-2 pathway.

Understanding the chemistry of atmospheric aerosol particle formation via observations of physical properties

It is widely accepted that the global climate is heating up due to human activities, such as burning of fossil fuels. Therefore we find ourselves forced to make decisions on what measures, if any, need to be taken to decrease our warming effect on the planet before any irrevocable damage occurs. Research is being conducted in a variety of fields to better understand all relevant processes governing Earth s climate, and to assess the relative roles of anthropogenic and biogenic emissions into the atmosphere. One of the least well quantified problems is the impact of small aerosol particles (both of anthropogenic and biogenic origin) on climate, through reflecting solar radiation and their ability to act as condensation nuclei for cloud droplets. In this thesis, the compounds driving the biogenic formation of new particles in the atmosphere have been examined through detailed measurements. As directly measuring the composition of these newly formed particles is extremely difficult, the approach was to indirectly study their different characteristics by measuring the hygroscopicity (water uptake) and volatility (evaporation) of particles between 10 and 50 nm. To study the first steps of the formation process in the sub-3 nm range, the nucleation of gaseous precursors to small clusters, the chemical composition of ambient naturally charged ions were measured. The ion measurements were performed with a newly developed mass spectrometer, which was first characterized in the laboratory before being deployed at a boreal forest measurement site. It was also successfully compared to similar, low-resolution instruments. The ambient measurements showed that sulfuric acid clusters dominate the negative ion spectrum during new particle formation events. Sulfuric acid/ammonia clusters were detected in ambient air for the first time in this work. Even though sulfuric acid is believed to be the most important gas phase precursor driving the initial cluster formation, measurements of the hygroscopicity and volatility of growing 10-50 nm particles in Hyytiälä showed an increasing role of organic vapors of a variety of oxidation levels. This work has provided additional insights into the compounds participating both in the initial formation and subsequent growth of atmospheric new aerosol particles. It will hopefully prove an important step in understanding atmospheric gas-to-particle conversion, which, by influencing cloud properties, can have important climate impacts. All available knowledge needs to be constantly updated, summarized, and brought to the attention of our decision-makers. Only by increasing our understanding of all the relevant processes can we build reliable models to predict the long-term effects of decisions made today.

Negotiating and Renegotiating Conversational Ground Rules : Formation of an Artifact Designed to Mediate Exploratory Collaboration

This study highlights the formation of an artifact designed to mediate exploratory collaboration. The data for this study was collected during a Finnish adaptation of the thinking together approach. The aim of the approach is to teach pulps how to engage in educationally beneficial form of joint discussion, namely exploratory talk. At the heart of the approach lies a set of conversational ground rules aimed to promote the use of exploratory talk. The theoretical framework of the study is based on a sociocultural perspective on learning. A central argument in the framework is that physical and psychological tools play a crucial role in human action and learning. With the help of tools humans can escape the direct stimulus of the outside world and learn to control ourselves by using tools. During the implementation of the approach, the classroom community negotiates a set of six rules, which this study conceptualizes as an artifact that mediates exploratory collaboration. Prior research done about the thinking together approach has not extensively researched the formation of the rules, which give ample reason to conduct this study. The specific research questions asked were: What kind of negotiation trajectories did the ground rules form during the intervention? What meanings were negotiated for the ground rules during the intervention The methodological framework of the study is based on discourse analysis, which has been specified by adapting the social construction of intertextuality to analyze the meanings negotiated for the created rules. The study has town units of analysis: thematic episode and negotiation trajectory. A thematic episode is a stretch of talk-in-interaction where the participants talk about a certain ground rule or a theme relating to it. A negotiation trajectory is a chronological representation of the negotiation process of a certain ground rule during the intervention and is constructed of thematic episodes. Thematic episodes were analyzed with the adapted intertextuality analysis. A contrastive analysis was done on the trajectories. Lastly, the meanings negotiated for the created rules were compared to the guidelines provided by the approach. The main result of the study is the observation, that the meanings of the created rules were more aligned with the ground rules of cumulative talk, rather than exploratory talk. Although meanings relating also to exploratory talk were negotiated, they clearly were not the dominant form. In addition, the study observed that the trajectories of the rules were non identical. Despite connecting dimensions (symmetry, composition, continuity and explicitness) none of the trajectories shared exactly the same features as the others.

Biophysical Characterization of Oxidized Phospholipids : Implications for Altered Membrane Structure and Function

The present study aims to elucidate the modifications in the structure and functionality of the phospholipid matrix of biological membranes brought about by free radical-mediated oxidative damage of its molecular constituents. To this end, the surface properties of two oxidatively modified phospholipids bearing an aldehyde or carboxyl function at the end of truncated sn-2 acyl chain were studied using a Langmuir balance. The results obtained reveal both oxidized species to have a significant impact on the structural dynamics of phospholipid monolayers, as illustrated by the progressive changes in force-area isotherms with increasing mole fraction of the oxidized lipid component. Moreover, surface potential measurements revealed considerable modifications in the electric properties of oxidized phospholipid containing monolayers during film compression, suggesting a packing state-controlled reorientation of the intramolecular electric dipoles of the lipid headgroups and acyl chains. Based on the above findings, a model describing the conformational state of oxidized phospholipid molecules in biological membranes is proposed, involving the protrusion of the acyl chains bearing the polar functional groups out from the hydrocarbon phase to the surrounding aqueous medium. Oxidative modifications alter profoundly the physicochemical properties of unsaturated phospholipids and are therefore readily anticipated to have important implications for their interactions with membrane-associating molecules. Along these lines, the carboxyl group bearing lipid was observed to bind avidly the peripheral membrane protein cytochrome c. The binding was reversed following increase in ionic strength or addition of polyanionic ATP, thus suggesting it to be driven by electrostatic interactions between cationic residues of the protein and the deprotonated lipid carboxyl exposed to the aqueous phase. The presence of aldehyde function bearing oxidized phospholipid was observed to enhance the intercalation of four antimicrobial peptides into phospholipid monolayers and liposomal bilayers. Partitioning of the peptides to monolayers was markedly attenuated by the aldehyde scavenger methoxyamine, revealing it to be mediated by the carbonyl moiety possibly through efficient hydrogen bonding or, alternatively, formation of covalent adduct in form of a Schiff base between the lipid aldehydes and primary amine groups of the peptide molecules. Lastly, both oxidized phospholipid species were observed to bind with high affinity three small membrane-partitioning therapeutic agents, viz. chlorpromazine, haloperidol, and doxorubicin. In conclusion, the results of studies conducted using biomimetic model systems support the notion that oxidative damage influences the molecular architecture as well as the bulk physicochemical properties of phospholipid membranes. Further, common polar functional groups carried by phospholipids subjected to oxidation were observed to act as molecular binding sites at the lipid-water interface. It is thus plausible that oxidized phospholipid species may elicit cellular level effects by modulating integration of various membrane-embedded and surface-associated proteins and peptides, whose conformational state, oligomerization, and functionality is known to be controlled by highly specific lipid-protein interactions and proper physical state of the membrane environment.

The role of catechol-O-methyltransferase (COMT) and the effects of COMT inhibition in brain and in cardiovascular and renal pathophysiology

Catechol-O-methyltransferase (COMT) metabolizes catecholamines such as dopamine (DA), noradrenaline (NA) and adrenaline, which are vital neurotransmitters and hormones that play important roles in the regulation of physiological processes. COMT enzyme has a functional Val158Met polymorphism in humans, which affects the subjects COMT activity. Increasing evidence suggests that this functional polymorphism may play a role in the etiology of various diseases from schizophrenia to cancers. The aim of this project was to provide novel biochemical information on the physiological and especially pathophysiological roles of COMT enzyme as well as the effects of COMT inhibition in the brain and in the cardiovascular and renal system. To assess the roles of COMT and COMT inhibition in pathophysiology, we used four different study designs. The possible beneficial effects of COMT inhibition were studied in double-transgenic rats (dTGRs) harbouring human angiotensinogen and renin genes. Due to angiotensin II (Ang II) overexpression, these animals exhibit severe hypetension, cardiovascular and renal end-organ damage and mortality of approximately 25-40% at the age of 7-weeks. The dTGRs and their Sprague-Dawley controls tissue samples were assessed with light microscopy, immunohistochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR) and high-pressure liquid chromatography (HPLC) to evaluate the tissue damages and the possible protective effects pharmacological intervention with COMT inhibitors. In a second study, the consequence of genetic and pharmacological COMT blockade in blood pressure regulation during normal and high-sodium was elucidated using COMT-deficient mice. The blood pressure and the heart rate were measured using direct radiotelemetric blood pressure surveillance. In a third study, the effects of acute and subchronic COMT inhibition during combined levodopa (L-DOPA) + dopa decarboxylase inhibitor treatment in homocysteine formation was evaluated. Finally, we assessed the COMT enzyme expression, activity and cellular localization in the CNS during inflammation-induced neurodegeneration using Western blotting, HPLC and various enzymatic assays. The effects of pharmacological COMT inhibition on neurodegeneration were also studied. The COMT inhibitor entacapone protected against the Ang II-induced perivascular inflammation, renal damage and cardiovascular mortality in dTGRs. COMT inhibitors reduced the albuminuria by 85% and prevented the cardiovascular mortality completely. Entacapone treatment was shown to ameliorate oxidative stress and inflammation. Furthermore, we established that the genetic and pharmacological COMT enzyme blockade protects against the blood pressure-elevating effects of high sodium intake in mice. These effects were mediated via enhanced renal dopaminergic tone and suggest an important role of COMT enzyme, especially in salt-sensitive hypertension. Entacapone also ameliorated the L-DOPA-induced hyperhomocysteinemia in rats. This is important, since decreased homocysteine levels may decrease the risk of cardiovascular diseases in Parkinson´s disease (PD) patients using L-DOPA. The Lipopolysaccharide (LPS)-induced inflammation and subsequent delayed dopaminergic neurodegeneration were accompanied by up-regulation of COMT expression and activity in microglial cells as well as in perivascular cells. Interestingly, similar perivascular up-regulation of COMT expression in inflamed renal tissue was previously noted in dTGRs. These results suggest that inflammation reactions may up-regulate COMT expression. Furthermore, this increased glial and perivascular COMT activity in the central nervous system (CNS) may decrease the bioavailability of L-DOPA and be related to the motor fluctuation noted during L-DOPA therapy in PD patients.

Mitochondrial Malfunction in Tumor Formation and Neuromuscular Diseases : Consequences of defects in fumarate hydratase and in the respiratory chain

The mitochondrion is an organelle of outmost importance, and the mitochondrial network performs an array of functions that go well beyond ATP synthesis. Defects in mitochondrial performance lead to diseases, often affecting nervous system and muscle. Although many of these mitochondrial diseases have been linked to defects in specific genes, the molecular mechanisms underlying the pathologies remain unclear. The work in this thesis aims to determine how defects in mitochondria are communicated within - and interpreted by - the cells, and how this contributes to disease phenotypes. Fumarate hydratase (FH) is an enzyme of the citrate cycle. Recessive defects in FH lead to infantile mitochondrial encephalopathies, while dominant mutations predispose to tumor formation. Defects in succinate dehydrogenase (SDH), the enzyme that precedes FH in the citrate cycle, have also been described. Mutations in SDH subunits SDHB, SDHC and SDHD are associated with tumor predisposition, while mutations in SDHA lead to a characteristic mitochondrial encephalopathy of childhood. Thus, the citrate cycle, via FH and SDH, seems to have essential roles in mitochondrial function, as well as in the regulation of processes such as cell proliferation, differentiation or death. Tumor predisposition is not a typical feature of mitochondrial energy deficiency diseases. However, defects in citrate cycle enzymes also affect mitochondrial energy metabolism. It is therefore necessary to distinguish what is specific for defects in citrate cycle, and thus possibly associated with the tumor phenotype, from the generic consequences of defects in mitochondrial aerobic metabolism. We used primary fibroblasts from patients with recessive FH defects to study the cellular consequences of FH-deficiency (FH-). Similarly to the tumors observed in FH- patients, these fibroblasts have very low FH activity. The use of primary cells has the advantage that they are diploid, in contrast with the aneuploid tumor cells, thereby enabling the study of the early consequences of FH- in diploid background, before tumorigenesis and aneuploidy. To distinguish the specific consequences of FH- from typical consequences of defects in mitochondrial aerobic metabolism, we used primary fibroblasts from patients with MELAS (mitochondrial encephalopathy with lactic acidosis and stroke-like episodes) and from patients with NARP (neuropathy, ataxia and retinitis pigmentosa). These diseases also affect mitochondrial aerobic metabolism but are not known to predispose to tumor formation. To study in vivo the systemic consequences of defects in mitochondrial aerobic metabolism, we used a transgenic mouse model of late-onset mitochondrial myopathy. The mouse contains a transgene with an in-frame duplication of a segment of Twinkle, the mitochondrial replicative helicase, whose defects underlie the human disease progressive external ophthalmoplegia. This mouse model replicates the phenotype in the patients, particularly neuronal degeneration, mitochondrial myopathy, and subtle decrease of respiratory chain activity associated with mtDNA deletions. Due to the accumulation of mtDNA deletions, the mouse was named deletor. We first studied the consequences of FH- and of respiratory chain defects for energy metabolism in primary fibroblasts. To further characterize the effects of FH- and respiratory chain malfunction in primary fibroblasts at transcriptional level, we used expression microarrays. In order to understand the in vivo consequences of respiratory chain defects in vivo, we also studied the transcriptional consequences of Twinkle defects in deletor mice skeletal muscle, cerebellum and hippocampus. Fumarate accumulated in the FH- homozygous cells, but not in the compound heterozygous lines. However, virtually all FH- lines lacked cytoplasmic FH. Induction of glycolysis was common to FH-, MELAS and NARP fibroblasts. In deletor muscle glycolysis seemed to be upregulated. This was in contrast with deletor cerebellum and hippocampus, where mitochondrial biogenesis was in progress. Despite sharing a glycolytic pattern in energy metabolism, FH- and respiratory chain defects led to opposite consequences in redox environment. FH- was associated with reduced redox environment, while MELAS and NARP displayed evidences of oxidative stress. The deletor cerebellum had transcriptional induction of antioxidant defenses, suggesting increased production of reactive oxygen species. Since the fibroblasts do not represent the tissues where the tumors appear in FH- patients, we compared the fibroblast array data with the data from FH- leiomyomas and normal myometrium. This allowed the determination of the pathways and networks affected by FH-deficiency in primary cells that are also relevant for myoma formation. A key pathway regulating smooth muscle differentiation, SRF (serum response factor)-FOS-JUNB, was found to be downregulated in FH- cells and in myomas. While in the deletor mouse many pathways were affected in a tissue-specific basis, like FGF21 induction in the deletor muscle, others were systemic, such as the downregulation of ALAS2-linked heme synthesis in all deletor tissues analyzed. However, interestingly, even a tissue-specific response of FGF21 excretion could elicit a global starvation response. The work presented in this thesis has contributed to a better understanding of mitochondrial stress signalling and of pathways interpreting and transducing it to human pathology.

Rab8 and Rab8-interacting proteins as players in cell polarization

Rab8 and its interacting proteins as regulators of cell polarization During the development of a multi-cellular organism, progenitor cells have to divide and migrate appropriately as well as organize their differentiation with one another, in order to produce a viable embryo. To divide, differentiate and migrate cells have to undergo polarization, a process where internal and external components such as actin, microtubules and adhesion receptors are reorganized to produce a cell that is asymmetric, with functionally different surfaces. Also in the adult organism there is a continuous need for these processes, as cells need to migrate in response to tissue damage and to fight infection. Improper regulation of cell proliferation and migration can conversely lead to disease such as cancer. GTP-binding proteins function as molecular switches by cycling between a GTP-bound (active) conformation and a GDP-bound (inactive) conformation. The Ras super-family of small GTPases are found in all eukaryotic cells. They can be functionally divided into five subfamilies. The Ras family members mainly regulate gene expression, controlling cell proliferation and differentiation. Ras was in fact the first human oncogene to be characterized, and as much as 30% of all human tumors may be directly or indirectly caused by mutations of Ras molecules The Rho family members mainly regulate cytoskeletal reorganization. Arf proteins are known to regulate vesicle budding and Rab proteins regulate vesicular transport. Ran regulates nuclear transport as well as microtubule organization during mitosis. The focus of the thesis of Katarina Hattula, is on Rab8, a small GTPase of the Rab family. Activated Rab8 has previously been shown to induce the formation of new surface extensions, reorganizing both actin and microtubules, and to have a role in directed membrane transport to cell surfaces. However, the exact membrane route it regulates has remained elusive. In the thesis three novel interactors of Rab8 are presented. Rabin8 is a Rab8-specific GEF that localizes to vesicles where it presumably recruits and activates its target Rab8. Its expression in cells leads to remodelling of actin and the formation of polarized cell surface domains. Optineurin, known to be associated with a leading cause of blindness in humans (open-angle glaucoma), is shown to interact specifically with GTP-bound Rab8. Rab8 binds to an amino-terminal region and interestingly, the Huntingtin protein binds a carboxy-terminal region of optineurin. (Aberrant Huntingtin protein is known to be the cause Huntington s disease in humans.) Co-expression of Huntingtin and optineurin enhanced the recruitment of Huntingtin to Rab8-positive vesicular structures. Furthermore, optineurin promoted cell polarization in a similar way to Rab8. A third novel interactor of Rab8 presented in this thesis is JFC1, a member of the synaptogamin-like protein (Slp) family. JFC1 interacts with Rab8 specifically in its GTP-bound form, co-localizes with endogenous Rab8 on tubular and vesicular structures, and is probably involved in controlling Rab8 membrane dynamics. Rab8 is in this thesis work clearly shown to have a strong effect on cell shape. Blocking Rab8 activity by expression of Rab8 RNAi, or by expressing the dominant negative Rab8 (T22N) mutant leads to loss of cell polarity. Conversely, cells expressing the constitutively active Rab8 (Q67L) mutant exhibit a strongly polarized phenotype. Experiments in live cells show that Rab8 is associated with macropinosomes generated at ruffling areas of the membrane. These macropinosomes fuse with or transform into tubules that move toward the cell centre, from where they are recycled back to the leading edge to participate in protrusion formation. The biogenesis of these tubules is shown to be dependent on both actin and microtubule dynamics. The Rab8-specific membrane route studied contained several markers known to be internalized and recycled (1 integrin, transferrin, transferrin receptor, cholera toxin B subunit (CTxB), and major histocompatibility complex class I protein (MHCI)). Co-expression studies revealed that Rab8 localization overlaps with that of Rab11 and Arf6. Rab8 is furthermore clearly functionally linked to Arf6. The data presented in this thesis strongly suggests a role for Rab8 as a regulator for a recycling compartment, which is involved in providing structural and regulatory components to the leading edge to participate in protrusion formation.

Genetic alterations in microsatellite-unstable colorectal cancer

Colorectal cancer (CRC) is the third most common cancer in Finland. Of all CRC tumors, 15% display microsatellite-instability (MSI) caused by defective cellular mismatch repair. Cells displaying MSI accumulate a high number of mutations genome-wide, especially in short repeat areas, microsatellites. When targeting genes essential for cell growth or death, MSI can promote tumorigenesis. In non-coding areas, microsatellite mutations are generally considered as passenger events. Since the discovery of MSI and its linkage to cancer, more that 200 genes have been investigated for a role in MSI tumorigenesis. Although various criteria have been suggested for MSI target gene identification, the challenge has been to distinguish driver mutations from passenger mutations. This study aimed to clarify these key issues in the research field of MSI cancer. Prior to this, background mutation rate in MSI cancer has not been studied in a large-scale. We investigated the background mutation rate in MSI CRC by analyzing the spectrum of microsatellite mutations in non-coding areas. First, semenogelin I was studied for a possible role in MSI carcinogenesis. The intronic T9 repeat of semenogelin I was frequently mutated but no evidence for selection during tumorigenesis was obtained. Second, a sequencing approach was utilized to evaluate the general background mutation rate in MSI CRC. Both intronic and intergenic repeats harbored extremely high mutation rates of ≤ 87% and intergenic repeats were more unstable than the intronic repeats. As mutation rates of presumably neutral microsatellites can be high in MSI CRC in the absence of apparent selection pressure, high mutation frequency alone is not sufficient evidence for identification of driver MSI target genes. Next, an unbiased approach was designed to identify the mutatome of MSI CRC. By combining expression array data and a database search we identified novel genes possibly related to MSI CRC carcinogenesis. One of the genes was studied further. In the functional analysis this gene was observed to cause an abnormal cancer-prone cellular phenotype, possibly through altered responses to DNA damage. In our recent study, smooth muscle myosin heavy chain 11 (MYH11) was identified as a novel MSI CRC gene. Additionally, MYH11 has a well established role in acute myeloid leukemia (AML) through an oncogenic fusion protein CBFB-MYH11. We investigated further the role of MYH11 in AML by sequencing. Three novel missense variants of MYH11 were identified. None of the variants were present in the population-based control material. One of the identified variants, V71A, lies in the N-terminal SH3-like domain of MYH11 of unknown function. The other two variants, K1059E and R1792Q are located in the coil-coiled myosin rod essential for the regulation and filament formation of MYH11. The variant K1059E lies in the close proximity of the K1044N that has been functionally assessed in our earlier work of CRC and has been reported to cause total loss of MYH11 protein regulation. As the functional significance of the three novel variants examined in this work remains unknown, future studies should clarify the further role of MYH11 in AML leukaemogenesis and in other malignancies.

Regulation of tumor suppressor protein p53 in cellular stress and tumorigenesis

Functional loss of tumor suppressor protein p53 is a common feature in diverse human cancers. The ability of this protein to sense cellular damage and halt the progression of the cell cycle or direct the cells to apoptosis is essential in preventing tumorigenesis. Tumors having wild-type p53 also respond better to current chemotherapies. The loss of p53 function may arise from TP53 mutations or dysregulation of factors controlling its levels and activity. Probably the most significant inhibitor of p53 function is Mdm2, a protein mediating its degradation and inactivation. Clearly, the maintenance of a strictly controlled p53-Mdm2 route is of great importance in preventing neoplastic transformation. Moreover, impairing Mdm2 function could be a nongenotoxic way to increase p53 levels and activity. Understanding the precise molecular mechanisms behind p53-Mdm2 relationship is thus essential from a therapeutic point of view. The aim of this thesis study was to discover factors affecting the negative regulation of p53 by Mdm2, causing activation of p53 in stressed cells. As a model of cellular damage, we used UVC radiation, inducing a complex cellular stress pathway. Exposure to UVC, as well as to several chemotherapeutic drugs, causes robust transcriptional stress in the cells and leads to activation of p53. By using this model of cellular stress, our goal was to understand how and by which proteins p53 is regulated. Furthermore, we wanted to address whether these pathways affecting p53 function could be altered in human cancers. In the study, two different p53 pathway proteins, nucleophosmin (NPM) and promyelocytic leukemia protein (PML), were found to participate in the p53 stress response following UV stress. Subcellular translocations of these proteins were discovered rapidly after exposure to UV. The alterations in the cellular localizations were connected to transient interactions with p53 and Mdm2, implicating their significance in the regulation of p53 stress response. NPM was shown to control Mdm2-p53 interface and mediate p53 stabilization by blocking the ability of Mdm2 to promote p53 degradation. Furthermore, NPM mediated p53 stabilization upon viral insult. We further detected a connection between cellular pathways of NPM and PML, as PML was found to associate with NPM in UV-radiated cells. The observed temporal UV-induced interactions strongly imply existence of a multiprotein complex participating in the p53 response. In addition, PML controlled the UV response of NPM, its localization and complex formation with chromatin associated factors. The relevance of the UV-promoted interactions was demonstrated in studies in a human leukemia cell line, being under abnormal transcriptional repression due to expression of oncogenic PML-RARa fusion protein. Reversing the leukemic phenotype with a therapeutically significant drug was associated with similar complex formation between p53 and its partners as following UV. In conclusion, this thesis study identifies novel p53 pathway interactions associated with the recovery from UV-promoted as well as oncogenic transcriptional repression.

Studies on metal complex formation of environmentally friendly aminopolycarboxylate chelating agents

Aminopolykarboksyylaatteja, kuten etyleenidiamiinitetraetikkahappoa (EDTA), on käytetty useiden vuosikymmenien ajan erinomaisen metalli-ionien sitomiskyvyn vuoksi kelatointiaineena lukuisissa sovelluksissa sekä analytiikassa että monilla teollisisuuden aloilla. Näiden yhdisteiden biohajoamattomuus on kuitenkin herättänyt huolta viime aikoina, sillä niiden on havaittu olevan hyvin pysyviä luonnossa. Tämä työ on osa laajempaa tutkimushanketta, jossa on tavoitteena löytää korvaavia kelatointiaineita EDTA:lle. Tutkimuksen aiheena on kuuden kelatointiaineen metalli-ionien sitomiskyvyn kartoitus. EDTA:a paremmin luonnossa hajoavina nämä ovat ympäristöystävällisiä ehdokkaita korvaaviksi kelatointiaineiksi useisiin sovelluksiin. Työssä tutkittiin niiden kompleksinmuodostusta useiden metalli-ionien kanssa potentiometrisella titrauksella. Metalli-ionivalikoima vaihteli hieman kelatointiaineesta riippuen sisältäen magnesium-, kalsium-, mangaani-, rauta-, kupari-, sinkki-, kadmium-, elohopea-, lyijy- ja lantaani-ionit. Tutkittavat metallit oli valittu tähtäimessä olevien sovellusten, synteesissä ilmenneiden ongelmien tai ympäristönäkökohtien perusteella. Tulokset osoittavat näiden yhdisteiden metallinsitomiskyvyn olevan jonkin verran heikompi kuin EDTA:lla, mutta kuitenkin riittävän useisiin sovelluksiin kuten sellunvalkaisuprosessiin. Myrkyllisten raskasmetallien, kadmiumin, elohopen ja lyijyn kohdalla EDTA:a heikompi sitoutuminen on eduksikin, koska se yhdistettynä parempaan biohajoavuuteen saattaa alentaa tutkittujen yhdisteiden kykyä mobilisoida kyseisiä metalleja sedimenteistä. Useimmilla tutkituista yhdisteistä on ympäristönäkökulmasta etuna myös EDTA:a pienempi typpipitoisuus.