Thesis (pdf) - Göteborgs universitet

REGULATION OF CELL-CELLADHESION AND TRANSCRIPTION INTHE OVARY:IMPLICATIONS FOR TUMORIGENESISKarin Sundfeldt

Department of Physiology and Pharmacology,University of Göteborg, SwedenGöteborg 1999REGULATION OF CELL-CELL ADHESION ANDTRANSCRIPTION IN THE OVARY: IMPLICATIONSFOR TUMORIGENESISAkademisk avhandlingSom för avläggande av medicine doktors examenvid Göteborgs Universitetkommer att offentligen försvarasi fysiologens stora aula, sal Inge Schiölertorsdagen den 20 maj 1998, kl. 09.00avKarin Sundfeldtlegitimerad läkareFakultetsopponent:Professor Joanne RichardsBaylor College of Medicine, Houston, Texas, USA

Avhandlingen baseras på följande arbeten:I. Karin Sundfeldt, Yael Piontkewitz, Håkan Billig and Lars Hedin. The E-cadherin/catenin complex in the rat ovary: Cell-specific expression duringfolliculogenesis and luteal formation. Submitted for publication.II. Karin Sundfeldt, Yael Piontkewitz, Karin Ivarsson, Ola Nilsson, Pär Hellberg,Mats Brännström, Per-Olof Janson, Sven Enerbäck and Lars Hedin. E-cadherinexpression in human epithelial ovarian cancer and the normal ovary. Int J Cancer, 74;275-280 (1997).III. Karin Sundfeldt, Karin Ivarsson, Katarina Rask, Magnus Haeger, Lars Hedinand Mats Brännström. Higher levels of soluble E-cadherin in cystic fluid ofmalignant/borderline tumors of the ovary than in benign cysts. Submitted forpublication.IV. Yael Piontkewitz, Karin Sundfeldt, Lars Hedin. The expression of c-Myc duringfollicular growth and luteal formation in the rat ovary in vivo. J Endocrinol, 152; 395-406 (1997).V. Karin Sundfeldt, Karin Ivarsson, Malin Carlsson, Sven Enerbäck, Per-OlofJanson, Mats Brännström, Lars Hedin. The expression of CCAAT/Enhancerbinding proteins (C/EBP) in the human ovary in vivo: Specific increase of C/EBPβduring epithelial tumour progression. Br J Cancer, 79; 1240-1248, (1999).REGULATION OF CELL-CELL ADHESION AND TRANSCRIPTION INTHE OVARY: IMPLICATIONS FOR TUMORIGENESISKarin Sundfeldt, Department of physiology and pharmacology, section ofendocrinology, University of Göteborg, SwedenABSTRACTIn this thesis the expression and localization of the epithelial (E)-cadherin/α - and β -catenincomplex was investigated during the normal ovarian cycle and in ovarian tumors of epithelialorigin. Furthermore, transcription factors (TFs) such as the proto-oncogene protein c-Myc wasinvestigated for its time- and cell-specific expression during follicular development and theexpression of CCAAT/enhancer binding proteins (C/EBPs) were investigated in ovariantumors of epithelial origin.The development of ovarian follicles is a continuous process. Cyclic stimulation by hormonesand growth factors will lead to proliferation and differentiation of granulosa cells (GCs) andtheca cells (TCs). This follicular development also involves dramatic changes in the cell’smorphology. Cells are held together by junctions i.e. tight-, adherens- (AJ) and gap-junctions.AJs, which are build up by the cadherin/catenin complex, are present between GCs of primaryfollicles but disappears in the fully developed preovulatory follicle. Regulation of E-cadherinat the transcriptional level could be managed by TFs such as c-Myc, C/EBPs and AP2.The ovarian surface epithelium (OSE) is the origin for approximately 90% of the malignantovarian tumors. The tumors arise from the inclusioncysts, localized in the ovarian stroma, and

grow solid, cystic or in mixed formations. Intra-abdominal spread of the ovarian cancer iscommon and this is a process closely connected with impaired cell-cell adhesion.In a rat model, where immature rats were stimulated by gonadotropins, normal developmentof follicles could be studied. Small primary follicles were found with positive E-cadherin andcatenin staining. During proliferation and differentiation of the follicle, E-cadherin was onlyfound in TCs and not in GCs, while the catenins were expressed in both celltypes. Just prior toovulation, the staining of E-cadherin/catenin complex was decreased in the TCs.The E-cadherin protein was not found in OSE of normal ovaries but in inclusioncysts, as wellas in benign and malignant ovarian tumors. The soluble form of E-cadherin was found inperipheral blood, cystic- and ascitic fluid. The concentrations were significantly increased incystic fluids from patients with borderline and malignant tumors when compared to patientswith benign adenomas.With a similar approach, as described for E-cadherin, the time and cell-specificexpression of c-Myc mRNA and protein was analyzed in rat ovaries. The levels of c-Mycwere found to be transiently elevated during the development of the preovulatory follicle. TheLH-surge resulted in a rapid increase in c-Myc mRNA (after 1h) and c-Myc protein (after 2h).The change in c-Myc expression was mainly localized to the GCs.The expression of C/EBPs (α , β , δ and ζ ) were mainly seen in the epithelialcells of the normal human ovary and ovarian tumors. The most prominent finding in thisstudy was the increased expression of C/EBPβ in the nucleus of cells in ovarianadenocarcinoma as compared to the benign adenoma and the normal ovary.Key words: E-cadherin, catenin, ovary, follicle development, ovarian surface epithelium,ovarian neoplasm, cystic fluid, proto-oncogene protein c-Myc, CCAAT/enhancer bindingprotein (C/EBP), cancerABSTRACTISBN 91-628-3604-8In this thesis the expression and localization of the epithelial (E)-cadherin/α - and β -catenincomplex was investigated during the normal ovarian cycle and in ovarian tumors of epithelialorigin. Furthermore, transcription factors (TFs) such as the proto-oncogene protein c-Myc wasinvestigated for its time- and cell-specific expression during follicular development and theexpression of CCAAT/enhancer binding proteins (C/EBPs) were investigated in ovariantumors of epithelial origin.The development of ovarian follicles is a continuous process. Cyclic stimulation by hormonesand growth factors will lead to proliferation and differentiation of granulosa cells (GCs) andtheca cells (TCs). This follicular development also involves dramatic changes in the cell’smorphology. Cells are held together by junctions i.e. tight-, adherens- (AJ) and gap-junctions.AJs, which are build up by the cadherin/catenin complex, are present between GCs of primaryfollicles but disappears in the fully developed preovulatory follicle. Regulation of E-cadherinat the transcriptional level could be managed by TFs such as c-Myc, C/EBPs and AP2.

The ovarian surface epithelium (OSE) is the origin for approximately 90% of the malignantovarian tumors. The tumors arise from the inclusioncysts, localized in the ovarian stroma, andgrow solid, cystic or in mixed formations. Intra-abdominal spread of the ovarian cancer iscommon and this is a process closely connected with impaired cell-cell adhesion.In a rat model, where immature rats were stimulated by gonadotropins, normal developmentof follicles could be studied. Small primary follicles were found with positive E-cadherin andcatenin staining. During proliferation and differentiation of the follicle, E-cadherin was onlyfound in TCs and not in GCs, while the catenins were expressed in both celltypes. Just prior toovulation, the staining of E-cadherin/catenin complex was decreased in the TCs.The E-cadherin protein was not found in OSE of normal ovaries but in inclusioncysts, as wellas in benign and malignant ovarian tumors. The soluble form of E-cadherin was found inperipheral blood, cystic- and ascitic fluid. The concentrations were significantly increased incystic fluids from patients with borderline and malignant tumors when compared to patientswith benign adenomas.With a similar approach, as described for E-cadherin, the time and cell-specificexpression of c-Myc mRNA and protein was analyzed in rat ovaries. The levels of c-Mycwere found to be transiently elevated during the development of the preovulatory follicle. TheLH-surge resulted in a rapid increase in c-Myc mRNA (after 1h) and c-Myc protein (after 2h).The change in c-Myc expression was mainly localized to the GCs.The expression of C/EBPs (α , β , δ and ζ ) were mainly seen in the epithelialcells of the normal human ovary and ovarian tumors. The most prominent finding in thisstudy was the increased expression of C/EBPβ in the nucleus of cells in ovarianadenocarcinoma as compared to the benign adenoma and the normal ovary.Key words: E-cadherin, catenin, ovary, follicle development, ovarian surface epithelium,ovarian neoplasm, cystic fluid, proto-oncogene protein c-Myc, CCAAT/enhancer bindingprotein (C/EBP), cancerISBN 91-628-3604-8Printed by Vasastadens Bokbinderi, Västra Frölunda, SwedenGENERAL BACKGROUND

THE OVARYOVARIAN EMBRYOLOGY AND ANATOMYThe gonads (ovary and testis) are formed from the mesodermic cell layer during fetaldevelopment. These tissues initially appear as genital ridges, which are formed byproliferation of the coelomic epithelium and condensation of the underlying mesenchyme.The coelomic epithelium will then penetrate into the mesenchyme and form cortical cords,which break into clusters with primitive germ cells (oogonia) close to the surface epithelium.Each germ cell will by this mechanism be surrounded by epithelial cells, which aredescendants of the surface epithelium. These cells will later form the follicular cells of theprimordial follicle.The outermost layer of the ovarian cortex is a single germinal epithelium, which is commonlyreferred to as the ovarian surface epithelium (OSE). These cells have either a flat or acuboidal appearance. This epithelium is often referred to as a mesothelial type of epithelialcells, since they share embryological background and some characteristics with themesothelium, which is lining the peritoneal cavity. Beneath this cell layer, the tunicaalbuginea, a poorly delineated layer of dense connective tissue, is situated. This stroma likelayer consists of connective tissue with fibroblasts, smooth muscle cells, endothelial cell andinterstitial cells including undifferentiated theca cells (TCs), degenerated follicular cells fromatretic follicles or regressed corpora lutea.OVARIAN PHYSIOLOGY AND HORMONAL REGULATIONAt the time of birth, the ovary contains small primordial follicles consisting of the oocyte anda single layer of flat epithelial/follicular cells. These follicles will rest in this position until theonset of puberty. Under the influence of the gonadotropins, follicle stimulating hormone(FSH) and luteinizing hormone (LH), these immature follicles will develop into primaryfollicles. The flat epithelial cells of the follicle proliferate and differentiate into granulosacells (GCs) with steroidogenic capacity. In the preovulatory (Graafian) follicle, the antrumand TC layers (interna and externa) are distinct structures. Theca cells also acquire steroidproducing functions. Since the blood and nerve supply to the follicle end in the theca interna a"blood-follicle barrier" between GCs and TCs has been suggested. The communicationbetween GC-GC and GC-oocyte is maintained mainly through gap junctions.

Figure 1. Overview over hormone levels in relation to ovarian eventsThe preovultory (Graafian) follicle undergoes extensive structural and functional changes inresponse to LH. The results of this LH-surge are maturation of the oocyte, ovulation andformation of a corpus luteum (luteinization). The non-ovulating large antral follicles willbecome atretic, a process characterized by apoptosis (Hsueh et al. 1996). Blood vessels will inresponse to the LH-surge become leaky and the thecal tissue will become edematous.Fibroblasts in the stroma layers will proliferate, dissociate and take on a looser connectivetissue-like appearance. Specific changes take place in the perifollicular tissue underlining theOSE i.e. focal reduction in tissue perfusion (Brannstrom et al. 1998) and inflammation-likeevents (Espey 1980). The cells of the OSE accumulate lysosomes and during these processesthese cells may secrete proteolytic enzymes responsible for OSE degradation (Bjersing et al.1974a, 1974b). The ovulation process, then leads to fragmentation and flattening of the OSEand accumulation of fluid between the cells, which break apart and retract away from theexpulsion area (Van Blerkom et al. 1978) (Nicosia et al. 1991).After the onset of the LH-surge, at the same time as the oocyte undergo maturation changesand the follicle prepares for rupture, the GCs and TCs start to proliferate and differentiate intoluteinized cells. This also involves vascularization of the previously avascular GC-layerfacilitated by degeneration of the GC-TC barrier. During this luteinization process the matureand the progesterone producing corpus luteum is generated.GENE EXPRESSION IN THE OVARYGonadotropins and locally produced growth factors bind to specific receptors on the follicularcells. This ligand-receptor bindings activate intracellular signaling pathways i.e. the proteinkinase A (PKA)-pathway with subsequent increases/decreases in transcription of the targetedgene (Richards et al. 1995). Different genes are expressed in a highly regulated and complexfashion during the events of follicular development, ovulation, corpus luteum formation, andcorpus luteum demise (luteolysis). The gene expression is further customized to the specificcells of the ovary. In general, most knowledge in this field comes from studies of the GCs(Richards 1994).There are now several well described transcription factors that are regulated by gonadotropinsin the ovary. In this thesis I will more specifically discuss the proto-oncogene protein C-myc,of the helix-loop-helix-leucin zipper family, and CCAAT/enhancer binding protein (C/EBP),of the leucin zipper family. Their specific expression patterns/regulation in the ovary andovarian tumors as well as possible target genes will be presented in the chapter entitledspecific background.CELL ADHESION

INTRODUCTIONDuring embryonic development, adhesive interactions play mayor roles in the triggering of avariety of morphogenic processes. Such interactions may involve either direct cell-cell contactor adhesion to extra-cellular matrix. The knowledge in this area has exploded since the firstcell-cell adhesion molecule (CAM) was discovered 20 years ago (Edelman 1976, Thiery et al.1977). In Figure 2, an overview of junctional mechanisms in epithelial cells is given.Figure 2. Overview of junctions and their corresponding cell-cell adhesion molecules.The adherens junction consists of a cell membrane-bound protein from the cadherin family ofCAMs. This type of junction is primarily found in plaque formations and adheres to othercells in a homophilic way. The intracellular tail binds cytoplasmic proteins. Thesecytoplasmic proteins belong to the family of catenins, which stabilizes the complex byconnection to the cytoskeleton. Details about the cadherin/catenin complex will be presentedin the chapter entitled specific background.The formation of adherens junctions might be an important mechanism to provide mechanicalstrength and to provide polarization and orientation of the cells within any organized tissue,while the non-junctional adhesion might promote locomotion of cells. The list of mechanismsthat are suggested to involve cadherin-mediated cell-cell adhesion is constantly growing.Today condensation and compaction of cells during embryogenesis, contact inhibition of cellgrowth, wound healing and tumorigenesis are some of the suggested processes in whichcadherins have been suggested to be important. In this thesis I will focus on the role for thecadherin/catenin-complex during tumorigenesis.CELL ADHESION DURING FOLLICULAR DEVELOPMENTIn the 1970´s Albertini et al (1974), by the use of freeze fraction analysis investigatedfollicular development and aspects of cell junctions. They found that during the early stages

of follicular development (primordial and primary follicles), contacts between GCs arecharacterized by adherens junctions, desmosomes and small gap junctions. Granulosa cells ofthese follicles are also small and tightly packed. As the follicle grows, gap junctions willincrease in size and numbers while adherens junctions will decrease (Albertini et al. 1974,Amsterdam et al. 1992). The GCs of preovulatory follicles resemble mesenchymal cells and aremore loosely attached (Albertini et al. 1975). The increase of gap junctions in GCs afterstimulation by gonadotropins and/or estrogen was later established, in contrast to thewhereabouts concerning changes in adherens junctions (Amsterdam et al. 1992, Grazul Bilska et al.1997). The group of Amsterdam et al (Ben Ze'ev et al. 1987) took on another approach by studyingthe intermediate filaments (i.e. actin and vimentin) connected to the adherens junctions. Theyfound that the presence of actin decreased after stimulation by gonadotropins, at the sametime as steroidogenesis increased in cultured GCs. The importance of junctional complexes infollicular development has recently become evident when it was found that connexin 37 -/-mice do not develop tertiary or Graafian follicles and do not ovulate (Simon et al. 1997). Thefollicles of these mice lacked any type of gap junction, but in contrast to follicles of normalmice they retained their adherens junctions.TUMORS OF THE OVARYINTRODUCTIONThe development of a tumor is the result of a series of events. Several mutations and a naturalselection process for the more competitive cell population lead to an uncontrolledproliferation of cells and a tumor is formed. Additional changes in oncogenes and/or tumorsuppressor genes, lead to loss of cell-cell adhesion, production of proteolytic enzymes andmetastasis through direct invasion or transport of the tumor cells through the lymphatic ductsor blood vessels.EPIDEMIOLOGY AND ETHIOLOGYThe epithelial-derived adenomas and adenocarcinomas account for 90% of the ovariantumors. In Scandinavia, the lifetime risk for females to develop ovarian cancer is 1-2%, whichis the highest lifetime risk for ovarian cancer in the world. In contrast, developing countriesand developed countries with a different lifestyle such as Japan has less than 0.5% risk (Katsoet al. 1997, Riman et al. 1998). The cause of ovarian cancer is poorly understood but some theorieswith support from both epidemiological and cellbiological studies have been put forward.The "incessant ovulation" theory (Fathalla 1971) hypothesis that the overall risk for developingovarian cancer is related to the number of ovulations that has taken place. Theepidemiological facts gathered, states that events or interventions that decrease the number ofovulations, such as childbirth, lactation and use of combined (estrogen/gestagen)contraceptives, lower the risk to develop epithelial ovarian tumors (Adami et al. 1994). Animalsdo not in general develop epithelial ovarian tumors, with the exception of hens thatinterestingly also have very high ovulation rates.The "gonadotropin" theory is based on the fact that ovarian tumors in general developpostmenopausally when the blood levels of gonadotropins are markedly elevated, whichresults from the loss of negative steroidogenic feed-back. Most epidemiological data speaksagainst this latter hypothesis, but the biological theories behind these hypotheses can serve as

explanations for each or both. Ovarian tumors often contain gonadotropin receptors andovarian cancer cells respond to gonadotropins with proliferation (Elbendary et al. 1996, Riman etal. 1998). At the time of ovulation, the OSE breaks apart and the cells will then becomeexposed to extremely high levels of estrogens and gonadotropins in the follicular fluid, whichpenetrates through the mesh-like exterior follicular wall during several hours before actualrupture (Zachrisson 1997). The repetitive proliferation of OSE, to cover the apex defect afterovulation, may increase the frequency and accumulation of spontaneous cancer-associatedmutations.Finally, there is the entrapment of OSE in the stroma of the ovary, which is the beginning ofthe inclusioncysts (Nicosia et al. 1991, Hamilton 1992, Scully 1995).HISTO-PATHOLOGYEpithelial-derived ovarian tumors develop from inclusioncysts in the stroma. Tumors can begrouped into benign adenomas, borderline tumors and malignant tumors (adenocarcinomas).There are several histopathological subgroups such as, serous (most common), mucinous,endometroid, clear cell and mixed or undifferentiated adenocarcinoma. The malignant tumorsgrow solid or in cyst formations and will typically invade the surrounding lower abdomen, setmetastasis in the peritoneal cavity by preference and more seldom give rise to peripheralmetastasis. The tumors are surgically staged (I-IV) and then graded (highly, moderately,poorly, or un-differentiated) according to the classification of the International Federation ofGynecology and Obstetrics (FIGO).MOLECULAR BIOLOGYIt is generally believed that ovarian cancers, like most other cancers, arise due to themutations or rearrangements of oncogenes and tumor suppressor genes. These genes arenormally involved in the regulation of cellular proliferation, differentiation and apoptosis.Inherited ovarian cancer (approximately 5-10%) have especially been connected to mutationsof the breast tumor related antigen (BRCA-1) tumor suppressor gene, since BRCA-1 carriershave a 63 % risk of developing ovarian cancer. The function for the BRCA-1 gene product isstill unknown. The majority of epithelial ovarian tumors are sporadic cancers and genes i.e.epidermal growth factor receptor (EGFr), Her-2/neu receptor tyrosine kinase, ras family of Gproteins, transcription factor c-Myc, retinoblastoma (Rb) and p53 (Boente et al. 1993,Berchuck et al. 1997) are known to be connected to the development and progression of thistumor. However, gene analysis shows a very heterogeneous picture with changes in theovarian tumor DNA at several locations and with great difference in-between patients.The mechanism of metastasis development of ovarian cancer from the ovary to theperitoneum is difficult to study mainly due to that suitable experimental models are lacking.One approach is to investigate the expression of genes and proteins involved in cell-cell, cellmatrixadhesion and matrix degrading functions.SPECIFIC BACKGROUNDIn this section there will be a description of cell-cell adhesion molecules involved in normalovarian cell adhesion and ovarian tumors with specific focus on the cadherin/catenin complex.Furthermore, the transcriptionfactor families of c-Myc and CCAAT/enhancer binding protein(C/EBP) will be introduced in general and also in an ovarian-related point of view.

THE EPITHELIAL (E)-CADHERIN, α - AND β -CATENIN - COMPLEXINTRODUCTIONCadherins belong to a family of single transmembrane, Ca 2+ -dependent, homotypic binding,cell-cell adhesion proteins. E-cadherin was discovered in the beginning of the 1980´s and hasbeen known under many names; Arc-1 (Imhof et al. 1983), uvomorulin (Urushihara et al.1980), L-CAM (Brackenbury et al. 1981), cell-CAM 120/80 (Damsky et al. 1983). The locusfor human E-cadherin has been mapped to 16q22.1. E-cadherin is synthesized from a 4.5-kbmRNA as a 135-kDa precursor polypeptide, which is processed within 2h by proteolyticcleavage to the mature 120 kDa form (Shore et al. 1991). The extracellular part of 80 kDa canlater be proteolytically cleaved (Damsky et al. 1983, Wheelock et al. 1987, Takeichi 1988).The classical cadherins are neuronal (N)-, placental (P)- and E-cadherin, which are all wellcharacterized. However, there has been a growing number of family members in recent yearsand all vertebrate cells seems to express one or more cadherin (Takeichi 1991). Thecytoplasmic tail is highly conserved and forms complexes with the catenins (see below).These proteins will in turn stabilize the cell-cell contact by binding to the cytoskeleton viaactin (Geiger et al. 1992, Gumbiner 1996, Yap 1998).It has been known for a long time that loss of epithelial differentiation in carcinomas, a typeof epithelial/mesenchymal conversion, is related to a more aggressive and invasive tumortype. This conversion involves loss of functional cadherin adhesion in normal epithelium anddown-regulation of E-cadherin in tumor cells (Van Roy et al. 1992, Birchmeier et al. 1994a).However, recently the ability of E-cadherin to induce desmosome formation and stratifiedepithelium in corneal fibroblast cells (Vanderburg et al. 1996, Tlsty 1998), a process ofmesenchymal/epithelial conversion was described. The expression of E-cadherin correlatedwith epithelial differentiation and invasive state of some carcinomas. Other studies haveshown intact E-cadherin in invasive cells, where either of the catenins was affected, aspossible explanations for reduced cell-adhesiveness (discussed in detail in the section namedGeneral Discussion). Even though the roles for the E-cadherin/catenin complex in tumorinvasion has been thoroughly investigated, the first studies which clearly show that E-cadherin loss actually leads to tumor formation or proliferation has recently been published(Guilford et al. 1998, Perl et al. 1998).

Figure 3.Model of the E-cadherin/catenin complex and the Wnt/wingless signaling pathwayThe catenins were first identified as a set of three (α , β and γ ) proteins that wouldimmunoprecipitate with E-cadherin (Gumbiner et al. 1993, Hinck et al. 1994, Yap 1998).Beta-catenin is known to bind directly to the cadherin cytoplasmic tail while α -catenin bindsto β -catenin and connects the complex to actin or actinin. Cells lacking α -catenin are unableto form stable contacts despite high expression levels of E-cadherin and β -catenin. It is nowwell established that β -catenin also interacts with other cytoplasmic proteins (BenZeev et al.1998). Thus, β -catenin has a role in the Wnt-wingless pathway, where stable complexes thatfacilitate degradation of β -catenin, are made with adenomatous polyposis coli (APC) geneproduct and glycogen synthase kinase-3β (GSK3β ) in normal cells (Rubinfeld et al. 1996)(Peifer 1996). In certain cancer cell lines with mutational changes in these proteins, a freepool of β -catenin is found and can bind to the transcription factor T-cell factor/Lymphocyteenhancer factor (Tcf/Lef-1) (Korinek et al. 1997, Morin et al. 1997, Rubinfeld et al. 1997)(Peifer 1997). Together they translocate to the cell nucleus (Behrens et al. 1996, Molenaar etal. 1996) and can initiate transcription of i.e. the c-Myc, E-cadherin and cylin D1 genes(Huber et al. 1996b, He et al. 1998, Tetsu et al. 1999). It is also of interest that catenins arepresent in cells with little or no cadherin (Geiger et al. 1992).THE CADHERIN/α - AND β -CATENIN – COMPLEX IN THE OVARYIn this section I will only present the findings in this particular area which had been publishedup to the time of the start of this PhD project. Thus, this was the background of papers I-III.More recent findings will be brought up in the general discussion.In 1989, it was shown that GCs from estrogen (DES)-primed animals expressed E-cadherinprotein (Farookhi and Blaschuk 1989). This observation was followed by a study who showedan increase of E-cadherin mRNA in whole ovaries of immature mice after systemic injectionof estradiol (E2) (MacCalman et al. 1994). Furthermore, E-cadherin expression was examinedin a murine ovarian tumor cell line (OV2944) with sublines of different metastatic activity(Hashimoto et al. 1989). Northern blot analysis showed high levels of E-cadherin mRNA inthe cell lines with low metastatic potential compared to low levels in cell lines with highmetastatic capacity. However, then it was found that E-cadherin was expressed in 9 out of 9immunohistochemically stained tumors of the human ovary (Inoue et al. 1991). In fact, inmutational studies of the E-cadherin gene in ovarian cancers (n=63), using single strand

conformation polymorphism (SSCP) technique, only one of the investigated ovarian tumorsexhibited a mutation (Risinger et al. 1994).N-cadherin had been described in cultured rat GCs and the expression increased afterstimulation by E2. Addition of FSH did not give any further increase and FSH alone had noeffect on N-cadherin expression (Blaschuk et al. 1989, Farookhi and Blaschuk 1991).OTHER CELL-CELL ADHESION MOLECULES IN THE OVARYTHE NEURAL CELL ADHESION MOLECULE (NCAM)A couple of studies have investigated the expression of NCAM mRNA and protein in theovary (Mayerhofer et al. 1991, 1994, Moller et al. 1991). The NCAM family consists of threemajor members generated by alternative splicing of a single gene (180 kDa, 140 kDa and 120kDa) (Edelman et al. 1991). Only the two smaller proteins are expressed in the rat ovary. Byimmunohistochemistry, NCAM was localized to the GCs and the luteal cells of ovaries fromboth rat and mouse (Mayerhofer et al. 1991). Granulosa cells from preovulatory follicles ofthe human ovary and cultured GCs that underwent luteinization in vitro, both expressedNCAM-140 mRNA and protein (Mayerhofer et al. 1994). The authors conclude from thesestudies that NCAM may be involved in corpus luteum formation. It is interesting to note thatNCAM-140 has been reported to downregulate the expression of matrix metalloproteinases(MMPs) i.e. interstitial collagenase (MMP-1) and 92-kDa gelatinase (MMP-9) (Edvardsen etal. 1993). MMPs are produced in the rat ovary and MMP-1 in particular is proposed to beimportant in ovulation (Reich et al. 1991, Tadakuma et al. 1993). In ovarian carcinoma,NCAM staining was seen in 7 out of 28 tumors that were investigated (Kaufmann et al.1997).INTEGRINSThe members of the cell-adhesion family of integrins build up cell-matrix connections and actas receptors for extracellular matrix proteins. This family of CAMs consists of severalmembers built up by different α and β chains. The mouse ovary was examined for theexpression of integrin α 6 (Fröjdman et al. 1995). This integrin was expressed in the ovaryduring embryogenesis and in adult mice α 6 was present in the OSE and in TCs of the largerfollicles. Several groups have found, that in particular integrin α 6 but also α 2, α 3 and β 1are expressed during follicular development and corpus luteum formation in time- and cellspecificmanners, indicating important roles for the integrins during these processes (Aten etal. 1995, Honda et al. 1995, Giebel et al. 1996, Nakamura et al. 1997, Fujiwara et al. 1998).There is clearly a role for integrins in cancer development or growth. Various tumor typesstop producing specific integrins, display new types of integrins or alter the distribution ofintegrins. These alterations are thought to facilitate migration of the tumor cells (Horwitz1997). In the human ovary, expression of integrins α 1, α 3, α 6 and β 4 were observed in the

OSE (Bartolazzi et al. 1993, Skubitz et al. 1996). In malignant primary tumors, loss ofintegrin α 1 was found (Bridges et al. 1995, Liapis et al. 1997, Buczek Thomas et al. 1998).In ascitic tumor cells, integrin β 4 and α 6 are downregulated in contrast to high expression ofthese integrins in both normal OSE and primary tumors. Defined functions for integrinsspecifically in ovarian tumor progression or invasion have not been described.GENE TRANSCRIPTION AND REGULATIONA transcriptionfactor, a set of transcriptionfactors, enhancers or control elements bind to thepromoter region of the targeted gene in a specific way so that transcription can start. Thetranscriptional control is quite complicated and the more crucial the transcript is for thesurvival of the cell, the more complex is the control machinery in relation to the genetranscriptionat the DNA level.The transcriptionfactors are grouped into different families depending on the way they bind toDNA. Transcriptional regulation and control of the E-cadherin gene was, based on results ofearly promoter studies, suggested to involve an E-box, a CCAAT-box and a GC-rich region aspossible DNA-binding sites (Hennig et al. 1996). The myc family was suggested to bind tothe E-box consensus site and the C/EBP-family was proposed to bind to the CCAAT-box. Itwas later found that the CCAAT-box represents only weak C/EBP binding sites. Still, thesefamilies of transcriptionfactors are both very interesting in the aspect of cell-proliferation andcell-differentiation and we were intrigued of the finding that also cell-cell adhesion might berelated to these events.THE PROTO-ONCOGENE C-MYCINTRODUCTIONThe c-Myc gene is very well preserved throughout evolution from Xenopus to humans. Thegene codes for an mRNA of approx. 2.2 to 2.4 kb, which translate into proteins of 64 and 67kDa. The c-Myc protein is a transcriptionfactor of the basic helix-loop-helix-leucin zipper(bHLH Zip) family (Marcu et al. 1992, Zörnig et al. 1996, Bouchard et al. 1998). The partnerprotein of c-Myc in vivo is another bHLH Zip called Max. Dimerization with Max is requiredfor DNA binding and transcriptional activation. The Myc-Max heterodimers bind mainly tothe DNA consensus sequence CACGTG, also referred to as the "E-box" (Grandori et al.1996).In the normal cell, c-Myc expression is tightly regulated in response to growth signals. Nonproliferating,quiescent cells express low or undetectable levels of c-Myc but this expressionis rapidly induced following mitotic stimulation. When the mitotic stimulus is withdrawn, c-Myc protein rapidly disappears. Both the mRNA and the protein are short lived, with t½ ofabout 30 min (Dean et al. 1986, Waters et al. 1991). C-Myc is also a potent inducer of apoptosis(Thompson 1998). Both reductions of c-Myc and inappropriate overexpression can be associatedwith cellular apoptosis. In human malignancies, c-Myc mutations that cause structurallynormal or certain mutant forms of c-Myc to be expressed continually are found or are at levelseven higher than those of normal cells. This overexpression is probably part of the multistep

oncogenic process and is often found in association with mutations of other proto-oncogenese.g. ras.The earliest response of activation of c-Myc is a rapid induction of cyclin-E-Cdk2 kinaseactivity. One potential target gene of c-Myc implicated in this activation is Cdc25A, a cellcycleregulatory protein. This is a G1-specific protein phosphatase that is required for theinitiation of the cell cycle and for its progression through G1 (Galaktionov et al. 1996). Otherknown target genes for c-Myc are, Cyclin A, Cyclin E, Cyclin D1, estrogen receptor, p53,C/EBPα and E-cadherin (Leone et al. 1997, Facchini et al. 1998). Regulation of c-Myc is primarilyon the mRNA and protein level by controlling their stability and regulation of their half-lives(Marcu et al. 1992) but transcriptional regulation has also been observed (Facchini et al. 1998).C-MYC IN THE OVARYIn the rat ovary, c-Myc mRNA and protein rapidly increase in GCs after gonadotropinprimingof the immature rat (Delidow et al. 1990). This increase most likely reflects proliferationof the cells since, H 3 thymidine incorporation increase in the stimulated cells. Peri-fusion ofimmature ovaries treated with FSH and insulin shows the same type of short-time increase ofc-Myc (Delidow et al. 1992). In other endocrine organs, such as the uterus, estrogen stimulatesexpression of c-Myc in the rat (Huet-Hudson et al. 1989) while progesterone lowers the oviductconcentration of c-Myc in the sheep (Fink et al. 1988).In ovarian tumors, the c-Myc gene is amplified in 30-50% of the tumors (Zhou et al. 1988,Sasano et al. 1990) and the protein is simultaneously overexpressed (Bauknecht et al. 1990,Sasano et al. 1992). In one report, it was demonstrated that the levels of c-Myc proteinincrease prior to development of aneuploidy during the progression of the ovarian malignantphenotype (Watson et al. 1987). In an ovarian cancer cell line (OVCAR3), derived from amoderately differentiated serous adenocarcinoma, estrogen stimulated the expression of c-Myc with subsequent proliferation of the cancer cells (Chien et al. 1994). In the same studytreatment with antisense c-Myc abolished proliferation of the estrogen stimulated cells.Recently the c-Myc mRNA levels was investigated to assess whether they were related toovarian tumor expression of estrogen receptors, metastasis, survival time, FIGO stage,histological grade and type. Approximately 30% of the 90 tumors contained detectable levelsof c-Myc mRNA but there was no correlation with any of the investigated parameters (Tanneret al. 1998).CCAAT/ENHANCER BINDING PROTEIN (C/EBP)INTRODUCTIONTo date, six members of the C/EBP family of transcription factors have been characterized.According to the nomenclature suggested by Cao et al (1991), these members are calledC/EBPα , β , δ , ε , γ and ζ . Similarities between the C/EBP family members suggest acommon evolutionary history of the gene, even though the different family members vary intissue specificity and transactivating ability (Lekstrom Himes et al. 1998). Moreover, all ofthem make homo- as well as hetero-dimers with each other (Descombes et al. 1990, Cao et al.1991, Ron et al. 1992) in order to activate transcription or direct the complex away from theDNA-binding site. There have also been descriptions of protein-protein interactions between

C/EBP-family members and NF-kβ proteins, p21 and Rb (LeClair et al. 1992, Matsusaka etal. 1993, Stein et al. 1993, Chen et al. 1996b, Timchenko et al. 1996). Interactions with thelatter two are pointing towards possible regulating functions during the cell-cycle progression.The first member of the C/EBP family which was cloned (C/EBPα ), was identified as aprotein binding to CCAAT-boxes (Landschulz et al. 1988). However, the optimal binding sitehas later been determined to ATTGCGCAAT (Agre et al. 1989). All C/EBP members havethe same DNA-binding motif except for C/EBPζ that lack DNA binding possibilities. Twoisoforms of C/EBPα are generated from the mRNA, one full-length protein of 42 kDa and ashorter form of 30 kDa. This shorter form has the same DNA-binding domain but an alteredtransactivation potential compared to the full-length C/EBPα (Lin et al. 1993, Ossipow et al.1993). C/EBPα was first found to be expressed in cells that are involved in liver metabolismand were specifically able to regulate the expression of genes involved in these metabolicprocesses (McKnight et al. 1989). In fact, C/EBPα knockout mice usually die about eighthours after birth due to impaired expression of glycogen synthase, which leads tohypoglycemia (Wang et al. 1995). C/EBPα also plays an important role in liver-, adipose- andintestinal tissue differentiation (Samuelsson et al. 1991, Chandrasekaran et al. 1993, Flodby etal. 1996).The C/EBPβ gene also codes for two protein isoforms. The full-length 38-34 kDa protein iscalled liver enriched activator protein (LAP) and a truncated 16-kDa form is called liverenriched inhibitory protein (LIP) (Descombes et al. 1991). The ratio of LAP-LIP heterodimerizationcomplexes has been suggested to be important for the rate of transcriptionalactivation (Raught et al. 1995). C/EBPβ was first referred to as an acute phase response genein cytokine-induced gene expression (Akira et al. 1990, Poli et al. 1990) and later as anactivator of proliferation in pre-adipose cells (Cao et al. 1991, Umek et al. 1991). Moreover,after partial hepatectomy C/EBPα was downregulated, and C/EBPβ and δ were activatedduring the regeneration period (Mischoulon et al. 1992, Flodby et al. 1993).The C/EBPδ gene codes for a 30-kDa protein and the expression have been detected inintestinal, adipose, lung and liver tissue. C/EBPδ function has, apart from a role in regulationof proliferation, also been suggested to involve the acute phase response gene and geneexpression (Lekstrom Himes et al. 1998).The last family member that I reflect upon in this thesis is C/EBPζ ,, which codes for aprotein of approximately 29 kDa. C/EBPζ hetero-dimerizes with the other C/EBP members,but the presence of two prolines in the DNA-binding region disrupts and prevents the dimercomplexto activate transcription. In this way, C/EBPζ could function as a dominant negativeinhibitor of C/EBP transcriptional activation (Ron et al. 1992). Induction of C/EBPζ hasactually been shown to block G1 to S-phase progression in adipocytes resulting in growtharrest (Barone et al. 1994, Constance et al. 1996).The role for C/EBPs during tumor development is largely unknown but preliminary studies oncell specific expression patterns of the different proteins suggest several possible functions forthese family members. C/EBPζ has been reported to be present in myxoid liposarcoma (Amanet al. 1992) and Ewing´s sarcoma (Crozat et al. 1993). The shorter form of C/EBPβ , LIP, wasdetected in human breast tumors (Zahnow et al. 1997). In liver carcinomas, the expression ofC/EBPα decreased in relation to tumor dedifferentiation (Xu et al. 1994). The antiproliferatingand anti-tumor properties of C/EBPα were further demonstrated by the induction

The methods used in this thesis are described in detail in the Material and Methods sections ofthe individual papers. Some of the aspects of these methods and techniques are discussed in amore general aspect below.ANIMALS AND EXPERIMENTAL PROTOCOLSThe different models used to initiate follicular growth, ovulation and corpusluteum formation are well established and have previously been characterized in detail(Richards 1975, Nordenström 1981, Billig et al. 1993). The reason for using several modelswas two-fold. Firstly, it was important to assure that the results and findings from one model(the PMSG-model) of folliculogenesis and corpus luteum formation could be repeated withthe second model (estradiol and FSH treated hypophysectomized rats) (paper IV). Secondly,the effect of estrogen on the expression of CAMs was of specific interest (paper I).HUMAN TISSUESTissue samples were collected from the patients that were admitted toSahlgrenska University Hospital for operations due to ovarian tumors (papers II, III and V).Normal ovarian tissue samples were taken from patients undergoing laparotomy for benignnon ovarian disease (papers II and V). The tumors were classified as benign adenomas,borderline tumors and adenocarcinomas according to well-accepted criteria (Kjellgren andAngström 1995). Furthermore, the tumors were staged and graded according to theInternational Federation of Gynecology and Obstetrics (FIGO) classification.TISSUE PREPARATIONThe dissection procedures of granulosa (GC)-, theca (TC) cells and residualovary (ROV), were performed according to well-established techniques (Piontkewitz et al. 1993).To minimize contamination of other types of cells in the different fractions, the dissectionprocedures were performed under microscope (Nordenström 1981). For each time-point, tissuesand cells from 3-5 animals were pooled.Ovarian tissues and cells were prepared for mRNA analysis by mechanical andchemical homogenization and RNA extraction with phenol-chloroform according toChomcynski and Sacchi. Levels of mRNA were related to the concentration of total nucleicacid (TNA). Tissue extracts from both human and rat was prepared for protein analysis bymechanical homogenization of the tissue in a buffer protecting the proteins from degradationby proteinases. The cells in the homogenate, were further lysed both chemically and bysonication before protein concentration was evaluated (Piontkewitz et al. 1997).SOLUTION HYBRIDIZATION ASSAYThis method is used to measure small quantities of mRNA. The mRNA ishybridized with a S 35 labeled antisense RNA probe and the individual concentrations of eachsample are related to a standard curve made from the probe’s sense RNA. This gives a reliablequantification of the mRNA. In this assay it is important to analyze the qualitative aspects ofthe labeled RNA (size of transcript) to ascertain probe specificity.NORTHERN BLOT ANALYSIS

With this technique, equal amounts of RNA can be separated by electrophoresis,blotted/transferred to a membrane and detected by hybridization to a 32 P labeled RNA orDNA probe. Hybridized bands are detected on X-ray films. This method provides informationof the size of the transcript that can be detected with the specific probe and if there are anysplice variants of different sizes present. In Northern blot analysis the amount of RNA neededis much larger than in solution hybridization assays and even though densitometric scanningenables you to compare in-between samples, the latter method is preferred in quantitativeanalysis.IMMUNOBLOTTINGThis method makes it possible to separate equal amounts of denaturated proteinsby electrophoresis and transfer them to a membrane. The protein is then detected withantibodies linked to alkaline phosphatase enzyme, which can emit chemiluminescence in thepresence of certain chemicals. The bands are visualized on X-ray film. With immunoblotting,protein levels can be determined and semi-quantitatively measured by densitometric scanning.By adding a standard probe of different sizes, this method also provides information ofprotein size.ASSAY OF SOLUBLE E-CADHERINThe method of enzyme-linked immunosorbent assays (ELISA) provides a sensitive way todetermine the concentration of proteins in liquid solutions. Even extremely low amounts of aprotein can be detected by this technique.IMMUNOHISTOCHEMISTRYIn this technique antigens in a tissue section is detected by the precise specificityof an antibody labeled with fluorescent dyes (FITC). With a fluorescence (UV-light)microscope evaluation of the cell-specific localization of a molecule can be achieved. Thesensitivity of antibodies is enhanced by a signal-amplification method that provides bindingof multiple secondary antibodies to the primary unlabeled antigen-antibody complex (i.e.biotin-streptavidin-FITC). This method can be used indirectly for quantification but is mostwidely used to specify the location of a protein.IN SITU DNA 3´-END LABELINGWhen a cell enters apoptosis, programmed cell death, the DNA breaks apart intosmaller fragments. This can be detected by an antibody directed to the 3´-end of the DNAfragments and visualized by colored dyes. With this sensitive method, it is possible to

determine which specific tissue and/or which specific cells that has entered the apoptoticpathway. You can also indirectly use this method as a quantitative assay.RESULTS AND COMMENTSIn this section, the results of papers I-V are summarized and commented on. In addition someunpublished data of relevance will be presented.THE CELL- AND TIME-SPECIFIC EXPRESSION AND LOCALIZATION OF E-CADHERIN, α -AND β -CATENIN IN THE RAT OVARY (PAPER I)Cell adhesion junctions in the ovary, and in particular among the granulosa cells(GCs), have been carefully examined by the use of electron microscopy. The profoundmorphological and functional changes that ovarian cells undergo during folliculogenesis,ovulation and corpus luteum formation suggest a role for cell-cell adhesion molecules(CAMs) in these processes. Not much was known about the specific location or the hormonalregulation of E-cadherin and the catenins during the ovarian cycle, when this study wasconducted. Ovaries were analyzed from gonadotropin- and estrogen-stimulated rats atdifferent time-points of the ovarian cycle (for details see paper I).By use of immunohistochemistry techniques, staining for E-cadherin were demonstrated intheca (TC)-, interstitial-cells and surface epithelium (OSE). E-cadherin was not detected inGCs except for GCs of small follicles located in the inner, cortical region of the ovary. Thistransient expression of E-cadherin in GCs could be of specific importance to the corticallylocated primordial and preantral follicles. The expression of E-cadherin protein was furtheranalyzed with immunoblotting techniques but no stimulatory effect could be demonstratedafter hormonal treatment. Apoptotic follicles undergoing atresia were also without E-cadherin.All of the ovarian cell compartments stained positively for α - and β -catenin. The expressionof α -catenin was affected by hormones, as demonstrated by a decrease in GCs after bothhuman chorion gonadotropin (hCG) and diethylstilbestrol (DES) treatment while theexpression of β -catenin was unaffected. Prior to ovulation, there was a less intense staining ofboth α - and β -catenin and to some extent also E-cadherin, which was restricted to the TCs.This interesting finding suggests that downregulation of cadherin/catenin complexes couldfacilitate the morphological changes that occur prior to ovulation.Corpus luteum (CL) of ovaries from cycling rats was investigated by immunohistochemistry.Positive staining of E-cadherin, α - and β -catenin was demonstrated in all of the investigatedCL. The intensity was, however, lower than in adjacent interstitial tissue or TCs of nearbyfollicles.EXPRESSION OF E-CADHERIN IN THE HUMAN OVARY AND OVARIAN TUMORS (PAPER IIAND III)

Even though the ovary is an organ of mostly mesoderm (mesenchymal) origin,the most common type of ovarian tumors is the epithelial type. These tumors are described asepithelial derived, since they morphologically resemble adenomas and adenocarcinomas andsince they also express characteristic epithelial markers. The cell-cell adhesion molecule E-cadherin has been described as a marker for epithelial cells that decreases in tissue levels withthe dedifferentiation from a highly to a poorly differentiated adenocarcinoma. In paper II, theexpression and cell specific localization of E-cadherin in the normal human ovary and ovariantumors of epithelial origin was investigated.By the method of immunohistochemistry, it was demonstrated that the ovariansurface epithelium (OSE) in general is without E-cadherin staining. However, E-cadherin wasup regulated and present in deep cleft formations of OSE and in inclusion cysts lined withOSE in the normal ovary. Preliminary results from primary cultures of normal human OSE(nOSE) and an immortalized OSE (iOSE) cell-line demonstrate cells without E-cadherinstaining or expression (Ivarsson, Sundfeldt et al unpublished results). Stimulation by growthfactors or cytokines such as insulin like growth factor 1 (IGF-1), interleukin-8 (IL-8), tumornecrosis factor α (TNFα ) and epidermal growth factor (EGF) did not induce E-cadherin inthe iOSE cells (Ivarsson, Sundfeldt et al unpublished results).The E-cadherin staining of the primary tumors of all grades and stages was epithelial-specificand strong except for in one undifferentiated adenocarcinoma, where the staining was morepunctuated. Two peritoneal metastasis (stage III) of moderately/poorly differentiatedadenocarcinoma were also analyzed and demonstrated areas of lesser E-cadherin staining ascompared to the primary tumor. By immunoblotting, an increase of E-cadherin expressionwas demonstrated in the more malignant ovarian tumors as compared to the more benigntumors. There was also expression of E-cadherin in an ovarian cancer cell line (OVCAR3-NIH) but not in a stroma-derived thecoma tumor. With immunoblotting, the lower levels of E-cadherin in metastasis could also be demonstrated.Since the ovarian tumors with acquired invasive capacity (stage III) and the poorly- andundifferentiated tumors still expressed E-cadherin, other components of this adhesioncomplex could be influenced. In a not yet completed study, we have looked at the expressionand cellular localization of α - and β -catenin in the normal ovary and ovarian tumors.Preliminary results demonstrate both α - and β -catenin in OSE, deep cleft formations and ininclusioncysts of the normal ovary (Figure 4 a, b and 5 a, b). The expression of α -catenin isstrong in the malignant tumors with a few exceptions, where it is either totally absent or lostin parts of the tumor (Figure 4 c, d, f and Figure 6 a-d). These particular tumor samples havebeen analyzed for mutational screening in collaboration with Dr. Nollet (University of Ghent,Belgium), with negative results (preliminary findings). Beta-catenin seems to be upregulatedin the more malignant tumors. Recently, a role for β -catenin in tumor progression wassuggested. Due to mutations, the tumor cells i.e. colon cancer cells, have large free pools ofcytoplasmic β -catenin that can interact with the transcription factor Tcf-Lef. The β -catenin/Tcf-Lef complex would then translocate to the cell nucleus and activate transcription.By separating tumor tissue into three different fractions (cell membrane, cytosol and nucleus),we could by immunoblotting demonstrate increased levels of β -catenin in the cytosol andnucleus of the malignant tumors (Figure 7) as compared to benign tumors and normal ovary(unpublished data).In paper III, we further analyzed the concentration of soluble (s) E-cadherin inperipheral blood, cystic-, and ascitic-fluids from patients with benign, borderline and

malignant cystic ovarian tumors. In light of the results of paper II, was an increase of sEcadherinin all fluids from the more malignant tumors, expected. Concentrations of sEcadherinin the fluids were measured by enzyme-linked immunosorbent assay (ELISA).Soluble E-cadherin was detected in all samples analyzed. The concentration of sE-cadherinwas higher in cystic fluid from borderline tumors and adenocarcinomas when compared tobenign cystadenomas. No differences in the levels of sE-cadherin were seen between thetumor groups regarding levels in ascites or peripheral blood. However, the ratios of cysticfluid/peripheral blood levels were also higher in borderline tumors and adenocarcinomascompared to benign cystadenomas. The results of this study suggest a possibility for sEcadherinto be beneficial in increasing the accuracy during preoperative diagnosis of adnexalmasses.THE CELL- AND TIME-SPECIFIC EXPRESSION AND LOCALIZATION OF THE PROTO-ONCOGENE C-MYC IN THE OVARY (PAPER IV)The proto-oncogene protein c-Myc is a transcription factor important forproliferation of cells, as it is believed to bring cells through the G1 checkpoint, whichsubsequently leads to initiation of mitosis. Both follicle and corpus luteum developmentinvolve proliferation of the cells, in particular the GCs of the follicle and endothelial cells ofthe corpus luteum. In paper IV, we could demonstrate an increase of both c-Myc mRNA andprotein during the formation of the preovulatory follicle. These findings were based on twodifferent rat models of induced folliculogenesis in immature prepubertal rats (for details seepaper IV). By separation of the follicle cells into GCs and residual ovary (ROV), we couldfurther show that the c-Myc increase was localized to the GCs at both mRNA and proteinlevels.After the injection of hCG, to mimic the LH-surge, c-Myc mRNA and protein levels wererapidly induced in both GCs and ROV suggesting an increased proliferation of the cellsduring the very initial steps of corpus luteum formation. The proliferative status of the cellswas further demonstrated by the expression of proliferating cell nuclear antigen (PCNA) byimmunoblotting which increased at the same time-points as c-Myc. PCNA has been shown tobe a marker of cells that are in S-phase of the cell cycle. The levels of c-Myc returned to thepreovulatory level just prior to ovulation. During the first days of pseudopregnancy c-Mycexpression resumed its high levels. Figure 8 gives a simplified view of the changes in c-Mycexpression during folliculogenesis, ovulation and corpus luteum formation, which arepresented in paper IV.

Figure 8. C-Myc mRNA and protein in expression in the rat ovary.EXPRESSION OF CCAAT/ENHANCER BINDING PROTEIN (C/EBP) FAMILY OFTRANSCRIPTION FACTORS IN THE HUMAN OVARY AND OVARIAN TUMORS (PAPER V)The knowledge about the C/EBP family of transcription factors has rapidlyincreased since C/EBP was first discovered in 1988. Their roles in proliferation anddifferentiation of normal ovarian cells have been well documented but possible functionsduring tumorigenesis are largely unknown. Since C/EBPs take part on the transcriptional levelin key processes concerning the cell cycle they can be regarded as potential oncogenes.In paper V, tissue samples from normal human ovaries, benign-, borderline- and malignantovarian tumors of different grades and stages were investigated for protein expression and cellspecific staining of the C/EBP family of transcription factors (α , β , δ and ζ ). Byimmunohistochemistry, we could localize the C/EBPs mainly to the normal epithelium andepithelial-derived tumor cells even though C/EBPδ and ζ to a lesser extent also were found inthe surrounding stromal tissue. The intracellular localization of C/EBPα was in the cytosol ofthe cells, in contrast to C/EBPβ and C/EBPδ , which stained the nucleus of all samples.C/EBPζ was predominantly localized in a perinuclear fashion in both stromal and epithelialcells.Semi-quantitative measurements of the protein levels with immunoblot technique could alsodemonstrate expression of the C/EBPs in a majority of the tissue samples. The amount ofC/EBPβ increased during epithelial tumor progression, while C/EBPα and δ levels were more

or less constant. A slight increase in the amount of C/EBPζ was also detected in the moremalignant samples of ovarian tumors. These results suggest a role for C/EBPβ during theproliferative process of epithelial-derived ovarian tumors and a role for the C/EBPβ levels asa potential malignant marker for these tumor cells. The fact that i.e. C/EBPα levels wereconstant and C/EBPζ levels even slightly increased, propose functions for these proteins asnegative regulators in tumorigenesis.Figure 4. Staining of α -catenin in human ovarian tumors and in normal ovaries byimmunohistochemical analysis of, a, normal ovary with OSE; b, ovary with inclusioncystslined with OSE; c, borderline type tumor, stage I; d, peritoneal metastasis from amoderately/poorly differentiated adenocarcinoma; e, moderately/poorly differentiatedadenocarcinoma; stage III; f, undifferentiated adenocarcinoma, stage III.Figure 5. Staining of β -catenin in human ovarian tumors and in normal ovaries byimmunohistochemical analysis of, a, normal ovary with OSE; b, ovary with inclusioncystslined with OSE; c, borderline type tumor, stage I; d, peritoneal metastasis from amoderately/poorly differentiated adenocarcinoma; e, moderately/poorly differentiatedadenocarcinoma; stage III; f, undifferentiated adenocarcinoma, stage III.Figure 6. Immunohistochemical staining of a moderately differentiated adenocarcinoma, stageIII with, a; cytokeratin (pos. control); b, E-cadherin (positive); c, α -catenin (negative); d, β -catenin (both positive and negative areas).Figure 7. Immunoblot expression of β -catenin in sub-cellular fractions of a normal ovary anddifferent ovarian tumors as indicated.

GENERAL DISCUSSIONCELL ADHESION MOLECULES (CAMs) IN FOLLICULOGENESIS, OVULATION AND CORPUSLUTEUM FORMATIONIn paper I it was found that E-cadherin was not expressed in granulosa cells(GCs) of growing antral follicles. Granulosa cells proliferate and differentiate with thedevelopment of the follicle from the antral to the preovulatory stage. Cadherins were earlierdescribed to increase (Blaschuk et al. 1989, Farookhi and Blaschuk 1989) in cultured GCs in vitrostimulated in the presence of estradiol or diethylstilbestrol (DES). In one study, anti-seraagainst avian N-cadherin, recognizing several cadherins, was used (Blaschuk et al. 1989) so thespecific cadherin expression could not be evaluated. However, N-cadherin was later described in GCs (Farookhiand Blaschuk 1991, Peluso et al. 1996, Farookhi et al. 1997, Trolice et al. 1997) and was regulated by estradiol(MacCalman et al. 1995). It is possible that the immunoreactivity detected was mostly due to the N-cadherinCAM. The other study (Farookhi and Blaschuk 1989) used an antibody specific for E-cadherin, but it should benoted that expression of E-cadherin was only found in GCs that were aggregated in culture and not indisaggregated cells. This observation does of course not reflect the in vivo situation, where the GCs willbecome more and more loosely packed in the growing follicle (Bjersing et al. 1974a, 1974b).Moreover, adherens junctions that are build up by E-cadherin are downregulated with follicular growth(Albertini et al. 1974, Amsterdam et al. 1992) at the same time as there are a growing number of gap junctions(Amsterdam et al. 1987, Grazul Bilska et al. 1997). Interestingly, N-cadherin is more often, compared E-cadherin, situated at non-junctional sites (Geiger et al. 1992, Salomon et al. 1992) and this could justify itspresence in GCs. In a preliminary report (Machell et al. 1997), the same group who previously described E-cadherin in GCs actually, by immunohistochemistry, describes the absence of E-cadherin in GCs but presence ofN- and K-cadherin. Moreover, they state later that N-cadherin is the only cadherin present in GCs (Harandian etal. 1998).There might be species differences though, since E-cadherin was described in GCs of porcineovaries (Ryan et al. 1996). The species differences are further noted by the expression E-cadherin in ovarian surface epithelium (OSE) of rat (Hoffman et al. 1993) paper I), mouse(Damjanov et al. 1986) and pig (Ryan et al. 1996), while normal human OSE was without E-cadherin expression (Auersperg et al. 1994, Maines-Bandiera et al. 1997, Davies et al. 1998,Wong et al. 1999)(paper II). In contrast there was no difference in expression of E-cadherintheca cells (TCs) or interstitial cells between porcine and rat ovaries (Ryan et al. 1996) paperI) and the localization to corpus luteum was similar in primates and rats (Khan-Dawood et al.1996a, 1996b) paper I). Thus, the cadherin group of proteins seems to be present in all celltypesof the ovary, though the expression patterns change within the different cell-types.However, the simultaneous expression of α - and β -catenin (Butz et al. 1995) and paper I)support a functional role of the cadherins, in the ovary. The catenins bind to the cytoplasmictail of cadherins and are necessary for the adhesive function (Ozawa et al. 1989, Gumbiner et

al. 1993) for both E-cadherin and N-cadherin (Wheelock et al. 1991). The cadherin/catenincomplex bind to the cytoskeleton via actin (Rimm et al. 1995a) and this has been suggested tobe vital for the strength and the function of cell-cell adhesion (Yap 1998). The expression ofcatenins in GCs, as described in paper I, supports a role in conjunction with cadherins in thesecells.In the in vivo study on normal follicular development in the rat (paper I), E-cadherin staining in epithelial cells/GCs of small follicles situated in the cortex of the ovarywas demonstrated. It was also noted that E-cadherin disappeared in GCs of antral follicles. Inline with this, fetal porcine ovaries had much higher levels of E-cadherin than ovaries fromfertile pigs (Ryan et al. 1996). However, both E- and N-cadherin mRNA levels in whole ovaries fromimmature mice was found to increase as a result of estradiol injection (MacCalman et al. 1994, 1995). Thechange of E-cadherin expression in primordial to antral follicles as described in paper I, could reflect a possiblevital function for this CAM during early stages of follicular development. Firstly, cells of small primordialfollicles are at a resting stage while GCs proliferate rapidly during follicle development (Richards 1980). Severalstudies have described increased cell proliferation after blocked or perturbed expression of cadherin in bothnormal (Hermiston et al. 1995a, 1995b) and tumor cells (Birchmeier et al. 1994a, Mareel et al. 1995, Perl et al.1998) (discussed in detail later). Secondly, both N- and E-cadherin may protect intestinal cells and GCs fromentering programmed cell death (Peluso et al. 1994, 1996, Hermiston et al. 1995a, Peluso 1997), suggesting asurvival-promoting role for the cadherins in these particular cells. Thirdly, the disappearance of E-cadherin couldbe just a reflection of the morphological changes of GCs in the growing follicle.The role for cell-adhesion in GCs during folliculogenesis was investigated by examining theintermediate filaments, i.e. actin, which was downregulated after gonadotropin stimulation ofrat GCs in vitro with a simultaneous increase in steroidogenesis (Ben Ze'ev et al. 1987).These results is supported by the findings of paper I, where it is clearly demonstrated that α -catenin decreased in GCs after human chorion gonadotropin (hCG) or DES treatment. Theinfluence of these hormones was specific for α -catenin since the expression of β -catenin wasunchanged. This strengthens the hypothesis that less adherens junction mediated adhesion isnecessary for GCs of the growing follicle.The expression of E-cadherin/catenin complex in the TCs could be mostly of mechanicalimportance (Adams et al. 1998) to maintain structural integrity of the growing follicle. The complex couldfurther help support the "blood-follicle barrier" (Powers et al. 1996) between GCs and TC interna cell layers, andprotect GCs from influences of the immune system until ovulation (Espey 1980). The downregulation of E-cadherin/catenin complex in TC, only hours before ovulation, as was found in paper I could most likely facilitatethe influx of immune cells, which promote the ovulatory process (Brannstrom et al. 1993). Morphologicalchanges that relate to the development of the corpora lutea might also be affected (Jablonka Shariff et al. 1993).The E-cadherin/catenin complex was demonstrated in corpus luteum of rat ovaries (paper II) and primates(Khan-Dawood et al. 1996a, 1996b). However the staining was less intense when compared to adjacentinterstitial cells and TCs of nearby follicles (paper II).NORMAL OVARIAN SURFACE EPITHELIUM (OSE): THE CELLULAR ORIGIN FORNEOPLASTIC INITIATION AND PROGRESSION?During fetal development the coelomic epithelium, which gives rise to the OSE,has the capacity to differentiate into many different cell types. This capacity probablycontributes to the large variety of phenotypes found among OSE-derived ovarian carcinomas(Nicosia et al. 1991, Blaustein 1995, Auersperg et al. 1995b). The adult OSE is also referredto as a germinal epithelium since it still shares characteristics with the mesothel lining theperitoneum and normal epithelium (Blaustein et al. 1979, Nicosia et al. 1991). However, inthe course of carcinogenesis, OSE become more committed to an epithelial phenotype than toundergo epithelial-mesenchymal conversion. Transformed OSE, and particularly ovarian

carcinoma cells, maintain some epithelial characteristics in culture longer than normal OSE(Auersperg et al. 1994). The epithelial characteristics of ovarian carcinomas tends to be notonly more stable but also more highly differentiated than in OSE, since more epithelialmarkers are expressed and a more complex epithelial morphology is seen (van Niekerk et al.1993, Dyck et al. 1996, Auersperg et al. 1996). This characteristic distinguishes ovariancarcinomas from most other cancers, which in the course of neoplastic progression, becomeless differentiated than the epithelium from which they arise. In this aspect, the finding that E-cadherin is absent in OSE (Shimoyama et al. 1989, Auersperg et al. 1994) paper II) is notsurprising. Mesothelial cells, mesothelioma cell lines and pleural mesotheliomas do notcontain E-cadherin, but express N-cadherin (Pelin et al. 1994, Peralta Soler et al. 1995,Schofield et al. 1997). N-cadherin has also been detected in human OSE (Trolice et al. 1997,Wong et al. 1999).The appearance of E-cadherin in cuboidal or tall cells, cleft formations and inclusion cysts ofthe normal ovary is quite intriguing (Maines-Bandiera et al. 1997, Davies et al. 1998) and paper II. Justrecently, E-cadherin was detected more frequent in cultured OSE from patients with a family history of ovariancancer (FHO) as compared to OSE from control patients (Wong et al. 1999). Mutations in the breast relatedcarcinoma gene-1 (BRCA1) were found in 10/14 of patients with FHO. Whether this induction of E-cadherinexpression is functionally significant in the neoplastic transformation of the ovarian epithelium or merelycontributes to or results from the process of metaplasia of ovarian epithelium is, however, not known. Actually,E-cadherin has been suggested to be a "master-gene", which can induce an epithelial phenotype whenupregulated (Birchmeier et al. 1991, Vanderburg et al. 1996). This mesenchymal/epithelial conversion of cellscaused by E-cadherin have been studied by cDNA transfection of fibroblastic cell-lines, i.e. L-cells or 3T3-cells(Nagafuchi et al. 1987, Nose et al. 1988) and metastatic tumor cell-lines (Frixen et al. 1991, Vleminckx et al.1991, Takeichi 1993, Birchmeier et al. 1994b). These studies described a number of epithelial characteristics,like assembly into epitheloid aggregates, apical basal polarity, appearance of junctions and desmosomes anddecreased invasiveness in tumor cell-lines, as a result of E-cadherin expression or re-expression. Tissue-specificoverexpression of E-cadherin in mouse intestinal epithelium also results in slower cell migration (Hermiston etal. 1996). On the other hand, when a dominant negative mutant N-cadherin that block E-cadherin expression wasused, epithelial morphology was changed and migration increased (Hermiston et al. 1995a).Based on histopathological studies, early malignant changes characteristically seem to occur in OSE-lined cleftsand inclusion cysts (Radisavljevic 1976, Scully 1995) and this sometimes occur in direct continuation from OSEto fully malignant tumors (Puls et al. 1992, Godwin et al. 1993). This suggests that upregulation of E-cadherin innormal OSE surrounded by stromal cells, could play a role in early events leading to the malignant phenotype.Genetic changes in normal cells, equivalent to the changes in the adjacent tumor, have been described in breastcarcinomas (Deng et al. 1996). When a dominant negative E-cadherin mutant was introduced to keratinocytes,cell adhesion was lost, proliferation was inhibited, and terminal differentiation was stimulated (Zhu et al. 1996).However, according to the few reports on E-cadherins role in tumor initiation (Hermiston et al. 1995b, Guilfordet al. 1998) or progression (Perl et al. 1998, Tlsty 1998) presented so far, a loss of E-cadherin rather than anupregulation would be the cause for tumor development.The absence of E-cadherin in OSE, likely reflects the immature nature of human OSE and/or that these cellsrepresent two different cell-types. One cuboidal/tall resting cell with CAM expression and one flat squamous cellthat associate with the site of ovulation (Gillett et al. 1991). The downregulation of E-cadherin has also beendescribed in wound healing of gastric ulcers (Hanby et al. 1996). Moreover, stimulation of growth factors whichare produced either by the underlying stroma (Kruk et al. 1994, Vigne et al. 1994, Karlan et al. 1995a) or theOSE itself (Kruk et al. 1992, Lidor et al. 1993, Ziltener et al. 1993, Auersperg et al. 1995a) could promote E-cadherin expression. In particular EGF, EGF plus IGF-I (Berchuck et al. 1993, Mareel et al. 1994) andtransforming growth factorα (TGFα ) (Jindal et al. 1994) increased proliferation while TGFβ (Berchuck et al.1992) inhibited and steroid hormones (estrogen and progesterone) (Karlan et al. 1995b) had no effect onproliferation of OSE in culture. The possible accumulation of growth factors and hormones in the inclusioncystfluid could have a tumor promoting effect, especially if the genome already has acquired neoplastic mutations.E-CADHERIN EXPRESSION DURING TUMOR PROGRESSION

The expression of E-cadherin was found in epithelial-derived benign adenomasand borderline tumors of the ovary (paper II, (Darai et al. 1997, Peralta Soler et al. 1997, Davies et al.1998). Protein and mRNA expression was further induced in well-, moderately- and poorly- differentiatedadenocarcinomas of the ovary (Inoue et al. 1991, Veatch et al. 1994, Fujimoto et al. 1997a) and paper II, whileDavies et al (1998) in their material found that all poorly differentiated tumors were without E-cadherin. Theextracellular part that can be proteolytically cleaved to a soluble form (Damsky et al. 1983) of E-cadherin (sEcadherin)was found in peripheral blood, cystic and ascites fluid from patients with ovarian cystic tumor masses(Darai et al. 1998) and paper III. Concentration of sE-cadherin was increased in cystic fluid from borderlinetumors and adenocarcinomas when compared to benign adenomas but no changes were seen in ascites (paper III)or peripheral blood (Darai et al. 1998) and paper III. In a genetic screening of 63 carcinomas of the ovary, onlyone mutation was detected in the E-cadherin gene (Risinger et al. 1994). However, decrease of E-cadherin wasdescribed in peritoneal metastasis and ascites cells when compared to the primary tumor (Veatch et al. 1994,Fujimoto et al. 1997b) and paper II.In contrast numerous reports have been published describing a decrease of E-cadherin inadenocarcinomas, which correlated to dedifferentiation and invasive capacity of the cancers of different organssuch as bladder and prostate (Umbas et al. 1992, Giroldi et al. 1994-95, Ross et al. 1995), colon (Dorudi et al.1995), ventricle (Shiozaki et al. 1991), cervix and endometrium (Sakuragi et al. 1994), breast (Takayama et al.1994, Rimm et al. 1995b, Schönborn et al. 1997, De Leeuw et al. 1997), head and neck (Schipper et al. 1991),basal cell of the skin (Pizarro et al. 1995) and thyroid (Brabant et al. 1993, von Wasielewski et al. 1997).Transfection of E-cadherin to dedifferentiated cell lines restored the adhesion and prevented invasion asdescribed above (section about OSE). This has put E-cadherin in the group of tumor suppressor genes (Fearon1998).It is also clear, however, that loss of differentiation of epithelial (carcinoma) cells can occurwhile E-cadherin expression is retained (Birchmeier et al. 1993, Shiozaki et al. 1996). The absence ofdownregulation during tumor progression like in ovarian carcinoma has also been described in studies of lungcancer (Shimoyama et al. 1989), colon cancer (Dorudi et al. 1993, Kinsella et al. 1993) and gastric cancer (Okaet al. 1992). However, in these studies E-cadherin was analyzed by immunological techniques. This does notexclude an altered function of E-cadherin (see discussion below).REGULATION OF E-CADHERIN EXPRESSIONAberrations that result in junctional defects and impaired cell-cell adhesion have beendescribed at many cell-biological levels.Expression of E-cadherin in vitro without the cytoplasmic tail (Nagafuchi et al. 1988, Ozawa et al.1990), extracellular domain (Kintner 1992) or point mutations in the Ca 2+ binding domain all result in nonfunctional adhesion (Birchmeier et al. 1994a, Berx et al. 1998). However, mutations and deletions in genescoding for the cadherin/catenin complex seem to be an exception rather than a rule in cancer cells in vivo.Screening of the E-cadherin gene has detected mutations only in a few cases (Berx et al. 1998)such as in some gastric cancers (Becker et al. 1994) and gastric cancer cell-lines (Oda et al. 1994), lobular breastcancer (Berx et al. 1995a), and endometrial cancer (Risinger et al. 1994). In the majority of tumors where E-cadherin expression was lost or distorted, mutations in the corresponding gene have not been found. In paper II,molecular weight of E-cadherin, analyzed by immunoblotting, did not differ between E-cadherin in normalovary, benign or malignant tumors. This suggests that at least in our material any occurrence of largerearrangements of the gene was not present. This is in line with a previous study where only one mutation wasdetected in 63 investigated ovarian carcinomas (Risinger et al. 1994).Deletions, mutations or truncations of either of the catenins have been described to disrupt interactions in thecadherin/catenin complex (Gumbiner et al. 1993, Huber et al. 1996a) and to alter adhesion in several cancer celllines of gastric (Oyama et al. 1994, Kawanashi et al. 1995), breast (Bullions et al. 1997, Tsukatani et al. 1997) orcolon origin (Breen et al. 1993, Vermeulen et al. 1995). Interestingly, E-cadherin expression remained strongand located to the cell membrane even though cell-cell adhesion was diminished or lost, but in some cases E-cadherin and either catenin were simultaneously downregulated (Shimoyama et al. 1992, Morton et al. 1993,Umbas et al. 1997). For endometrioid (3/6) but not serous (0/5) adenocarcinomas of the ovary, mutations havebeen found in the β -catenin gene (Palacios et al. 1998). The mutational screening of ovarian carcinomas with

decreased α -catenin expression (unpublished results) was without positive findings (Nollet,personal communication). Expression of α - and β -catenin mRNA was not affected duringovarian tumor progression (Fujimoto et al. 1997a) but in peritoneal metastasis (Fujimoto et al. 1997b).Furthermore, the α - and β -catenin protein was lost in 18% and 21% respectively in ovariancancers (Davies et al. 1998). We have also found a loss of α - and/or β -catenin in some ovariancarcinomas (unpublished results), that was not related to stage or grade. However, in sometumors the expression of β -catenin was increased. These findings might indicate an alteredfunction of the cadherin/catenin complex albeit the presence of E-cadherin.Transcriptional regulation of the E-cadherin gene was also reported. The E-cadherin promoterhas been cloned in the mouse (Behrens et al. 1991, Ringwald et al. 1991) and the human (Bussemaker etal. 1994, Berx et al. 1995b, Ji et al. 1997). Promoter assays of several cell lines confirmed that decreased mRNAexpression was due to alterations of E-cadherin transcriptional regulation (Bussemaker et al. 1994, Hennig et al.1995, Ji et al. 1997). The promoter of the murine E-cadherin had a GC-rich region, a CCAAT-box, an E-pal site,containing two E-box consensus binding sites and two AP-2 sites. The two latter regions were not fullyconserved in the human sequence. (Hennig et al. 1996) Hennig et al (1996) proposed that transcription factorsfrom the helix-loop-helix family i.e. the myc family could bind to the E-pal site, members of the C/EBP familycould bind the CCAAT-box while the AP-2 protein could bind the AP-2 sites. However, the optimal binding sitefor C/EBP has later been determined to ATT GCG CAA T (Agre et al. 1989). In preliminary studies of the E-cadherin promoter by electrophoretic-mobility-shift assay (EMSA), we found binding to both the CCAAT-boxand to a closely (87%) related sequence (CTT GCG GAA G) to the optimal C/EBP site, with nuclear extractsfrom an ovarian cancer cell-line (NIH-OVCAR3) (Rask, Sundfeldt, unpublished results). However, only thealmost optimal C/EBP site in the E-cadherin promoter and not the CCAAT-box could super-shift with C/EBPαand C/EBPβ specific anti-serum (Rask, Sundfeldt, unpublished results). Recently, it was alsoshown that c-Myc, AP2 and the retinoblastoma gene product (RB) could activate transcriptionof the E-cadherin gene (Batsché et al. 1998). Moreover as Tcf-Lef/β -catenin was found to bind theE-cadherin promoter an autocrine-regulatory-loop was suggested (Huber et al. 1996b). Whethertranscriptional regulation of E-cadherin is altered in ovarian tumors remains to be elucidated.Alternatively, other mechanisms may have overcome the invasive suppressor function of E-cadherin. Lateralclustering has, in cell culture experiments, demonstrated to influence cell adhesion strength without changes inexpression of E-cadherin (Adams et al. 1998, Yap 1998). This occurs during compaction of blastomeres and canalso be seen in cell culture systems (Adams et al. 1998). The significance of clustering and lateral dimerizationin tumor progression has not been investigated but it could impair the adhesive function in dedifferentiatedtumors, such as ovarian carcinomas, without changes in E-cadherin expression. The crystallographic structure ofthe extracellular cadherin has recently been elucidated and showed that cadherins bind in dimers to the cadherindimers of the neighboring cell (Shapiro et al. 1995). This involves the specific histidine-alanine-vaneline (HAV)sequence (Blaschuk et al. 1990, Shapiro et al. 1995) and synthetic decapeptides containing HAV inhibited E-cadherin mediated activities (Blaschuk et al. 1990). This finding suggests that the extracellular proteolyticallycleaved sE-cadherin also might have the ability to influence E-cadherin functions through its HAV sequence.The functional role of the 80 kDa form is not clear (Damsky et al. 1983, Takeichi 1988). It was demonstrated tohave biological activities, since sE-cadherin was able to disrupt cell-cell adhesion in epithelial cell cultures(Wheelock et al. 1987). The increased concentration of sE-cadherin in blister fluids from patients with variouscutaneous diseases further supported this theory (Matsuyoshi et al. 1995). The potential thus exists for a highturnover of E-cadherin in ovarian adenocarcinomas, with an increased proteolytic cleaving or shedding of sEcadherinto the cystic fluid (paper III). This could in turn lead to a more invasive phenotype. The 80 kDa producthas also been proposed to recruit more E-cadherin to the cellmembrane (Näthke et al. 1993), which in ovariantumor cells could turn out to induce a viscous circle.E-cadherin/catenin complexes are also substrates for kinases (Daniel et al. 1997). Several groups havedemonstrated an inhibitory influence on cell-cell adhesion by tyrosine phosphorylation of E-cadherin. The firstexperiments, where cellular tyrosine phosphorylation was increased, reduced cadherin mediated adhesivestrength (Masuyoshi et al. 1992, Hamaguchi et al. 1993, Behrens et al. 1993) but did not effect the level of E-cadherin expression. When tyrosine phosphorylation was blocked, cell adhesion was restored. Observations havesuggested that regulation by phosphorylation were effectuated through β -catenin (Shibamoto et al. 1994,Daniel et al. 1997, Yap 1998) but also via p120 cas (Shibamoto et al. 1995) that binds directly to the cytoplasmic

tail of E-cadherin (Reynolds et al. 1994, Daniel et al. 1995). A thorough review about cadherin/cateninregulation, phosphorylation via growth factors and cell signaling was presented by Daniels et al (1997). Recentfindings of β -catenins role in the Wnt signaling pathway suggests modifications of this proteinby other intracellular partners, such as glycogen synthase kinase 3β (GSK3β ) (BenZeev et al.1998) affecting the amount of free β -catenin in the cytosol. In fact, Wnt signaling increased the freepool of β -catenin by dephosphorylation, which then could bind to the transcription factor T-cell factor-Leukocyte enhancer factor (Tcf-Lef) (BenZeev et al. 1998). The β -catenin/Tcf-Lefcomplex translocated to the nucleus and could regulate transcription of E-cadherin (Huber et al.1996b), c-Myc (He et al. 1998) and cyclin D1 (Tetsu et al. 1999), the only targeted genes found so far. In ourimmunohistochemical analysis of ovarian carcinomas, β -catenin were observed mainly in the plasmamembrane and not in the cytosol or nucleus (Figure 5). However, the ovarian cancer cell line(NIH-OVCAR3) and immunoblotting analysis of ovarian carcinoma cell fractionsdemonstrated β -catenin expression in all cell compartments (Figure 7) which then could be aresult of changes in the Wnt signaling pathway.The strong expression of E-cadherin in ovarian carcinomas could be a reflection of the earlierchange from the mesenchymal OSE to the epithelial carcinoma and the fact that ovariancarcinomas retain many epithelial characteristics (se discussion about OSE) even at lowdifferentiated grades. It could also be speculated that the ovarian carcinomas might benefitfrom expressing CAM during progress until the point or the location where invasion starts. Atransient up- and downregulation of E-cadherin could actually promote proliferation of tumorcells and allow repeated steps of invasion (Giroldi et al. 1994-95, Mareel et al. 1995). As discussed indetail above, many findings suggest that functional adhesion may be impaired even when E-cadherin isexpressed. If functional adhesion is impaired during ovarian carcinogenesis remains to be elucidated.E-CADHERIN IN CLINICAL PRACTICEE-cadherin as a prognostic marker for ovarian carcinomas seem less plausible since there wasno correlation between expression of the protein or mRNA with dedifferentiation or invasivecapacity (paper II) (Fujimoto et al. 1997a, Davies et al. 1998). The tumor-promoting role of E-cadherinincreasein OSE from patients with BRCA1 mutations (Wong et al. 1999) and expression in inclusioncysts of thenormal ovary remains to be elucidated. It would be of great interest to find out whether patients with E-cadherinexpression are prone to develop ovarian cancers or if it is just a reflection of a mesenchymal/epithelialconversion?In paper III, the levels of the soluble part of E-cadherin (sE-cadherin) in peripheral blood, cystic and ascites fluidfrom patients with benign, borderline and malignant ovarian cystic tumors were investigated. The sE-cadherin isproteolytically cleaved and this cleavage was hypothesized to increase during tumor progression. This wasdemonstrated to be true in plasma from patients with a number of different cancers (Katayama et al. 1994) butnot from patients with gastric or ovarian carcinoma (Velikova et al. 1997, Darai et al. 1998). Increasedconcentrations of sE-cadherin were also described in urine from patients with bladder cancer and in blister fluidsfrom patients with cutaneous diseases (Banks et al. 1995, Matsuyoshi et al. 1995). In cystic fluid from ovariantumors, concentrations of sE-cadherin were increased in borderline tumors and adenocarcinomas as compared tobenign adenomas (Darai et al. 1998)(paper III). In paper III, no differences in concentrations between the patientgroups were seen in peripheral blood or ascites fluid. The finding could however be useful in attempts to find adiagnostic tool, which can differentiate between benign cystic masses and borderline/malignant cystic massesbefore surgical intervention is decided upon (Chapron et al. 1996). Thus low concentrations of sE-cadherinwould predict a benign cystadenoma rather than a borderline or malignant tumor. Cystic fluids from ovariantumors have been found to contain several factors i.e. interleukin-8 (IL-8) (Ivarsson et al. 1998), IL-6 (van derZee et al. 1995), and insulin-like growth factor-I (IGF-I) (Karasik et al. 1994) suggested as possible markers ofovarian tumor progression. However, a prospective study with a larger material of cystic fluids would berequired before adapting these "markers" to clinical practice. In addition cyst fluids from simple cysts shouldalso be included in such study.THE PROTO ONCOGENE C-MYC AND FOLLICLE CELL PROLIFERATION

In the primordial follicle the single layer of GCs is quiescent (Hirshfield 1991).After a couple of slowly dividing cell cycles, GCs start to proliferate rapidly as aresponse to follicle stimulating hormone (FSH) and luteinizing hormone (LH) (Richards1980, Richards et al. 1995). Proliferation of GCs in rat during this phase is demonstratedby the concomitant increase in 3 H-thymidine (Rao et al. 1978), 5-bromodeoxyuridine(BrdU) (Gaytan et al. 1996) or proliferating nuclear cell antigen (PCNA) (Chapman etal. 1994)(paper IV). This latter protein is involved in DNA synthesis and repair (Shivji etal. 1992). PCNA is commonly used as a marker of proliferation. In human ovariesproliferation in follicles was also shown by increased labeling indexes of Ki67, PCNAand silver staining of nuclear organizer regions (AgNORs) (Funayama et al. 1996).Proliferation of GCs during follicular development were further demonstrated in cellcycle cyclin D2 (-/-) mice, were GC was unable to respond with normal proliferationafter FSH stimulation (Sicinski et al. 1996). Moreover, cyclin D2 (Robker et al. 1998b)and cyclin E (Robker et al. 1998a) were hormonally induced during follicular growthand localized to proliferating granulosa cells. These two cyclins are positive regulators ofcell cycle progression during the G1-phase (Sherr 1994).In paper IV, we demonstrated that c-Myc, which is low in quiescent cells and increase rapidlyafter mitotic stimuli (Bouchard et al. 1998), is transiently increased in GCs after PMSGstimulation in the immature rat. By immunohistochemistry this increase was alsoprimarily detected in the GCs. This response to PMSG had been demonstrated earlierfor intact animals and for ovaries in peri-fusion (Delidow et al. 1990, 1992). In paper IV,there was a transient increase of c-Myc after PMSG. However according to Hirshfield etal (reviewed 1991)) the GCs in the antral follicles from the small immature ovary, belongto the 8 th or 9 th generation. This means that the proliferative potential of GCs in the PMSGmodelis limited, since the preovulatory follicle contains GCs of the 10 th generation(Hirschfield 1991). This could explain the transient and limited increase of c-Myc afterPMSG in paper IV. Moreover, we could demonstrate an induction of c-Myc in ovaries fromimmature rats, 25 days of age, as compared to c-Myc levels in ovaries from immature rats, 18days of age. Both c-Myc mRNA and protein were present in all follicles but primordialfollicles in human ovaries (Putowski et al. 1997) and in GC and corpus luteum of primates(Fraser et al. 1995). C-Myc was not present in corpus albicans or in ovaries frompostmenopausal women (Putowski et al. 1997). This strengthen the hypothesis that c-Myc is important during the proliferative stages of ovarian cell cycle but also that c-Mycmight play a role in apoptosis since atretic follicles and corpus luteum expressed thisproto-oncogene (Fraser et al. 1995, Putowski et al. 1997, Thompson 1998)The GCs of the developing antral follicle not only proliferate but alsodifferentiate into steroid producing cells and acquire LH-receptors (Uilenbroek et al. 1979,Richards 1980). The LH-surge reprograms the steroidogenic cells to produce mainly progesterone and todifferentiate by luteinization. Both TCs, GCs and endothelial cells join in the formation of the mature corpusluteum which has been proposed to be among the fastest growing tissues known (Jablonka Shariff et al. 1993).The staining in the corpora lutea by BrdU-technique was localized to the vascular endothelium (Jablonka Shariffet al. 1993, Gaytan et al. 1996). The steroid producing cells in the corpora lutea were not stained.These two studies were performed in cycling rats and sheeps and did not describe the initialresponse to LH i.e. prior to ovulation. Incorporation of 3 H-thymidine, as a marker ofproliferation, was decreased in GCs 24h and 48h after hCG (LH-surge) stimulation (Rao et al.1978) but the decrease was less pronounced in TCs. In paper IV, however, we can show anincrease of PCNA in GCs as early as 2h after hCG-stimulation followed by a constantdecrease during the first 24h (CL day 1). At 24h after hCG, PCNA was elevated in theresidual ovary compared to corpus luteum , which could reflect the proliferation of non-

steroid producing cells as described before (Jablonka Shariff et al. 1993, Gaytan et al. 1996). Apronounced increase of c-Myc mRNA and protein was also observed 1h and 2h after hCG,respectively, both in GCs (Agarwal et al. 1996) (paper IV) and residual ovaries (paper IV). Suggesting thatthere is a proliferative response of the steroidogenic cells maybe concentrated to the TCs (Rao et al. 1978).However cyclin D2 protein decreased as early as 4h after hCG while cyclin E protein was notdownregulated until 48 h after hCG (Robker et al. 1998a, 1998b). Interestingly, cyclin E was shown to bea downstream target of c-Myc (Leone et al. 1997, Bouchard et al. 1998), so the actual increased levels of cyclinE after hCG could be a result of the induction by c-Myc in GC. The cyclins and cell cycle progression areinhibited by i.e. p27 Kip1 . This protein was found in very low levels 4h after hCG and thenincreased to high levels 24h and 48h after hCG (Robker et al. 1998b). This high concentration ofp27 Kip1 might be the signal to effectuate a total stop in cell cycle mediated division of GCs.CCAAT/ENHANCER BINDING PROTEINS (C/EBPs) IN TUMORIGENESISOriginally detected in liver (Diehl 1998) and adipose (Darlington et al. 1998) tissues, theC/EBP transcriptionfactor proteins have now been found in cells of several other origins(Lekstrom Himes et al. 1998) like breast (Robinson et al. 1998, Seagroves et al. 1998), lung(Flodby et al. 1996), ovary (Piontkewitz et al. 1993, Sirois et al. 1993), gastro-intestinal tract(Chandrasekaran et al. 1993, Blais et al. 1995) and hematopoetic cells (Zhang et al. 1997).Although the C/EBPs play a role in the acute phase response (Poli 1998), as they both canactivate cytokines i.e. interleukin-6 (IL-6) and IL-8 or be activated by IL-6, I will in thissection concentrate on C/EBPs role in regulation of the cell cycle.As was discussed in the section of Specific Background the different proteins ofthe C/EBP family of transcription factors either take part in differentiation (C/EBPα ) orproliferation (C/EBPβ and δ ) or block transcription (C/EBPζ ). The C/EBPs has actuallybeen found to interact with several regulators of cell cycle progression. Firstly, C/EBPα wasfound to activate transcription of and stabilizing p21(WAF-1) protein (Timchenko et al. 1996,1997). This protein is an inhibitor of cyclin-dependent kinase. Secondly, the retinoblastomagene (RB), which is suggested to be a gatekeeper for entry into the S-phase of cell division,interact with C/EBP during adipocyte differentiation (Chen et al. 1996a). Thirdly, the protooncogene protein c-Myc, that facilitate G1-progression of the cell cycle, inhibits ordownregulates the expression of C/EBPα (Freytag et al. 1992, Antonson et al. 1995, Mink etal. 1996, Piontkewitz et al. 1996b). Moreover, cyclin D2, a cell cycle activating protein hasconsensus binding sites for C/EBPs in its promoter (Jun et al. 1997). These studies providesome clues to the earlier findings of C/EBPα as an activator of terminal differentiation inadipocytes (Samuelsson et al. 1991), since cell division can be halted through interaction withseveral known key regulators in the cell cycle.In line with this C/EBPα should be downregulated or inactivated during tumor progression. Inovarian (paper V) and colorectal (Rask et al. 1999) unpublished results) tumors C/EBPα wasfound in all tumor samples and did not change with tumor progression. However, C/EBPαwas localized to the cytosol of the cells, which might indicate that it was inactivated. In livercarcinomas C/EBPα was found in both nucleus and cytoplasm of the tumor cells anddecreased in preneoplastic nodules (Flodby et al. 1995) and dedifferentiated tumors (Xu et al.1994). Moreover, induction of C/EBPα demonstrated anti-proliferating functions i.e. growtharrest, in tumor cell-lines (Watkins et al. 1996).During liver regeneration C/EBPβ and δ was found to be activated (Flodby et al.1993). In fact, increased C/EBPβ DNA-binding activity during G1 phase of the hepatocyte cell

cycle was demonstrated in liver regeneration in vivo (Rana et al. 1995). A role for C/EBPβ inbreast was also shown as normal lobuloalveolar proliferation during mammary glanddevelopment was disturbed in C/EBPβ -/- mammary glands (Robinson et al. 1998, Seagroves et al.1998). Moreover, ovarian follicles from C/EBPβ -/- mice (Sterneck et al. 1997) or treated withantisense C/EBPβ (Pall et al. 1997), do not develop normally, have a markedly decreasedovulation rate and the C/EBPβ -/- mice have no corpus luteum. More recently another role forC/EBPβ was shown, as treatment of colorectal cancer cells with antioxidants could lead toapoptosis by induction of p21(Waf1) through a mechanism involving C/EBPβ (Chinery et al.1997). Both the involvements C/EBPβ in cell proliferation and apoptosis are interesting in theaspect of tumorigenesis. This transcription factor has been found in the nucleus of ovarian(Paper V) and colon (Rask et al. 1999), unpublished results) cancer cells and increased duringovarian tumor progression in correlation with dedifferentiation and invasion. Interestingly, theshorter form of C/EBPβ (the LIP protein) was detected only in the malignant ovarian tumors(paper V). A specific increase of LIP was also found in human breast tumors and LIP wassuggested as a marker for the identification of patients with poor prognosis (Zahnow et al. 1997).However, LIP is probably not functional as an activator of transcription since the activatingdomain in the N-terminal part of the protein is missing (Descombes et al. 1991). LIP can insteadform inactive dimers with the full-length C/EBPβ (LAP) and the LAP/LIP ratio have beensuggested to be of importance for the transcriptional activity (Raught et al. 1995).Cancer invasion, that is, synthesis of proteolytic enzymes and transport throughthe extracellular matrix are thought to be an interplay between the cancer cells and the nonneoplasticstromal cells (Johnsen et al. 1998). The increased degradation of collagen in theextracellular matrix, which facilitates the migration of tumors cells, is mediated by matrixmetalloproteinases (MMPs) (Shapiro 1998). The MMPs are predominantly produced bysurrounding host stromal and inflammatory cells in response to factors released by tumors butalso by the tumor cell itself (Shapiro 1998). In this aspect the recent finding that C/EBPβ inducetranscription of the collagenase-1 (MMP-1) gene is very interesting (Doyle et al. 1997). MMP-1seems to be the enzyme that is principally responsible for collagen turnover in most humantissues. The increased expression of C/EBPβ in ovarian tumor cells described in paper V,could in this way also be an activator of cancer invasion.ACKNOWLEDGEMENTSI would like to thank all the people at the Department of Physiology, University of Göteborgand at "Spec Labb", Department of Gynecology and Obstetrics, Sahlgrenska Hospital for alltheir friendship, help and fruitful discussions.Special thanks to;Lars Hedin, my supervisor and teacher in science, to whom I which to express my deepestgratitude for his great knowledge, never ending flow of ideas and untiring support duringthese last years. Mats Brännström, my co-supervisor for his enormous enthusiasm andpositive personality and for his ability to get everything done before due time. ProfessorStaffan Edén, for trying a wild card and constantly encouraging and supporting it. ProfessorP-O Janson, for taken part in my work and for interesting conversations. Göran Bergström,for introducing me to the world of research. Sven Enerbäck, for his energetic and stimulating

discussions. Yael Piontkewitz, for teaching me the art of immunohistochemistry. KarinIvarsson, for sharing workload and for your admirable patience with the OSE cultures.Katarina Rask, for following up all the loose ends and pursuing the cell-adhesion trace.Håkan Billig, Ola Nilsson, Pär Hellberg and Magnus Haeger for interesting and fruitfulcollaboration. Anna Wickman, for her comfort and advice. Malin Carlsson, and all the otherstudents in the lab that has contributed to this work. Ann Wallin, for excellent statistical help.Lena Olofsson and Gunnel Larsson, without you this thesis would not have been possible.I also like to thank all of my friends and especially the "BAUTA-gang" for their friendshipin both sorrow and happiness. Martin, Saskia and Nina for many god laughs and friendship.Mother, for being my best mother at all times but also as an example to follow in life.Father, for your constant love and believe in me. Petra, Erik and Frida for letting me knowwhat life is all about and at last Mikael, for giving me endless amounts of freedom, love anddreams.This research project was also supported by grants from The Swedish Medical ResearchCouncil (to Lars Hedin: grant 10375), the Göteborg Medical Society, King Gustav V JubileeClinic Cancer Research Foundation and the foundations of the Royal Society of Arts andSciences in Göteborg, Hjalmar Svensson, Assar Gabrielsson, Ollie and Elof Ericsson, Knutand Alice Wallenberg, Axel and Margaret Ax:son-Johnson, Lundberg, Magnus Bergvall, apostdoctoral fellowship to Yael Piontkewitz from the Wennergren foundation and the MedicalFaculty, University of Göteborg.REFERENCESADAMI, H.O., HSIEH, C.C., LAMBE, M., TRICHOPOULOS, D., LEON, D.,PERSSON, I., EKBOM, A. and JANSON, P.-O. Parity, age at first childbirthand risk of ovarian cancer. Lancet., 344, 1250-1254 (1994).ADAMS, C.L. and NELSON, J.W. Cytomechanics of cadherin-mediated cellcelladhesion. Curr Opin Cell Biol., 10, 572-577 (1998).AGARWAL, P., PELUSO, J.J. and WHITE, B.A. Steroidogenic factor-1expression is transiently repressed and c-myc expression and deoxyribonucleicacid synthesis are induced in rat granulosa cells during the periovulatoryperiod. Biol Reprod., 55, 1271-1275 (1996).

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