Lethal weapons : novel approaches for receptor-targeted cancer cell elimination

The currently used forms of cancer therapy are associated with drug resistance and toxicity to healthy tissues. Thus, more efficient methods are needed for cancer-specific induction of growth arrest and programmed cell death, also known as apoptosis. Therapeutic forms of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) are investigated in clinical trials due to the capability of TRAIL to trigger apoptosis specifically in cancer cells by activation of cell surface death receptors. Many tumors, however, have acquired resistance to TRAIL-induced apoptosis and sensitizing drugs for combinatorial treatments are, therefore, in high demand. This study demonstrates that lignans, natural polyphenols enriched in seeds and cereal, have a remarkable sensitizing effect on TRAIL-induced cell death at non-toxic lignan concentrations. In TRAIL-resistant and androgen-dependent prostate cancer cells we observe that lignans repress receptor tyrosine kinase (RTK) activity and downregulate cell survival signaling via the Akt pathway, which leads to increased TRAIL sensitivity. A structure-activity relationship analysis reveals that the γ-butyrolactone ring of the dibenzylbutyrolactone lignans is essential for the rapidly reversible TRAIL-sensitizing activity of these compounds. Furthermore, the lignan nortrachelogenin (NTG) is identified as the most efficient of the 27 tested lignans and norlignans in sensitization of androgen-deprived prostate cancer cells to TRAIL-induced apoptosis. While this combinatorial anticancer approach may leave normal cells unharmed, several efficient cancer drugs are too toxic, insoluble or unstable to be used in systemic therapy. To enable use of such drugs and to protect normal cells from cytotoxic effects, cancer-targeted drug delivery vehicles of nanometer scale have recently been generated. The newly developed nanoparticle system that we tested in vitro for cancer cell targeting combines the efficient drug-loading capacity of mesoporous silica to the versatile particle surface functionalization of hyperbranched poly(ethylene imine), PEI. The mesoporous hybrid silica nanoparticles (MSNs) were functionalized with folic acid to promote targeted internalization by folate receptor overexpressing cancer cells. The presented results demonstrate that the developed carrier system can be employed in vitro for cancer selective delivery of adsorbed or covalently conjugated molecules and furthermore, for selective induction of apoptotic cell death in folate receptor expressing cancer cells. The tested carrier system displays potential for simultaneous delivery of several anticancer agents specifically to cancer cells also in vivo.

Cellular responses to stress and kinase signaling activation : apoptosis and differentiation

Stressignaler avkänns många gånger av membranbundna proteiner som översätter signalerna till kemisk modifiering av molekyler, ofta proteinkinaser Dessa kinaser överför de avkodade budskapen till specifika transkriptionsfaktorer genom en kaskad av sekventiella fosforyleringshändelser, transkriptionsfaktorerna aktiverar i sin tur de gener som behövs för att reagera på stressen. En av de mest kända måltavlorna för stressignaler är transkriptionsfaktor AP-1 familjemedlemen c-Jun. I denna studie har jag identifierat den nukleolära proteinet AATF som en ny regulator av c-Jun-medierad transkriptionsaktivitet. Jag visar att stresstimuli inducerar omlokalisering av AATF vilket i sin tur leder till aktivering av c-Jun. Den AATF-medierad ökningen av c-Jun-aktiviteten leder till en betydande ökning av programmerad celldöd. Parallellt har jag vidarekarakteriserat Cdk5/p35 signaleringskomplexet som tidigare har identifierats i vårt laboratorium som en viktig faktor för myoblastdifferentiering. Jag identifierade den atypiska PKCξ som en uppströms regulator av Cdk5/p35-komplexet och visar att klyvning och aktivering av Cdk5 regulatorn p35 är av fysiologisk betydelse för differentieringsprocessen och beroende av PKCξ aktivitet. Jag visar att vid induktion av differentiering fosforylerar PKCξ p35 vilket leder till calpain-medierad klyvning av p35 och därmed ökning av Cdk5-aktiviteten. Denna avhandling ökar förståelsen för de regulatoriska mekanismer som styr c-Jun-transkriptionsaktiviteten och c-Jun beroende apoptos genom att identifiera AATF som en viktig faktor. Dessutom ger detta arbete nya insikter om funktionen av Cdk5/p35-komplexet under myoblastdifferentiering och identifierar PKCξ som en uppströms regulator av Cdk5 aktivitet och myoblast differentiering.

Unconventional integrin-ligand interactions

Integrins are cell surface adhesion and signaling receptors. Cells use integrins to attach to the extracellular matrix and to other cells, as well as for sensing their environment. In addition to adhesion and migration, integrins have been shown to be important for many biological processes including apoptosis, cell proliferation, and differentiation into specific tissues. Many important next generation biological drugs inhibit integrin functions. Thus, research into interactions between integrins and their ligands under different physiological and pathological conditions is not only of academic interest, but is also important for the field of drug discovery. In this Ph.D. project, the functions of integrin-ligand interactions were studied under different physiologically interesting conditions including 1) human echovirus 1 binding to integrin α2β1, 2) integrin α2β1 binding to collagen under flow conditions, 3) integrin α2β1 binding to a ligand in the presence of the angiogenesis inhibitor histidine rich glycoprotein (HRG) and 4) integrin binding to posttranslationally citrullinated ligands. As a result of the project, we could show that for each condition the integrin-ligand interaction is somewhat unconventional. 1) Echovirus 1 binds only to non-activated conformations of integrin α2β1. 2) Surprisingly, the non-activated conformation is also the primary conformation of integrin α2β1 when it binds to collagen under flow conditions, like when platelets adhere to subendothelial collagen in vascular injuries. In addition, the pre-activation of integrin α2β1 does not increase adhesion under flow. 3) HRG binds to integrin α2β1 through a low-affinity interaction that inhibits integrin binding to collagen. This shows that low affinity interactions could be biologically relevant and possibly regulate angiogenesis. 4) The citrullination of collagen, a posttranslational modification reported to occur in rheumatoid arthritis, specifically inhibits the binding of integrin α10β1 and α11β1, but does not affect the binding of α1β1 ja α2β1. On the other hand, the citrullination of isoDGR in fibronectin and RGD in pro-TGF- β:n inhibit integrin binding completely. Citrullination seems to be an inflammation related process and integrin ligands become citrullinated frequently in vivo. This Ph.D. thesis suggests that unconventional interaction mechanisms between integrins and their ligands, such as posttranslational modifications, low affinity interactions, and non-activated integrin conformations, can have an important role in pathological processes. The study of these kinds of integrin-ligand interactions is important for understanding biological phenomena more deeply. The research might also be beneficial for the development of integrin based therapies for treating diseases.

Osteoclastic tartrate-resistant acid phosphatase 5b. Diagnostic use and biological significance in bone physiology

The skeleton undergoes continuous turnover throughout life. In women, an increase in bone turnover is pronounced during childhood and puberty and after menopause. Bone turnover can be monitored by measuring biochemical markers of bone resorption and bone formation. Tartrate-resistant acid phosphatase (TRACP) is an enzyme secreted by osteoclasts, macrophages and dendritic cells. The secreted enzyme can be detected from the blood circulation by recently developed immunoassays. In blood circulation, the enzyme exists as two isoforms, TRACP 5a with an intact polypeptide chain and TRACP 5b in which the polypeptide chain consists of two subunits. The 5b form is predominantly secreted by osteoclasts and is thus associated with bone turnover. The secretion of TRACP 5b is not directly related to bone resorption; instead, the levels are shown to be proportional to the number of osteoclasts. Therefore, the combination of TRACP 5b and a marker reflecting bone degradation, such as C-terminal cross-linked telopeptides of type I collagen (CTX), enables a more profound analysis of the changes in bone turnover. In this study, recombinant TRACP 5a-like protein was proteolytically processed into TRACP 5b-like two subunit form. The 5b-like form was more active both as an acid phosphatase and in producing reactive oxygen species, suggesting a possible function for TRACP 5b in osteoclastic bone resorption. Even though both TRACP 5a and 5b were detected in osteoclasts, serum TRACP 5a levels demonstrated no change in response to alendronate treatment of postmenopausal women. However, TRACP 5b levels decreased substantially, demonstrating that alendronate decreases the number of osteoclasts. This was confirmed in human osteoclast cultures, showing that alendronate decreased the number of osteoclats by inducing osteoclast apoptosis, and TRACP 5b was not secreted as an active enzyme from the apoptotic osteoclasts. In peripubertal girls, the highest levels of TRACP 5b and other bone turnover markers were observed at the time of menarche, whereas at the same time the ratio of CTX to TRACP 5b was lowest, indicating the presence of a high number of osteoclasts with decreased resorptive activity. These results support the earlier findings that TRACP 5b is the predominant form of TRACP secreted by osteoclasts. The major source of circulating TRACP 5a remains to be established, but is most likely other cells of the macrophage-monocyte system. The results also suggest that bone turnover can be differentially affected by both osteoclast number and their resorptive activity, and provide further support for the possible clinical use of TRACP 5b as a marker of osteoclast number.

Conformational Regulation of alpha2beta1 Integrin in Ligand Binding

Integrins are heterodimeric cell adhesion receptors involved in cell-cell and cell-extracellular matrix (ECM) interactions. They transmit bidirectional signals across the cell membrane. This results in a wide range of biological events from cell differentiation to apoptosis. alpha2beta1 integrin is an abundant collagen receptor expressed on the surface of several cell types. In addition to ECM ligands, alpha2beta1 integrins are bound by echovirus 1 (EV1) which uses alpha2beta1 as a receptor to initiate its life cycle in the infected cell. The aim of this thesis project was to provide further insight into the mechanisms of alpha2beta1 integrin ligand recognition and receptor activation. Collagen fibrils are the principal tensile elements of the ECM. Yet, the interaction of alpha2beta1 integrin with the fibrillar form of collagen I has received relatively little attention. This research focused on the ability of alpha2beta1 integrin to act as a receptor for type I collagen fibrils. Also the molecular requirements of the EV1 interaction with alpha2beta1 were studied. Conventionally, ligand binding has been suggested to require integrin activation and the binding may further trigger integrin signalling. Another main objective of this study was to elucidate both the inside-out and outside-in signalling mechanisms of alpha2beta1 integrin in adherent cells. The results indicated that alpha2beta1 integrin is the principal integrin-type collagen receptor for type I collagen fibrils, and alpha2beta1 may participate in the regulation of pericellular collagen fibrillogenesis. Furthermore, alpha2beta1 integrin inside-out activation appeared to be synergistically regulated by integrin clustering and conformational activation. The triggering of alpha2beta1 integrin outside-in signalling, however, was shown to require both conformational changes and clustering. In contrast to ECM ligands, EV1 appeared to take advantage of the bent, inactive form of alpha2beta1 integrin in initiating its life cycle in the cell. This research together with other recent studies, has shed light on the molecular mechanisms of integrin activation. It is becoming evident that large ligands are able to bind to the bent form of integrin, which has been previously considered to be physiologically inactive. Consequently, our understanding of the conformational modulation of integrins upon activation is changing.

Prediction of 3D structure and ligand-binding properties : ABC transporters and flavodiiron proteins from Synechocystis sp. strain PCC 6803

Cyanobacteria are unicellular, non-nitrogen-fixing prokaryotes, which perform photosynthesis similarly as higher plants. The cyanobacterium Synechocystis sp. strain PCC 6803 is used as a model organism in photosynthesis research. My research described herein aims at understanding the function of the photosynthetic machinery and how it responds to changes in the environment. Detailed knowledge of the regulation of photosynthesis in cyanobacteria can be utilized for biotechnological purposes, for example in the harnessing of solar energy for biofuel production. In photosynthesis, iron participates in electron transfer. Here, we focused on iron transport in Synechocystis sp. strain PCC 6803 and particularly on the environmental regulation of the genes encoding the FutA2BC ferric iron transporter, which belongs to the ABC transporter family. A homology model built for the ATP-binding subunit FutC indicates that it has a functional ATPbinding site as well as conserved interactions with the channel-forming subunit FutB in the transporter complex. Polyamines are important for the cell proliferation, differentiation and apoptosis in prokaryotic and eukaryotic cells. In plants, polyamines have special roles in stress response and in plant survival. The polyamine metabolism in cyanobacteria in response to environmental stress is of interest in research on stress tolerance of higher plants. In this thesis, the potd gene encoding an polyamine transporter subunit from Synechocystis sp. strain PCC 6803 was characterized for the first time. A homology model built for PotD protein indicated that it has capability of binding polyamines, with the preference for spermidine. Furthermore, in order to investigate the structural features of the substrate specificity, polyamines were docked into the binding site. Spermidine was positioned very similarly in Synechocystis PotD as in the template structure and had most favorable interactions of the docked polyamines. Based on the homology model, experimental work was conducted, which confirmed the binding preference. Flavodiiron proteins (Flv) are enzymes, which protect the cell against toxicity of oxygen and/or nitric oxide by reduction. In this thesis, we present a novel type of photoprotection mechanism in cyanobacteria by the heterodimer of Flv2/Flv4. The constructed homology model of Flv2/Flv4 suggests a functional heterodimer capable of rapid electron transfer. The unknown protein sll0218, encoded by the flv2-flv4 operon, is assumed to facilitate the interaction of the Flv2/Flv4 heterodimer and energy transfer between the phycobilisome and PSII. Flv2/Flv4 provides an alternative electron transfer pathway and functions as an electron sink in PSII electron transfer.