To investigate the tumor fraction with cancer stem/tumor initiating cell (CSC/TIC) characteristics, we tested the human cervical carcinoma cell lines A431, Caski and SiHa, by growth as non-adherent spheres in specific media and aldehyde dehydrogenase (ALDH) enzymatic activity. A good correlation between the two parameters was observed and the highest levels were observed in A431 cell line that was selected for characterization of the CSC/TIC fraction. A431 parental cells already displayed characteristics common to CSC/TIC, such as sphere forming efficiency, adherent holoclone formation and high ALDH activity. Non-adherent spheres maintained or increased these properties, and, in particular, ALDH-positive fraction increased from 46 to 65% and a transient induction of stem cell markers such as Nanog, Nestin and Oct4 was observed. Furthermore, a significant increase of paraclone forming cells was observed, suggesting that differentiation took place inside sphere cell populations. As compared to parental cells, spheres were characterized by: (1) a ten-fold higher verapamil-sensitive side population fraction; (2) the appearance of a podoplanin-positive subpopulation characterized by a small cell size; (3) the ability to propagate tumors in nude mice at a lower cell dose. The global gene expression analysis demonstrated a strong and reversible modulation of 'sphere' phenotype in comparison to parental and sphere cells re-induced to adherent conditions. All together our results indicated that the growth of A431 cells as a non-adherent sphere was not sufficient by itself to define a stem-like population, but it was essential for the emergence of a small population of tumor cells with CSC properties.

Cadherin-mediated adhesion plays an important role in maintaining cell-cell contacts and reducing tumor metastasis. However, neo-expression of E-cadherin in ovarian carcinoma does not prevent the release and spread of cells from the primary tumor. Because caveolin-1 is down-regulated concomitantly with Ecad expression, we investigated whether the stability of adherens junctions in ovarian carcinoma was affected by caveolin-1 expression. We used IGROV1 cells transfected with caveolin-1 (IGtC3), mock-transfected control cells (IGtM87), and SKOV3 cells that endogenously express caveolin-1. Simultaneous expression of caveolin-1 and E-cadherin favored membrane distribution of E-cadherin and its associated catenin (p120ctn), even when caveolin-1 was only focally associated with adherens junctions. Silencing of caveolin-1 induced intracellular E-cadherin redistribution in IGtC3 and SKOV3 cells. Treatment with the specific src kinase inhibitor PP1 increased E-cadherin expression in IGtM87 and SKOV3 cells and enhanced membrane localization of both E-cadherin and p120ctn. However, PP1 could not completely reverse the detrimental effects on cell-cell adhesion induced by Ca Caveolin-1 (cav-1) is a 22-to 24-kd integral membrane protein present in numerous tissue types, which localizes in membrane subdomains called caveolae. Interaction of cav-1 with a variety of protein and nonprotein molecules results in pleiotropic effects on numerous cellular events such as signal transduction, gene regulation, lipid metabolism, and vesicular traffic.1-3 Down-regulation of cav-1 in human tumor samples of different histological origin has been documented, suggesting that this protein has a negative regulatory role in tumor development and acts as an onco-suppressor.4 -10 Although cav-1-null mice are no more prone to tumor development than are the cav-1-expressing counterparts, the null mice reveal an increased susceptibility to dysplastic mammary 11 or epidermal lesions 12 in the presence of an initial oncogenic stimulus. However, in a few tumor histotypes, cav-1 up-regulation has been associated with tumor progression and enhanced metastatic capability. 13,14In vitro, cav-1 expression is up-regulated in confluent cells, and the protein distributes in areas of cell-cell contacts, 15 in which molecules involved in intercellular cell adhesion, such as E-cadherin (E-cad), are localized. Ecad mediates cell-cell adhesion through calcium-dependent homophilic interaction of the extracellular domains forming cis-dimers on the same cell, which interact with cis-dimers on neighboring cells to generate trans-interactions. Stable cell-cell interactions require binding of catenins (cat) to the cytoplasmic domain of E-cad. Commonly, ␤-or ␥-cat bind the carboxy-terminal sequence of E-cad via armadillo-repeats and simultaneously interact with cytoplasmic ␣-cat to link the entire complex to the Supported by grants from Associazione Italiana Ricerca sul Cancro and the Cariplo Foundation.

IntroductionUrokinase-type plasminogen activator (uPA) is a serine protease involved in normal tissue remodeling and also in pathological events such as tumor invasion and metastasis. 1 Several human tumors have been found to overexpress uPA, and inhibition of the expression or of the enzymatic activity of this protease reduces the invasive and metastatic phenotype. 1 A number of physiological, pathological, and chemical stimuli activate transcription of the uPA gene. 2 Their effect is mediated by the uPA enhancer, located approximately 2000 bp upstream of the transcriptional start site. 3 This element contains an Ets-2 site juxtaposed to an octameric AP-1 A site at the 5Ј end and an eptameric AP-1 B site at the 3Ј end. 4 The region between the AP-1 sites is defined as cooperativity mediator (COM) and is necessary for the combined action of the AP-1-binding transcription factors. 5 The COM region is subdivided, in turn, in an upstream COM (uCOM) and a downstream COM (dCOM). By electrophoretic mobility shift assays, it was shown that uCOM can form 4 different complexes 6-8 with individual proteins, such as Oct-1, 9 with heterodimers of the Prep-1 and Pbx proteins 10,11 and with an as-yet-unidentified UEF-1 protein.The dCOM region appears to bind specifically only UEF-1 (M.P. unpublished data, May 2000). Interestingly, although some of the factors binding to the uCOM region are known transcriptional activators (ie, Oct-1 12 and Prep-1/Pbx with HoxB1 11,13 ), they do not display any transactivating activity in the uPA enhancer context. 6 However, both uCOM and dCOM are required for full enhancer activity. 6 It is thus the proteins binding to the AP-1 sites that behave as transactivators in the uPA enhancer. AP-1 family transcription factors activate many genes in response to a large number of stimuli. 14 Since members of the c-Jun family can form homodimers or heterodimers with members of the c-Fos family and with ATF-2, 15 it is evident that the number of transcription factors potentially involved in transcriptional activation is rather large. In the case of the uPA enhancer, Cirillo et al 16 have shown that, besides the transcription factor ATF-2, at least 2 members of the c-Jun family and 2 of the c-Fos family are involved in the response of HepG2 cells to induction by interleukin 1 (IL-1) and tetradecanoyl phorbol acetate (TPA). Furthermore, it was shown that the phosphorylation state of these proteins also is relevant to transcriptional activation. 16 Traditionally, the proximal promoter of the human uPA gene has been considered as spanning approximately 86 bp upstream of the transcription start site. 3 This region contains 5 high-(3 ϫ GGGCGG) and low-(2 ϫ GGGAGG) affinity binding sites for the Sp1 family transcription factors, immediately upstream of the TATA box. This family is composed of 4 members, with a high degree of structural but not functional similarity. 17 Sp1 and Sp4 are transcriptional activators, whereas Sp3 represses Sp1-mediated transcription. [18][19][20][21][22] Sp3 contains a portable repr...