ACTAUNIVERSITATISUPSALIENSISUPPSALA2006

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 143

Taking Pressure of AnaplasticThyroid Carcinoma

Molecular Studies of Apoptosis and InterstitialHypertension

PERNILLA ROSWALL

ISSN 1651-6206ISBN 91-554-6538-2urn:nbn:se:uu:diva-6804

List of papers

This thesis is based on the following papers, which will be referred to in the text by their Roman numerals:

I Roswall, P., Bu, S., Rubin, K., Landström, M., Heldin, N.-E. 2-Methoxyestradiol Induces Apoptosis in Cultured Human Anaplastic Thyroid Carcinoma Cells. Thyroid, 16:143-150, 2006

II Lammerts, E., Roswall, P., Sundberg, C., Gotwals, P.J., Kote-liansky, V.E., Reed, R.K., Heldin, N.-E., Rubin, K. Interference with TGF- 1 and - 3 in Tumor Stroma Lowers Tumor Interstitial Fluid Pressure Independently of Growth in Experimental Carcinoma. Int. J. Cancer, 102:453-62, 2002

III Salnikov, AV., Roswall, P., Sundberg, C., Gardner, H., Hel-din, N.-E., Rubin, K. Inhibition of TGF- Modulates Macrophages and Vessel Maturation in Parallel to a Lowering of Interstitial Fluid Pres-sure in Experimental Carcinoma. Lab. Invest., 85:512-21, 2005

IV Roswall, P., Hermansson, A., Liljegren, U., Zhang, X.-Q., Heldin, N.-E. Generation of a Transgenic Mouse with Thyrocyte-specific Inducible Expression of Cre Recombinase Manuscript

Reprints were made with permission from the publishers.

Contents

Introduction.....................................................................................................7

Background.....................................................................................................8The thyroid gland .......................................................................................8

Physiology and function ........................................................................8Anaplastic thyroid carcinoma (ATC) ....................................................9Diagnosis and treatment of ATC .........................................................10

Cancer ...........................................................................................................12Tumor stroma ...........................................................................................12

Composition and function....................................................................12Tumor interstitital fluid pressure (IFP) ....................................................14Transforming growth factor- (TGF- ) and cancer .................................16

Apoptosis ......................................................................................................18Apoptosis related genes/proteins..............................................................182-Methoxyestradiol (2-ME) .....................................................................21

Site-specific recombination ..........................................................................24Tissue-specific Cre recombinase..............................................................24Inducible cre/loxP systems.......................................................................25

Present investigation .....................................................................................26Aims .........................................................................................................26Results and discussion..............................................................................27

Paper I: 2-ME-induced apoptosis in ATC cells ...................................27Paper II and III: Lowering of tumor IFP by TGF- inhibition ............28Paper IV: Thyroid-specific expression of Cre recombinase ................30

Populärvetenskaplig sammanfattning ...........................................................32

Acknowledgements.......................................................................................34

References.....................................................................................................36

Abbreviations

-SMA -smooth-muscle actin APAF-1 Apoptotic protease activating factor-1 ATC Anaplastic thyroid carcinoma COMT Catechol-O-methyltransferase DISC Death-induced signaling complex DR Death receptor ECM Extracellular matrix EGFR epidermal growth factor receptor EMT Epithelial to mesenchymal transition ER Estrogen receptor FasL Fas ligand 5-FU 5-fluorouracil 17 -HSD 17 -hydroxysteroid dehydrogenase HIF-1 Hypoxia-inducible factor-1 IFP Interstitial fluid pressure IL-1 Interleukin-1IL-1 Ra IL-1 receptor antagonist JNK c-Jun NH2-terminal kinase LBD Ligand-binding domain MAPK Mitogen-activated protein kinase 2-ME 2-Methoxyestradiol NIS Sodium iodide symporter P Pendrin PGE1 Prostaglandin E1rh-IL-1 Ra Recombinant human IL-1 Ra SOD Superoxide dismutase SV40 Simian virus 40 T3 Tri-iodo-thyronine T4 Thyroxine TAM Tumor-associated macrophage Tg Thyroglobulin TGF- Transforming growth factor-TNF- Tumor necrosis factor-TPO Thyroperoxidase TRAIL TNF-related apoptosis-inducing ligand TSH Thyroid-stimulating hormone TSHR TSH receptor VEGF Vascular endothelial growth factor

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Introduction

Each time a cell divides it opens up for a number of errors, this process is however strictly regulated through control mechanisms. DNA aberration or damage is usually taken care of through repair machinery or otherwise the cell is forced into programmed cell death (apoptosis). Cancer formation is the consequence of an accumulation of genetic alterations in the cell, due to failure in the cellular control system. According to Hanahan and Weinberg, six essential alterations are required for a cell to achieve a malignant growth pattern (Hanahan and Weinberg 2000). To become a cancer cell it needs to become self-sufficient in growth signals, insensitive to growth-inhibitory signals, evolve limitless replicative potential, escape from apoptosis, sustain angiogenesis, and to achieve invasion and metastatic ability. Lately there has been an increased focus towards studies of the adjacent stromal cells. Carci-noma cells of a solid tumor recruit normal fibroblasts, endothelial blood vessel cells, and cells from the immune system, creating an environment suitable for malignant growth. The surrounding tumor stroma is essential for the growth and metastasis of solid tumors and this is one subject that has been investigated in this thesis.

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Background

The thyroid gland Physiology and function The epithelial cells of the thyroid gland, the thyrocytes, are organized in follicles (Figure 1). The function of the thyrocytes is to produce thyroid hormones, tri-iodo-thyronine (T3) and thyroxine (T4). T3 and T4 are impor-tant regulators of growth, development and differentiation. These hormones are also very important regulators of the metabolic rate. Between the folli-cles there are individual or small groups of parafollicular cells or C cells. These cells secrete calcitonin when there is an increase in serum calcium level. The most important regulator of thyrocyte function is the thyroid-stimulating hormone (TSH), which is synthesized and released by the pitui-tary gland. TSH binds to the TSH receptor (TSHR) on the basolateral side of the thyrocytes and regulates hormone production. Iodide is taken up by the cells via the sodium iodide symporter (NIS), on the apical side pendrin (P) transport iodide out of the cell into the follicular lumen (Royaux et al. 2000). Iodide is converted into iodine catalyzed by thyroperoxidase (TPO), and incorporated into thyroglobulin (Tg), which is stored as colloid inside the follicle. The colloid is then endocytosed and degraded, and T3 and T4 are released into the blood (Figure 1).

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Tg

T3/T4

TPO

Tg+I2 Tg-I

Tg-I

T3/T4Basolateral side

Apical side

TSH

TSHR

I-

P

NIS

TG

Figure 1. Synthesis of tri-iodo-thyronine (T3) and thyroxine (T4) by the thyrocyte. Iodide (I-), iodine (I2), pendrin (P), sodium iodide symporter (NIS), thyroglobulin (Tg), thyroid-stimulating hormone (TSH), TSH receptor (TSHR), thyroperoxidase (TPO).

Anaplastic thyroid carcinoma (ATC) Thyroid carcinomas originating from the thyrocytes can be divided into dif-ferentiated and undifferentiated (anaplastic) thyroid carcinomas. The differ-entiated tumors, papillary thyroid carcinoma (PTC) and follicular thyroid carcinoma (FTC), account for 65-80% and 5-15% respectively of all thyroid tumors (DeGroot et al. 1990; LiVolsi and Asa 1994). ATC is generally con-sidered to develop from more differentiated thyroid carcinomas and repre-sents about 2% of all thyroid tumors (Carcangiu et al. 1984; Gilliland et al. 1997; Landis et al. 1998). ATC is an aggressive neoplasm, and patients usu-

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ally die from suffocation due to local tumor invasion, with a median survival time of about six months (Carcangiu et al. 1985). These tumors seldom ex-press thyroid specific markers, such as TSHR, TPO and Tg, and is therefore classified as undifferentiated (Monaco et al. 1984). The genetic mechanisms involved in the generation of thyroid cancer are not fully understood but several genetic alterations have been reported (Fig. 2). Among these are the activating mutation in RAS and BRAF, as well as RET/PTC and TRK rear-rangements and the formation of the PAX8-PPAR 1 fusion proteins after chromosomal translocation (Kroll et al. 2000; Kimura et al. 2003; Sherman 2003). Inactivating mutations of TP53 are frequently observed in ATC (Ito et al. 1992). -Catenin (CTNNB1) play a crucial role in E-cadherin-mediated cell-cell adhesion, and somatic alterations were found in 60% of the ana-lyzed ATC patients (Garcia-Rostan et al. 1999). A recent report showed a constitutive activation of NF- B particularly in undifferentiated carcinomas (Pacifico et al. 2004).

Normal thyrocyte

Follicularadenoma

Follicular carcinoma

Anaplastic carcinoma

Occultpapillary

carcinoma

Papillary carcinoma

RAS

RET/PTCTRK TP53

TP53

CTNNB1

BRAF

Figure 2. Development of anaplastic thyroid carcinoma. Modified version of the hypothesis presented by Wynford-Thomas (1993).

Diagnosis and treatment of ATC Thyroid cancer incidence increases with age and is more common in women than in men (Kravdal et al. 1991; Glattre and Kravdal 1994). Typical presen-tation of thyroid tumors is a thyroid nodule or neck node enlargement. Pa-tients with ATC usually show both local invasion and distant metastases in lung, bone and brain (Aldinger et al. 1978; Carcangiu et al. 1985). Hoarse-ness, dysphagia, cough, and shortness of breath suggest advanced disease. Fine-needle aspiration cytology is the single most informative investigation followed by histological examination (Ravetto et al. 2000). Today a combi-

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nation of external radiation, chemotherapy, and surgical resection are the common treatment of ATC tumors (Haigh et al. 2001). A large treatment study on patients with ATC showed an increased effect after combinatory treatment with hyperfractionated radiotherapy, doxorubicin and surgery (Tennvall et al. 2002).

Alternative therapeutic approaches for ATC have been tested in several experimental studies. Doxorubicin is today the most effective single cyto-toxic drug on ATC and the mechanisms behind Doxorubicin-induced apop-tosis in ATC cells were shown to be through hyperacetylation of histone 3 (Rho et al. 2005). Gene therapy, with the introduction of wildtype TP53, in combination with Doxorubicin treatment led to tumor regression of ATC in nude mice (Nagayama et al. 2000). The ONYX-015 adenovirus enhanced the anti-tumor effects of Doxorubicin and Paclitaxel (inhibitor of microtubu-lar function), and delayed the growth of xenograft ATC in nude mice in combination with radiotherapy (Portella et al. 2002; Portella et al. 2003). A combination of the farnesyltransferase inhibitor, Manumycin, and Paclitaxel has been shown to inhibit angiogenesis and tumor growth in in vivo studies of ATC in nude mice (Xu et al. 2001). In a follow-up report the above sub-stances were combined with a third substance, Minocycline, which inhibits matrix metalloproteinases (MMPs) that are required for endothelial migra-tion during angiogenesis (She and Jim Yeung 2005). This triple combination of drugs increased the survival length of nude mice with xenograft ATC. Another substance, Gefitinib, blocked activation of epidermal growth factor receptor (EGFR) by EGF, and had significant antitumor activity in an ATC tumor model in nude mouse (Schiff et al. 2004).

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Cancer

Tumor stromaComposition and function Loose connective tissues are present in all organs and surround peripheral blood vessels. Their basic structure is similar in most organs and they are rich in extracellular matrix (ECM) components with a relatively low amount of cells, such as fibroblasts, immune and inflammatory cells, and nerve cells. Fibroblasts are the primary stromal cell type that produces ECM (reviewed by (Liotta and Kohn 2001). The ECM consists of a collagen fiber scaffold that contains a gel phase made up of hyaluronan, proteoglycans, and other glycoproteins. The main function of the collagen fibers is to absorb stress, while the gel distributes the hydraulic pressure. Fibroblasts and ECM in normal tissue have a restrictive influence on epithelial cells, regarding their morphogenesis and proliferation, thereby suppressing transformation (Maffini et al. 2004).

Tumor stroma represents a pathologic loose connective tissue with impor-tant functions for the growing carcinoma. Pathologists early discovered that the number of fibroblasts was strongly increased in for instance breast and colon carcinoma, in certain cases constituting up to 90% of the tumor mass, compared to normal loose connective tissue (Schurch et al. 1981; Sappino et al. 1990). There is a striking similarity between the stromal response during tumor progression and wound repair. Tumors have been referred to as “wounds that do not heal” (reviewed by (Dvorak 1986)). During tumori-genesis fibroblasts lose their ability to revert their phenotype back to normal, compared to wound healing when the alterations are reversible. This might be due to adjacent carcinoma cells, which constantly stimulate the fibro-blasts.

In order to grow, solid tumors require blood vessels for oxygen and nutri-tion supply. Angiogenesis is the generation of new capillaries from already existing vessels. This is a critical process during development, wound heal-ing, and various diseases such as cancer (Folkman and Shing 1992). Angio-genesis are mediated by the stromal components and permits tumor growth (Carmeliet 2000). Due to imbalance or lack of communication between fac-

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tors involved in angiogenesis, and uncontrolled growth, the tumor blood vessels become irregular and leaky. They often have defects in pericyte cov-erage and function (Abramsson et al. 2002; Baluk et al. 2003). The blood vessel abnormality leads to insufficient blood supply to the tumor. Together with the finding that lymphatic vessels are absent in tumors this leads to non-functional drainage of the fluid (Alitalo and Carmeliet 2002; Padera et al. 2002). Due to this insufficient blood supply especially large solid tumors have a central part characterized by tissue death, or necrosis. The insufficient blood supply also gives rise to areas with low oxygen level (hypoxia), and this has been shown to induce dedifferentiation of human neuroblastoma cells into a more malignant phenotype (Jogi et al. 2002).

The stromal components and especially the fibroblasts do not only pro-vide structural support and protection of the vasculature but also play a ma-jor role in the maintenance and growth of the tumor (Atula et al. 1997). An increased number of activated fibroblasts changes the ECM by a higher pro-duction of collagens, fibronectins, glycosaminoglycans and proteoglycans (Mahfouz et al. 1987). In many cases the tumor stroma appears as a fibrotic tissue e.g. in ATC (Dahlman et al. 2000; Dahlman et al. 2002). The higher collagen content makes the tumor stroma “stiffer” than normal loose connec-tive tissues (Lagace et al. 1985; Minamoto et al. 1988). During invasion the stroma is involved in both matrix degradation and tumor cell migration (Sappino et al. 1990; Gregoire and Lieubeau 1995).

Monocytes are continously recruited to tumors and infiltrate the stroma where they differentiate into tumor-associated macrophages (TAMs). These macrophages may play a cytotoxic role in the defense mechanisms towards tumor cells but also promote tumor progression (reviewed by (Sunderkotter et al. 1994; Pepper 2001)). In most studies a high number of infiltrated TAMs is correlated to a more aggressive tumor progression (Shimura et al. 2000; Bingle et al. 2002). TAMs seem to accumulate in hypoxic areas and they are probably recruited by chemoattractants, such as vascular endothelial growth factor (VEGF) and endothelin-2, released from hypoxic tumor or stromal cells (Leek et al. 1999; Lewis et al. 2000; Grimshaw et al. 2002). Hypoxia induces a pro-tumor phenotype of the macrophages. Once situated in hypoxic areas, TAMs stimulate angiogenesis through VEGF and angio-poietin production, and also through production of proteases necessary for the endothelial cell movement into the tumor tissue (Arias et al. 2005; Lewis and Murdoch 2005).

Activation of macrophages may be defined as the process by which the monocyte/macrophage acquires competence to perform a complex function, usually at a high intensity (Adams and Hamilton 1984). The process of acti-vation is generally transient and includes the regulation of multiple genes. This is distinct from the process of differentiation which involves permanent, stepwise irreversible, changes in gene expression (Cohn 1978). Markers often used for detection of activated macrophages include the expression of

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the cell-surface antigens CD23b, CD25, MHC class II, and LFA-1 (CD11a). Activated TAMs produce mitogens, growth factors and enzymes, beneficial for tumor growth and metastasis (Munn and Armstrong 1993).

Lymph vessel

Normal tissue

TAM

Fibroblast

Pericyte

Collagen fibers

Microvessel

Tumor tissue

Tumor cell

Figure 3. Illustration of differences between normal and tumor stroma. Adapted from (Heldin et al. 2004). Tumor associated macrophage (TAM).

Tumor interstitital fluid pressure (IFP) The interstitium is the space between cells in a tissue and the body fluid re-tained in this is called the interstitial fluid. Fluid constantly filtrates through the capillary wall out into the surrounding loose connective tissues. The Starling forces determine the net filtration pressure over the capillary wall (Figure 4). The pressure gradient is generated from differences in the capil-lary blood pressure (PC), the capillary osmotic pressure ( C), the interstitial osmotic pressure ( IF), and finally the IFP. These forces keep a constant in-terstitial volume and fluid transport over the capillary wall under normal circumstances (Aukland and Reed 1993). In normal tissue the resulting pres-sure gradient is slightly negative leading to a transcapillary flow into the tissue. High molecular weight compounds are transported through convec-tion, i.e. carried by the fluid over the membrane, compared to low molecular weight compounds that are transported by diffusion (reviewed by (Rippe and Haraldsson 1994)).

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C

IF

PC

IFP

Loose connective tissue

Endothelialcell

Microvessel

Figure 4. Fluid flux according to Starling relation. Filtration rate (JV), constant (K), plasma protein reflection coefficient ( ), capillary pressure (PC), capillary osmotic pressure ( C), interstitial fluid osmotic pressure ( IF), interstitial fluid pressure (IFP).

Most solid tumors have a high IFP (Gutmann et al. 1992; Less et al. 1992; Nathanson and Nelson 1994). Several studies have shown a correlation be-tween high tumor IFP and poor prognosis (Milosevic et al. 2001; Rofstad et al. 2002). The high tumor IFP forms or reflects a barrier for transport of drugs into the tumor. Transport of low molecular weight compounds into tumors benefits from acute and transient lowering of tumor IFP, probably through an additional transport by convection (Salnikov et al. 2003).

The mechanisms behind the generation of the high tumor IFP are not fully understood. One theory is that it is due to high capillary permeability com-bined with non-functional lymphatic drainage, which gives an excess of fluid and molecular compounds in the tumor tissue (Leu et al. 2000). The tumor stroma is believed to be in a constantly contracted state. This is possibly mediated through contacts between fibroblasts and the collagen microfibril-lar network through integrins (Rodt et al. 1996; Heuchel et al. 1999; Reed et al. 2001). This contraction prevents swelling of hyaluronan and proteogly-cans in the ECM, which usually is the response to an increase in the amount of fluid (Meyer 1983). Several factors that induce a lowering of tumor IFP have been identified (summarized in Table 1).

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Table 1. Factors shown to lower tumor IFP.

Factor/substance Effect Reference Dexamethason slow (Kristjansen et al. 1993) Nicotinamide rapid (Lee et al. 1992) Prostaglandin E1 (PGE1) rapid (Rubin et al. 2000) STI 571 (Glivec) slow (Pietras et al. 2001) TGF- 1 inhibitor slow (Paper II) Tumor necrosis factor- (TNF- ) rapid (Kristensen et al. 1996) VEGF monoclonal antibodies (Avastin) slow (Lee et al. 2000) 1 Transforming growth factor-

The timing for the onset of the effect differ between them, some have a rapid effect (within minutes our hours) whereas others have a longer period (days) before the effect appears. The difference in onset and duration for lowering of tumor IFP, indicates that there are separate mechanisms involved. Some of these substances have been investigated in combination with chemothera-peutic agents, such as 5-fluorouracil (5-FU), and they all show an increased uptake and efficacy of the cytostatics (reviewed by (Heldin et al. 2004)). Interestingly, blocking of VEGF with Avastin has been performed in clinical studies and this resulted in improved effect of 5-FU with prolonged survival of patients with metastatic colorectal cancer (Kabbinavar et al. 2005; Kabbi-navar et al. 2005).

Transforming growth factor- (TGF- ) and cancer TGF- is a multifunctional cytokine that is involved in wound healing and tissue repair. Upon tissue injury, TGF- increases the production and de-creases the degradation of ECM protein (reviewed by (Branton and Kopp 1999)). TGF- is produced by and can affect virtually all types of cells. It signals through binding into type I (T R-I) and type II (T R-II) receptors on the cell surface (reviewed by (Shi and Massague 2003)). TGF- interacts with a dimer of T R-II and T R-I is recruited and phosphorylated. Upon phosphorylation T R-I transfers the signal to downstream signaling mole-cules called Smads (Smad2 and -3). These molecules form complexes with Smad4 and enter the nucleus to be able to interact with DNA and induce a gene response.

TGF- seems to play a dual role in carcinogenesis (Siegel et al. 2003). Due to its ability to inhibit cellular proliferation it suppresses tumor devel-opment in its early stages, whereas it in later tumor stages promotes metasta-sis and further progresses the malignancy (Derynck et al. 2001). Mecha-nisms behind the escape from the TGF- inhibitory effect in late tumor stages are probably many, but mutations in the signaling molecules (Smads),

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the TGF- receptors, as well as reduced expression levels of the receptors have been shown (Markowitz et al. 1995; Eppert et al. 1996; Guo et al. 1997). TGF- has been shown to promote epithelial to mesenchymal transi-tion (EMT) (Ellenrieder et al. 2001; Grande et al. 2002). EMT is believed to mediate an increased cellular motility and invasiveness and thus is suggested to promote tumor metastases.

By reestablishment of the TGF- signaling in a lung tumor mice model, by reintroduction of functional T R-II, the malignant behavior of cells was reverted (Anumanthan et al. 2005). ATC cell lines, which are investigated in this thesis, seem to have decreased sensitivity to TGF- (Heldin et al. 1999). This has also been confirmed in other reports indicating a decreased expres-sion of T R-II in thyroid tumors (Wyllie et al. 1991; Coppa et al. 1997).

There are different ways of inhibiting TGF- signaling, either by target-ing the receptors or the Smads, or indirectly by binding to the ligands. Hu-man pancreatic cancer cells transfected with a soluble T R-II inhibitor were injected s.c. in athymic mice, and this resulted in a decreased growth of tu-mors in comparison to control cell-transfected tumors (Rowland-Goldsmith et al. 2002). Long-term treatment of an experimental metastatic mammary tumor model with a soluble T R-II inhibitor reduced tumor cell motility and inhibited metastases (Muraoka et al. 2002).

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Apoptosis

Apoptosis is a naturally occurring process which is crucial for proper em-bryogenesis in humans as well as animals (Kerr et al. 1972). The apoptotic process is usually divided into two major pathways (Figure 5), the extrinsic and the intrinsic pathway, involves activation of different subsets of aspar-tate specific cystein proteases (caspases) (reviewed by (Afford and Rand-hawa 2000)). During development and immune-system-mediated tumor re-moval, the extrinsic pathway is responsible for the apoptosis. The intrinsic pathway is responsible for apoptosis initiated by ionizing radiation, chemo-therapeutic drugs and mitochondrial damage. The intrinsic pathway is usu-ally described with engagement of proapoptotic factors released from mito-chondria whereas the extrinsic pathway is described as mitochondria-independent.

The onset of apoptosis is characterized by condensation of the nuclear material, synthesis of apoptosis regulatory proteins, increased mitochondrial activity and caspase activation. After shrinkage of the cytoplasm DNA, RNA and proteins are fragmented and specific cell surface molecules are ex-pressed and signal to phagocytes. A loss of membrane asymmetry leads to exposure of phosphatidylserines at the surface of the cell, which also func-tions as a signal to neighboring phagocytic cells. Finally, the cell is frag-mented and buds off, in membrane-surrounded vesicles, which are rapidly engulfed by phagocytic cells (Kerr et al. 1972; Koopman et al. 1994).

Apoptosis related genes/proteins Caspases are responsible for the degradation of the apoptotic cell. At present there are 13 known caspase-family members, and these form an intracellular proteolytic cascade modulating many cellular events in the apoptotic path-way, including activation of transcription factors (reviewed by (Afford and Randhawa 2000)). Caspases are classified as initiators or executioners de-pending on when they enter the apoptotic cascade. These are stored as latent precursors called procaspases and these require an activation event. Earlier this activation was thought to be mediated by cleavage but recent studies reveal that cleavage is neither needed nor sufficient for activation of the ini-tiator caspases. Intriguingly, the initiator caspases becomes active upon dimerization (Boatright et al. 2003; Donepudi et al. 2003).

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Mitochondria are central in the intrinsic apoptotic pathway. An increase in the mitochondrial membrane permeability, as a response to calcium re-lease from the endoplasmic reticulum (ER), leads to the release of key effec-tor proteins including cytochrome c and smac/DIABLO which induce or enhance caspase activation (Kluck et al. 1997; Yang and Cortopassi 1998). The bcl-2 family are involved in the regulation of apoptotic pathways that involve the mitochondria and some members appear to regulate this process through their localization at the ER (Nutt et al. 2002; Scorrano et al. 2003). The bcl-2 family contains at least 15 members that can homo- or het-erodimerize (Cory 1995; Chao and Korsmeyer 1998). Members of the bcl-2 family can be divided into two groups, one anti-apoptotic group (e.g. bcl-2, bcl-xL) and one pro-apoptotic group (e.g. bax, bid) (Tsujimoto et al. 1985; Reed 1994). The ratio of anti- and pro-apoptotic proteins, determine if the cell is surviving or not (Yin et al. 1994; Srinivas et al. 2000). For instance overexpression of bcl-2 or bcl-xL blocks the release of cytochrome c whereas overexpression of bax promotes cytochrome c release (Kluck et al. 1997; Pastorino et al. 1998). Regulation on protein level has also been implicated, as phosphorylation of bcl-2 has been reported to inactivate the ability of bcl-2 to promote survival (Haldar et al. 1995). Bcl-xL, bax and bak have been shown to interact with voltage-dependent anion channel (VDAC), situated in the mitochondrial membrane. Bax was found to stimulate the release of cy-tochrome c from liposomes reconstituted with VDAC (Shimizu et al. 1999). Another study, however, used liposomes without VDAC and in this experi-ment bax molecules themselves formed a channel, causing a release of cyto-chrome c (Saito et al. 2000).

The extrinsic pathway is mediated through death receptors (DRs) that are cell surface receptors, which transmit apoptotic signals after interaction with their specific death ligands (Ashkenazi and Dixit 1998). There are six mem-bers of this family (DR1-DR6) and these belong to the TNF receptor super-family. TNF, Fas ligand (FasL) and TNF-related apoptosis-inducing ligand (TRAIL) are cytokines that can act as death ligands. Two signaling pathways have been demonstrated in Fas-mediated apoptosis (Scaffidi et al. 1998; Sun et al. 1999). In one pathway binding of FasL to Fas receptor, is followed by the formation of a death-inducing signaling complex (DISC). The initiator caspase, procaspase-8, is recruited to DISC and after cleavage and dimeriza-tion, effector caspases are activated. In the other pathway DISC is also formed but to a lesser extent, leading to a lower amount of activated and dimerized caspase-8, causing a cleavage of bid that translocates to mito-chondria promoting a release of cytochrome c (Luo et al. 1998). Modified bid seems to induce a conformational change in bax that leads to pore forma-tion in the mitochondrial membrane (Petak and Houghton 2001). Released cytochrome c forms a complex with apoptotic protease activating factor-1 (APAF-1) and dimerized caspase-9 is activated, which further activate effec-tor caspases (Zou et al. 1999).

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The TP53 protein called “the guardian angel of the genome” is a central protein in the apoptotic process. This protein is a tumor-suppressor, i.e. it encodes for a protein whose normal function is to inhibit cell transformation and whose inactivation is advantageous for tumor cell growth and survival (Lane 1992). The most studied function of TP53 is its role as a transcription factor. TP53 activate apoptosis through transcriptional activation of pro-apoptotic genes, as well as transcriptional repression of anti-apoptotic genes (Vousden and Lu 2002; Fridman and Lowe 2003). Mutations in TP53 are the most common genetic alterations found in human cancers and more specifi-cally as previously described also in ATC (Hollstein et al. 1991; Ito et al. 1992; Greenblatt et al. 1994).

Nearly all cell signal transduction are mediated through one or more of the mitogen-activated protein kinases (MAPKs) (Marshall 1994). c-Jun NH2-terminal kinase (JNK), p38 MAPK and extracellular signal-regulated kinase (ERK) are subgroups of the large MAPK family. JNK and p38 MAPK, are mainly activated through environmental stress while ERK is activated by different cell growth and differentiation stimuli (Matsuzawa and Ichijo 2001). In a study on ATC cell lines JNK activation was essential for induc-tion of Taxol-induced apoptosis (Pushkarev et al. 2004). The activated MAPKs regulate the activity of different transcription factors such as c-Jun, ATF2 and elk-1, which control gene expression (Marshall 1994).

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caspase-3 procaspase-3

caspase-9APAF-1

cytochrome cVDAC

bax

bidbcl-xL

caspase-8

procaspase-8p38 MAPK

JNK

DR

DISC

death ligand

STRESS

APOPTOSIS

bcl-2

Figure 5. Schematic illustration of apoptotic pathways. Apoptotic protease activat-ing factor-1 (APAF-1), c-Jun NH2-terminal kinase (JNK), death-inducing signaling complex (DISC), death receptor (DR), voltage-dependent anion channel (VDAC), mitogen-activated protein kinase (MAPK).

2-Methoxyestradiol (2-ME) 2-ME is a naturally occurring estrogen metabolite (Seegers et al. 1989), which has been shown to induce apoptosis in many types of tumors (Fotsis et al. 1994; Klauber et al. 1997). During pregnancy, when serum estrogen lev-els are high, 2-ME is present in blood, urine and most tissues reaching mi-cromolar concentrations (Berg et al. 1983; Wang et al. 2000). Estradiol is hydroxylated to 2- or 4-hydroxyestradiol by cytochrome P450, this mainly takes place in the liver. 2-ME is then formed from 2-hydroxyestradiol by catechol-O-methyltransferase (COMT), which is present in many organs (Amin et al. 1983). Interestingly, women with low COMT activity suffer

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from a higher incidence of breast carcinoma (Lavigne et al. 1997). The bioavailability of 2-ME has been analyzed and the level of the enzyme in-volved in 2-ME metabolism, 17 -hydroxysteroid dehydrogenase (17 -HSD) type 2, is correlated to the resisting amount of 2-ME (Newman et al. 2006). The authors claim that synthetic modified compounds of 2-ME would be much more potent because of their resistance to metabolism by 17 -HSDtype 2.

Most studies of 2-ME indicate that the apoptotic response is only seen in transformed cells and not in normal cells. Studies of 2-ME showed that it inhibited the transcription of superoxide dismutases (SODs), which protects cells from damage by superoxide (a free radical) (Huang et al. 2000; Gao et al. 2005). The fact that tumor cells are more subjected to free radicals than normal cells, and thus are more dependent on SODs, might be one reason for 2-ME targeting only cancer cells the authors claim. The principal phar-macological action of 2-ME appears to be the disturbance of microtubular function, leading to cell cycle arrest in G2/M-phase (D'Amato et al. 1994; Lin et al. 2000). The effect of 2-ME on cell cycle regulation in prostate can-cer cells was investigated. This study showed that 2-ME increased the amount of cyclin B1 protein and its associated kinase activity, followed by a later decrease in cyclin A-dependent kinase activity, which arrested the cells in G2/M and further induced apoptosis (Perez-Stable 2006).

Several studies suggest that cell cycle arrest caused by 2-ME is not medi-ated through the classic estrogen receptors (ERs), since 2-ME has low affin-ity for ERs (Merriam et al. 1980; LaVallee et al. 2002). Recent studies, how-ever, indicate that 2-ME besides having a strong antiproliferative and apop-totic effect at pharmacological concentration, also have a moderate ER-dependent mitogenic effect at relatively low concentrations on ER-positive breast carcinoma cells (Liu and Zhu 2004; Kim et al. 2005). In a wider per-spective 2-ME has been shown to have an antiangiogenic effect, i.e. inhibi-tion of angiogenesis, in solid tumors (Fotsis et al. 1994). In a mouse breast cancer model 2-ME-treatment downregulated and blocked the nuclear accu-mulation of the hypoxia-inducible factor-1 (HIF-1), which is known to be involved in the induction of VEGF, thereby inhibiting angiogenesis (Mabjeesh et al. 2003; Escuin et al. 2005).

The mechanism of the cellular response to 2-ME-stimulation is not com-pletely understood and it seems to differ depending on cell types and con-centration. 2-ME has been shown to phosphorylate and inactivate bcl-2, through a rapid upregulation of phosphorylated JNK in leukaemia cells and prostate cancer cells (Attalla et al. 1998; Bu et al. 2002). The intrinsic apop-totic pathway was further suggested after the findings that 2-ME-treatment of multiple myeloma cells resulted in a release of cytochrome c, followed by a cleavage of procaspase-9 and procaspase-3 (Chauhan et al. 2002). In a study of human prostate cancer cells Smad7, which is required for activation of p38 MAPK in the TGF- signaling pathway, seem to be important for the

23

regulation of the proapoptotic bcl-2-family protein bim in the 2-ME-induced apoptosis (Davoodpour and Landstrom 2005). A recent study showed an activation of the extrinsic pathway, through an up-regulation of DR5 and cleavage of procaspase-8 (LaVallee et al. 2003). In addition to this bcl-xLphosphorylation, cleavage of bid, translocation of bax to the mitochondria and release of cytochrome c have been observed in prostate and pancreatic cancer cells after 2-ME-treatment (Qanungo et al. 2002; Basu and Haldar 2003).

2-ME has been licensed by EntreMed under the name Panzem . In clini-cal trials (Phase I and Phase II) orally administered 2-ME demonstrated anti-cancer activity in patients with breast cancer, prostate cancer and multiple myeloma (Sweeney et al. 2005). It has been reported that patients have shown a tumor response with a stabilizing disease, and 2-ME was well toler-ated.

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Site-specific recombination

Tissue-specific Cre recombinase Cre recombinase is a site-specific recombinase identified in P1 bacterio-phage (Hoess et al. 1982). By introduction of 34 bp long specific sequence (loxP recognition sites) into the genome, flanking the gene of interest, the gene could either be deleted, integrated or inversed depending on the relative orientation of these sites. Cre recombinase is self-sufficient and requires no accessory host factors (Reviewed by (Sauer 1998)).

Today a gene can be investigated in a specifically determined tissue using Cre recombinase under the control of a tissue-specific promoter. In 1992 two reports described crossing of two transgenic mice, one carrying cre in a tis-sue-specific manner and the other carrying a loxP flanked STOP cassette followed by a transgene. In the first report an oncogenic transgene, the sim-ian virus 40 (SV40) large tumor-antigen, was expressed after Cre recombi-nase activity and this resulted in tumor development. The other report showed expression of the lacZ gene product, -galactosidase, as a result of Cre recombinase activity (Lakso et al. 1992; Orban et al. 1992). This was the starting point for the widely used cre/loxP system for selective gene inacti-vation (Figure 6).

TGcreERT2 loxP-gene A-loxP

TGcreERT2/loxP-gene A-loxP

Thyroid-specificgene A inactivation

+ Tamoxifen

X

Figure 6. Schematic illustration of gene inactivation in the thyroid using cre/loxPsystem.

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Inducible cre/loxP systems To be able to control the activity of Cre recombinase, a number of alterna-tives have evolved, either concerning the onset of gene expression or the protein activity. Regulation on transcriptional level has been done using the tetracycline responsive binary system, where cre is expressed in the absence but not in the presence of tetracycline (Gossen and Bujard 1992; St-Onge et al. 1996). In a similar manner, inducible transcription of a transgene is achieved through addition of drugs or steroids, such as Ecdysone (No et al. 1996). Another way to control Cre recombinase is to introduce a ligand-dependency on protein level by fusion of ligand-binding domains (LBDs) of human receptors for progesterone, glucocorticoid and estrogen. To avoid binding and activation through endogenous steroids, the LBDs are mutated and only respond to the synthetic ligands, like RU 486, Dexamethasone and Tamoxifen (Metzger et al. 1995; Kellendonk et al. 1996; Brocard et al. 1998; Indra et al. 1999). In the absence of ligand Cre recombinase is believed to be covered with heat shock proteins and when present the ligand liberates the protein, which thereby regains its function (Figure 7).

Heat shock protein

ER LBD

Inactive

+ Tamoxifen

Cre recombinase

Tamoxifen

ER LBDCre recombinase

Active

Figure 7. Schematic illustration of Tamoxifen-induced activation of Cre recombi-nase; liberation from steric hindrance caused by heat shock proteins. Estrogen recep-tor (ER) ligand-binding domain (LBD).

26

Present investigation

AimsThe overall aim of the thesis was to investigate molecular mechanisms in the regulation of growth, apoptosis and interstitial tumor pressure in anaplastic thyroid carcinoma.

Specific aims to study the growth inhibitory potential and mechanism of action of the natural occurring estrogen metabolite 2-ME on ATC (paper I). to investigate the effect of a TGF- inhibitor on tumor stroma and tumor IFP, in order to elucidate the mechanisms behind the genera-tion of the high tumor IFP that exists in a solid tumor (paper II and III).to generate a transgenic mouse with thyroid-specific expression of Cre recombinase (paper IV).

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Results and discussion

Paper I: 2-ME-induced apoptosis in ATC cellsAnaplastic thyroid carcinoma is an aggressive cancer and current treatment is mainly of palliative character. 2-ME has been shown to have a growth inhibitory effect in many transformed cells (Fotsis et al. 1994). In the present study the growth of five of six ATC cell lines was strongly inhibited after the addition of 2-ME. The KAT-4 cell line, however, showed almost no re-sponse. The sensitive cell lines seemed to be arrested in G2/M-phase after 2-ME-treatment. After 24h of 2-ME-stimulation, 63% of the HTh7 cells were in this fraction compared to only 17% in the control. Furthermore, the sub G1-fraction, which often is referred to as apoptotic cells, increased 4-fold. Apoptotic assays showed that this increase in sub G1-fraction was indeed due to apoptotis. Earlier studies have shown that 2-ME arrest cells in G2/M, e.g. endothelial and hepatoma cells, and further subject the cells into apop-tosis (Fotsis et al. 1994; Lin et al. 2000). In vivo studies have also shown growth inhibition, for instance in transplanted human breast carcinoma in immunodeficient mice (Klauber et al. 1997; Zhu and Conney 1998). The effect of 2-ME probably involves disturbance of microtubular function (D'Amato et al. 1994; Fotsis et al. 1994).

Induction of apoptosis by 2-ME appears to be initiated both via the intrin-sic and the extrinsic pathway. An increase in the expression of DR5 was reported by LaVallee et al (LaVallee et al. 2003). In our study we saw a significant upregulation of both DR4 and DR5 on mRNA level in HTh7 and C643 cells, but not in KAT-4 cells. A slight increase in the protein expres-sion of DR5 was observed in both HTh7 and KAT-4 cells. DR5 is known to cleave and activate caspase-8, and we could see a minute sequential cleavage of caspase-8 in HTh7 cells after 4 to 12h of 2-ME-treatment. The effect of this activation, however, is probably minor since a caspase-8 specific inhibi-tor did not attenuate the apoptotic effect of 2-ME. Furthermore, initiator caspases has been shown to require dimerization to become activated (Boatright et al. 2003; Donepudi et al. 2003). This would be interesting to investigate to elucidate the importance of the cleavage of caspase-8.

Previous studies on 2-ME have shown involvement of the instrinsic pathway through a rapid activation of JNK (Yue et al. 1997). In our study, JNK is phosphorylated in the responding cell lines. In KAT-4 cells no acti-vation of JNK could be detected. p38 MAPK was also activated, but not only in the responding cell lines but also in the KAT-4 cell line. By using inhibi-tors towards the MAPKs, it was shown that both JNK and p38 MAPK activ-ity appeared to be necessary for the 2-ME-induced apoptosis, in the investi-gated ATC cell lines.

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Since the activation of caspases is involved in almost all apoptotic path-ways the effect of 2-ME on the activation of caspase-3 was investigated (Alnemri et al. 1996; Kornblau et al. 1999). A proteolytic cleavage of pro-caspase-3 was seen in HTh7 cells after 2-ME-treatment whereas KAT-4 cells showed no such cleavage. The number of cells in apoptosis after 2-ME-treatment in combination with a caspase-3 inhibitor was significantly re-duced, which indicates that caspase-3 is involved in 2-ME-induced apop-tosis. This finding is supported by other studies for instance in hepatoma cells and prostate cancer cells (Lin et al. 2000; Kumar et al. 2001).

Taken together we show that 2-ME induces apoptosis in ATC cell lines. The most prominent pathway seems to be the stress-induced mitochondrial pathway.

Paper II and III: Lowering of tumor IFP by TGF- inhibition Solid tumors have a high IFP and this reflects a barrier for the delivery of anti-tumor drugs from the blood vessels. A xenograft tumor mouse model has been used to investigate the effect of a TGF- inhibitor on tumor IFP. In this model KAT-4 cells were injected s.c.. The inhibitor is a soluble fusion protein of murine TGF- receptor type II and the Fc-region of IgG, which specifically binds TGF- 1 and - 3 (Smith et al. 1999). Administration of the TGF- inhibitor to KAT-4 tumors resulted in a 50% reduction of tumor IFP after 10 days of treatment compared to IgG2A-treated control animals.

The TGF- inhibitor-treatment induced tumor growth during the first 10 days of treatment. This might be explained by the dual role of TGF- in tumors. TGF- has been shown to be growth inhibitory in early stages and growth stimulatory in late tumor stages (Siegel et al. 2003). Histologically no difference could be seen in how the cells were organized in control tu-mors and inhibitor-treated tumors respectively. Two zones could be seen, one central zone with tissue destruction and one peripheral with viable cells. However, a wider peripheral zone could be seen in tumors treated with the TGF- inhibitor compared to the control tumors. Both the amount of cells in apoptosis and proliferation were higher in TGF- inhibitor-treated tumors compared to control tumors. All together this suggests an increased carci-noma cell turnover. The TGF- inhibition caused an increase in the protein level of the cell cycle inhibitor p27KIP1. Longer treatment times with the TGF- inhibitor (29 days), with a second injection of the inhibitor at day 14, also showed a reduction in tumor growth rate in the TGF- inhibitor treated tumors compared to control tumors. These latter results are in concordance with a recent study on mice carrying transgenic mammary carcinomas, where long-term treatment with the same TGF- inhibitor, increased the level of apoptosis (Muraoka et al. 2002).

In vitro studies on proliferation and activation of Smad2, showed that KAT-4 cells were insensitive to TGF- and/or the TGF- inhibitor. The

29

amount of collagen in the tumors was reduced after TGF- inhibition. These observations suggest that the in vivo effect observed after treatment with the TGF- inhibitor is due to a modulation of the surrounding stromal cells.

Affymetrix microarrays on total RNA from animals treated with the TGF- inhibitor or IgG2A were performed in order to investigate differences in

mouse (host) genes. A group of genes known to be expressed by macro-phages were downregulated after treatment with the TGF- inhibitor. Inter-leukin-1 (IL-1 ) and IL-1 receptor antagonist (IL-1 Ra) were among the downregulated genes. An analysis of the number of macrophages after stain-ing on cryosections, reveled a 30% reduction, in TGF- inhibitor-treated tumors. This is in concordance with the result that TGF- 1 functions as a chemoattractant for monocytes (Wahl et al. 1987). The number of activated macrophages was investigated using expression of MHC class II antigen as a marker. There was a 3-fold decrease in the number of activated macrophages in the TGF- inhibitor-treated tumors compared to control-treated tumors. That is inhibition of TGF- leads to deactivation of macrophages or lowers the recruitment of macrophages.

Macrophages are known to play a role in angiogenesis, so we decided to investigate the vasculature. The microvessel density was however not af-fected by the TGF- inhibition. The blood vessels are in normal tissue cov-ered by supportive muscle cells. Mature arteries and veins are surrounded by one or more layers of vascular smooth muscle cells, whereas the smaller microvessels are covered with pericytes (Gerhardt and Betsholtz 2003). A decrease in the NG2 (chondroitin sulfate proteoglycan) protein expression, a marker of activated pericytes (Stallcup 2002), could be seen. The number of CD31-positive vessels that were covered with -smooth-muscle actin ( -SMA)-positive cells increased. These observations probably reflect a change in the pericyte phenotype towards the one covering and supporting normal or mature microvessels. Perfused tumor vessels were visualized with FITC-labeled dextran to be able to investigate the leakiness of the microvessels. The vascular protein-permeability was assessed after TGF- inhibition by determining the leakage of Evans-blue dye (EBD), which binds to albumin in the blood. Semiquantitative analysis revealed that the protein leakage was decreased after treatment with the TGF- inhibitor. These data suggest that treatment with the TGF-inhibitor normalize vascular function. Some reports have shown that this normalization of tumor vasculature increases the deliv-ery of chemotherapeutic drugs into the tumor tissue (Inai et al. 2004; Tong et al. 2004; Willett et al. 2004). In our investigation this normalization after inhibition of TGF- 1 and- 3 indeed increased the delivery and efficacy of doxorubicin. The microarray result regarding genes expressed by macro-phages, prompted us to further investigate the role of macrophages in gener-ating the elevated tumor IFP. Treatment of mice with KAT-4 tumors with a recombinant human IL-1 Ra (rh-IL-1 Ra) reduced the tumor IFP to the same degree as the TGF- inhibitor. However combined treatment with the TGF-

30

inhibitor had no additive effect, thus indicating common mechanisms. Inhi-bition of IL-1 did not affect, or at least to a lesser extent, the investigated parameters for microvessel maturation. The latter finding might open for a possibility that the two inhibitors lower tumor IFP by different mechanisms. Or that IL-1 is not the only cytokine expressed by macrophages that are in-volved in the change of the vasculature.

In earlier studies VEGF has been shown to stimulate protein leakage from blood vessels and has also activated pericytes (Veikkola et al. 2000; Witmer et al. 2004). VEGF expression level seem to correlate with the number of TAMs (Salvesen and Akslen 1999). This, together with the findings that inhibitors of the VEGF-system resulted in a reduced tumor IFP and a nor-malization of the vascular function (Tong et al. 2004; Willett et al. 2004), made VEGF a candidate effector protein. However neither the TGF- nor the IL-1 inhibitor reduced the number of microvessels. The mRNA and pro-tein levels of VEGF (mouse and human) were not changed in the experimen-tal tumors, and not in cultured tumor cells, indicating that VEGF does not participate in the modulation of tumor IFP in these tumors. The VEGF pro-tein level from cultured KAT-4 cells was, however, very high and might conceal in vivo participation of VEGF. An analysis of the distribution of VEGF would be interesting, to further investigate possible VEGF involve-ment.

In summary, our data identify TGF- as a potential target for novel anti-cancer therapies, by a lowering of tumor IFP and an increased uptake and efficacy of anti-tumor drugs. TGF- affects tumor growth by changing the composition of the tumor stroma. The participation of macrophages in the mechanisms behind elevated tumor IFP is further suggested, and this is in-teresting since some substances known to lower IFP are inhibitors of growth factors or cytokines produced by macrophages (e.g. TGF- , IL-1, PDGF and VEGF).

Paper IV: Thyroid-specific expression of Cre recombinase To study the regulation of growth and function of the thyroid gland, as well as mechanisms behind thyroid tumorigenesis and tumor progression, a mouse model for controlled gene inactivation would be of great value. This paper describes the generation of a model based on the cre/loxP system. Using this transgenic mouse, genes of interest could be spatio- and tempo-rally knocked out. The first task was to choose a gene promoter specific for the thyroid, to obtain an expression of Cre recombinase exclusively in the thyrocytes. We have chosen the Tg promoter, since this promoter has been used and shown to be tissue-specific in the generation of several other trans-genic mice models (Ledent et al. 1990; Feil et al. 1997). The Tg promoter was ligated upstream of an inducible cre recombinase, creERT2. By fusion of a mutated LBD of ER to cre, the CreERT2 protein has been made inducible

31

through the exogenous addition of the synthetic ligand tamoxifen. This sys-tem has been successively used in the generation of other transgenic mice (Imai et al. 2001; el Marjou et al. 2004). The generated Cre recombinase plasmid was sequenced to control its accuracy. The TGcreERT2 fragment was cleaved out from the plasmid and microinjected into fertilized one-cell eggs from B6CBAF1 background. These eggs were implanted in pseudopregnant B6CBAF1 females. PCR screening on mouse tail DNA showed that 12 founder mice had incorporated the transgene. Reverse transcriptase PCR (RT-PCR) on total RNA extracted from thyroid glands from these mice showed cre mRNA expression in just a few of these. A creERT2 fragment could be amplified in two founders called founder 248 (F248) and founder 300 (F300). To further investigate mRNA expression and the size of the creERT2 transcript, Northern blot analysis was performed on total RNA as above. Expression of a 3.5kb creERT2 mRNA could be seen in thyroids from F248 and F300, this correlated to the expected size. No expression could be seen in the other mouse tissues investigated, thus indicating a tissue-specific mRNA expression.

Functional activity of the Cre recombinase was analysed through crossing of F300 derived mice with reporter mice, ROSA26-LacZ, carrying an inacti-vated lacZ-gene (Soriano 1999). Upon tamoxifen-activation Cre recombi-nase is able to excise the intervening cassette and promote expression of the lacZ-gene product, -galactosidase. Double heterozygote mice for cre and lacZ genes, together with wildtype mice, were treated with Tamoxifen or vehicle. A blue staining of the whole thyroid gland could be seen in PCR-positive mice whereas wildtype mice and vehicle treated mice showed no staining. However the staining observed was only light blue compared to positive control mice, TPO-Cre (Kusakabe et al. 2004), which might reflect the number of transgene insertions. However, Metzger and Chambon argues that the intracellular level of tamoxifen could be more limiting than the ac-tual amount of CreERT protein (Metzger and Chambon 2001). In this paper the excision efficiency varied quite a lot between different investigated tis-sues, with tail and skin showing highest recombination rate whereas thymus and heart turned out to have a very low amount of recombined DNA.

We are currently further investigating the protein expression and Cre ac-tivity in the offspring of the two founder mice, hoping to clearly state that we have generated a spatiotemporally controlled transgenic mouse.

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Populärvetenskaplig sammanfattning

Min avhandling baserar sig på studier av sköldkörtelcancer. Sköldkörtelcan-cer uppträder i olika former och olika grader av aggressivitet. Den som stu-derats i avhandlingen är den mest aggressiva formen, anaplastisk thyroidea-carcinom, som framförallt drabbar personer mellan 60-80 år. Det är vanliga-re att kvinnor drabbas men orsaken till det är inte känd. I dagsläget finns inga botemedel mot sjukdomen utan den behandling som erbjuds är framför-allt av lindrande karaktär.

En cancercell har förmågan att dela sig i oändlighet. Avsikten har varit att undersöka cancercellerna vad gäller tillväxt och tillväxthämning. Detta har gjorts på cellkulturer med tillsatts av en naturligt förekommande östrogen-metabolit, 2-metoxyöstradiol. Vid tillsatts har cellerna gått in i s.k. apoptos, eller självmordsfas. Det här leder till en process som inte lämnar några spår, såsom inflammation och skadar därigenom inte omgivningen. Eftersom 2-metoxyöstradiol endast attackerar celler som delar sig, såsom cancerceller, skulle metaboliten kunna användas för att behandla olika cancerformer.

En tumör karakteriseras av ett högt tryck, som kan fungera som en barriär mot t.ex. tillförsel av cancermediciner, eller cytostatika. Cytostatika trans-porteras via blodet och tar sig på så vis ut i vävnaden i kroppen. Genom att det höga trycket hindrar läkemedel från att ta sig in och utöva dess effekt, så har vi här undersökt en hämmare som vi har visat har förmågan att sänka trycket i tumören på en musmodell med insprutade tumörceller. Ett flertal andra studier med olika hämmare eller andra ämnen har också visat sig kun-na reglera tumörtrycket. Ett annat fynd var att en typ av immunförsvarscell, makrofagen, verkar vara involverad i genereringen av det höga tumörtrycket. Vi och andra har också visat på både ett ökat upptag och en ökad effekt av cancermediciner.

Slutligen har vi försökt att skapa en musmodell som ska kunna användas för studier av bl.a. sköldkörtelsjukdomar. Musen har en gen insatt som styrs av en promotor som bara ger uttryck i sköldkörtelceller, på så vis slipper alla celler i musen få ändringen. Den här genen har förmågan att känna igen andra märkta gener och klippa bort dessa. Så genom att korsa musen med andra modifierade möss, som har en sådan märkt gen, kan man ta bort en gen i taget och undersöka vilken effekt det har på utvecklingen av olika sjukdomar. För att göra det ännu mer kontrollerat så har vi gjort systemet inducerbart genom en tillsatts av ett ämne, härigenom kan vi bestämma när processen ska ske.

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Sammantaget så visar vi i avhandlingen mekanismen bakom olika be-handlingsförsök både på cancerceller i odling och på cancerceller i en mus-modell. Förhoppningsvis kan den ökade kunskapen bidra till förbättrad be-handling av sköldkörtelcancer i framtiden.

34

Acknowledgements

The present study was carried out at the Department of Genetics and Pathol-ogy, and the Department of Medical Biochemistry and Microbiology, Upp-sala University. This work was supported by grants from the Swedish Can-cer Foundation, the Swedish Research Council, the Gustaf V:s 80-årsfond, the Selanders Foundation, the Åke Wiberg Foundation, the Gunnar Nilsson Cancer Foundation, and Göran Gustavsson Foundation.

I would like to thank all of you that in anyway have helped me with this thesis, and in particular a few persons that have helped me a little bit more:

My supervisor Nils-Erik Heldin for being the only supervisor in the building who performs laborations with his students, for encouraging me to attend at so many meetings, and for his pedagogical teaching of all the “stone age” techniques and lately a few modern kits.

My co-supervisor Kristofer Rubin, for all philosophical and theoretical help during the progress of our tricky tumor IFP projects and the writing of this thesis.

My favourite professor Bengt Westermark for putting up with me even in late afternoons screaming at “never-working” printers, and for professional guidance through my phD-study.

Annika Hermansson for teaching me everything about cell culturing and different cellular techniques, and with all help during lab sessions and with the mice. Also for being a perfect support during my first official talk in Warsaw.

Ann-Marie Gustafson for always being prepared to offer all kinds of help working with the mice.

Maréne Landström for all the help with apoptotic and 2-ME issues.

My co-authors, especially Alexei Salnikov, Christian Sundberg, Ellen Lam-merts, Ulrika LiIjegren, Xiao-Qun Zhang.

35

Åsa Franzén for rookie-invitation into lab-sessions and for being like a big sister during our Washington trip (September eleven).

Henrik Viberg for professional teaching of gavauge-technique.

Rose-Marie Lindgren for PCR-expertise, support and without whom these years had been much more boring.

Anna Lindkvist for being such great fun, as well as skilled photograph dur-ing our attendance at the thyroid conference in LA, and super super support through all the years at Rudbeck.

Fredrik Johansson for keeping up with all questions, being the best support one can wish for, and great companionship/fathership/pharmaceuticalship during our Keystone trip.

Lene for being the best personal coach ever during our trip to Keystone.

Double Fredrik and Andy for your super-friendly attitude and enormous help with everything from flow cytometry to tricky immunological questions.

Alexei Salnikov for laughings during endless cryosection sessions and sup-port in immunohistochemical staining.

Anna Dimberg, Ingrid Nilsson and Peetra Magnusson for always being glad to help in different laboratory issues.

All past and present members at the lab, and especially Marianne Kastemar, Maria Ferletta, Helena Wensman, Eva Hellmén, Ylva Paulsson, Linda Wendt, Cecilia Engblom, Evelina Larsson.

Finally I want to thank people that perhaps haven´t helped me directly with my thesis, but in other ways have supported me:

Friends/handboll/rugby teams for keeping me on the right track.

Ludvigs family, for “bathroom-building”, helping out with Stina, Rasmus … and Ludvig.

My family, for all support, helping out with Stina and Rasmus, believing in me. You are the best!

And finally Ludvig, Stina and Rasmus for endless love and being someone to really long home for… Jag älskar er!

36

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