Temperature-Sensitive Src-Transformed Mdck Cells nn 3d Cultures as a Model of Malignant Transformation of Epithelial Cells

The equilibrium between cell proliferation, differentiation, and apoptosis is crucial for maintaining homeostasis in epithelial tissues. In order for the epithelium to function properly, individual cells must gain normal structural and functional polarity. The junctional proteins have an important role both in binding the cells together and in taking part in cell signaling. Cadherins form adherens junctions. Cadherins initiate the polarization process by first recognizing and binding the neighboring cells together, and then guiding the formation of tight junctions. Tight junctions form a barrier in dividing the plasma membranes to apical and basolateral membrane domains. In glandular tissues, single layered and polarized epithelium is folded into tubes or spheres, in which the basal side of the epithelial layer faces the outer basal membrane, and the apical side the lumen. In carcinogenesis, the differentiated architecture of an epithelial layer is disrupted. Filling of the luminal space is a hallmark of early epithelial tumors in tubular and glandular structures. In order for the transformed tumor cells to populate the lumen, enhanced proliferation as well as inhibition of apoptosis is required. Most advances in cancer biology have been achieved by using two-dimensional (2D) cell culture models, in which the cells are cultured on flat surfaces as monolayers. However, the 2D cultures are limited in their capacity to recapitulate the structural and functional features of tubular structures and to represent cell growth and differentiation in vivo. The development of three-dimensional (3D) cell culture methods enables the cells to grow and to be studied in a more natural environment. Despite the wide use of 2D cell culture models and the development of novel 3D culture methods, it is not clear how the change of the dimensionality of culture conditions alters the polarization and transformation process and the molecular mechanisms behind them. Src is a well-known oncogene. It is found in focal and adherens junctions of cultured cells. Active src disrupts cell-cell junctions and interferes with cell-matrix binding. It promotes cell motility and survival. Src transformation in 2D disrupts adherens junctions and the fibroblastic phenotype of the cells. In 3D, the adherens junctions are weakened, and in glandular structures, the lumen is filled with nonpolarized vital cells. Madin-Darby canine kidney (MDCK) cells are an epithelial cell type commonly used as a model for cell polarization. Its-src-transformed variants are useful model systems for analyzing the changes in cell morphology, and they play a role in src-induced malignant transformation. This study investigates src-transformed cells in 3D cell cultures as a model for malignant transformation. The following questions were posed. Firstly: What is the role of the composition and stiffness of the extracellular matrix (ECM) on the polarization and transformation of ts v-src MDCK cells in 3D cell cultures? Secondly: How do the culture conditions affect gene expression? What is the effect of v-src transformation in 2D and in 3D cell models? How does the shift from 2D to 3D affect cell polarity and gene expression? Thirdly: What is the role of survivin and its regulator phosphatase and tensin homolog protein (PTEN) in cell polarization and transformation, and in determining cell fate? How does their expression correlate with impaired mitochondrial function in transformed cells? In order to answer the above questions, novel methods of culturing and monitoring cells had to be created: novel 3D methods of culturing epithelial cells were engineered, enabling real time monitoring of a polarization and transformation process, and functional testing of 3D cell cultures. Novel 3D cell culture models and imaging techniques were created for the study. Attention was focused especially on confocal microscopy and live-cell imaging. Src-transformation disturbed the polarization of the epithelium by disrupting cell adhesion, and sensitized the cells to their environment. With active src, the morphology of the cell cluster depended on the composition and stiffness of the matrix. Gene expression studies revealed a broader impact of src transformation than mere continuous activity of src-kinase. In 2D cultures, src transformation altered the expression of immunological, actin cytoskeleton and extracellular matrix (ECM). In 3D, the genes regulating cell division, inhibition of apoptosis, cell metabolism, mitochondrial function, actin cytoskeleton and mechano-sensing proteins were altered. Surprisingly, changing the culture conditions from 2D to 3D affected also gene expression considerably. The microarray hit survivin, an inhibitor of apoptosis, played a crucial role in the survival and proliferation of src-transformed cells.

Kudosten normaalin toiminnan kannalta solujen jakaantumisen, erilaistumisen ja ohjelmoidun solukuoleman tasapaino on tärkeää. Syövissä erilaistuneen kudoksen rakenne muuttuu. Tämä johtuu pitkälti solujen jakaantumisen ja ohjelmoidun solukuoleman säätelyn häiriintymisestä, joka mahdollistaa rauhastiehyiden ja rauhasten täyttymisen elävillä soluilla. Kudostasolla rauhasen täyttymis-ilmiötä pidetäänkin yhtenä syövän ensimerkeistä. Syöpää on tutkittu pitkään käyttämällä erilaisia soluviljelymalleja. Nykytieto pohjautuu suurimmaksi osaksi tuloksiin, jotka on saatu käyttämällä kaksiulotteisia soluviljelymalleja eli viljelemällä soluja joko soluviljelymaljalla tai siihen tarkoitetussa pullossa. Kyseisen mallin etuja ovat mm. sen helppokäyttöisyys ja edullisuus. Huonoina puolina ja käyttöä rajoittavana tekijänä voidaan mainita, ettei se ei kuvaa luotettavasti solujen käyttäytymistä mutkikkaammassa elimistössä. Kolmiulotteisten soluviljelymallien kehitys on mahdollistanut solujen tutkimisen enemmän luonnollisessa ympäristössään. Tutkimuksen tarkoituksena on tarkastella src-syöpägeenin aiheuttamaa rauhassolujen muuntumista syöväksi kokeellisissa kolmiulotteisissa soluviljelymalleissa. Työtä varten kehitettiin uusia soluviljely- ja kuvantamismenetelmiä. Erityinen mielenkiinto oli säilyttää kolmiulotteinen rakenne konfokaalimikroskooppia käyttämällä ja kehittämällä elävien solujen kuvantamismenetelmä. Src-syöpägeeni häiritsee solun erilaistumista ja herkistää ne ympäristönsä vaikutukselle. Geenien aktiivisuusmäärityksissä src-syöpägeenin vaikutus oli enemmän kokonaisvaltainen kuin pelkkä src-kinaasin jatkuva aktiivisuus. Myös viljely-ympäristön muuttaminen kaksiulotteisesta kolmiulotteiseksi muuttaa geenien aktiivisuutta. Ohjelmoidun solukuoleman estäjä surviviini-proteiinilla näyttäisi olevan merkittävä rooli solujen jakaantumisen ja ohjelmoidun solukuoleman välttämisen säätelyssä. Tutkimus on tuonut myös tärkeää uutta tietoa eri viljelymallien vaikutuksesta tutkittavien solujen käyttäytymiseen.