EDUCACIÓN Y VALORES
3. R ELACIÓN INTELIGENCIA VOLUNTAD AFECTIVIDAD EN EL PROCESO FORMATIVO
inactive GDP-bound state to regulate actin cytokeleton organisation. This
molecular switch is regulated by guanine nucleotide exchange factors (GEF)
which accelerates the intrinsic exchange activity to favour the Rac-GTP bound
form. In contrast, GTPase activating proteins (GAP) stimulate the intrinsic GTP
hydrolysis activity of Rac to form Rac-GDP. (Adapted from Ridley, 2011; Wert-
heimer et al., 2012).
GDP Rac1 GTP Rac1 GEF Guanine nucleotide exchange factor GAP GTPase activating proteinsChapter 1: Introduction
1993). EGF also disrupts adhesion of weakly metastatic A431 cells by rounding up and ruffling (Chinkers et al., 1981). Non-malignant human mammary epithelial cells acquire dose dependent motility upon EGF stimulation (Joslin et al., 2007). The similar motogenic effects of EGF in both oncogenic and non-tumorigenic systems highlight the subversion of a normal cell feature into an oncogenic property.
1.4 Physiological contexts of interdigitations in non-tumorigenic systems Interdigitations are common features in other complex epithelial cells and have been reported in kidney podocytes, keratinocytes and endothelial cells (Baluk et al., 2007; Hoelzle and Svitkina, 2012; Mundel et al., 1995; Vasioukhin et al 2000). Ultrastructural studies have described well-developed cytoplasmic interdigitations of mammary epithelial cells isolated from human donor post weaning breast milk (Russo et al., 1975). During early embryonic development and puberty, the elongated tips of mammary buds form multi-layered epithelia, and cells within the interior layers show prominent interdigitation and high levels of desmosomes (Ewald et al., 2012).
Podocytes in kidney have been characterised to form interdigitating foot processes that attach to glomerular basement membrane (GBM) but remain connected via slit membranes (Mundel and Kriz, 1995). The gaps between digits allow ions and solutes to filter through the glomerulus. In keratinocytes and endothelial cell cultures, interdigitations form prior to the assembly of stable adherens junctions and cell-cell contact (Hoelzle and Svitkina, 2012; Vasioukhin et al., 2000). These structures require actin reorganisation and reflect basal phenomena in subconfluent cell cultures without ligand stimulation. Oak leaf-shaped lymphatic endothelial cells display striking interdigitations decorated by cadherins and tight junctions (Baluk et al., 2007; Yao and McDonald, 2014). These were reported to reside specifically at the initial rather than the distal lymphatics to facilitate flow of fluids especially in newborn mice (Pflicke et al., 2009; Yao and McDonald, 2014). In induced inflammation, digits regressed and fluid flow was disrupted. Despite burgeoning evidence of interdigitation in physiological systems, molecular pathways that control the decision between smooth and interdigitated cell boundaries have not been dissected in vitro.
Chapter 1: Introduction
1.5 Non-tumorigenic mammary epithelial cells 1.5.1 MCF10A cells
MCF10A are non-tumorigenic mammary epithelial cell, which were originally derived from subcutaneous mastectomy tissue of a 36 year-old parous, premenopausal woman diagnosed with benign fibrocystic disease (Tait et al., 1990; Soule et al., 1990). These cells were spontaneously immortalised and were characterised as pseudodiploid luminal ductal cells (Tait et al., 1990). MCF10A cells are negative for oestrogen receptors, and have been classified as both basal and luminal of origin, depending on the method of classification. Histocytochemical staining of relevant keratins in MCF10A favours the luminal classification (Tait et al., 1990). On the other hand, technologies using DNA, CGH (Complete Genome Hybridisation) arrays and their phenotype in 3D culture tend to classify MCF10A as basal breast epithelial cells (Debnath et al., 2003; Neve et al., 2006; Charafe-Jauffret et al., 2006; Simpson et al., 2008).
Modern single-nucleotide polymorphism (SNP) arrays revealed that immortalisation of MCF10A cells was due to the amplification of Myc on chromosome 8q24 and deletion of CDKN2A (cyclin-dependent kinase inhibitor 2A) gene on chromosome 9q21.3 (Kadota et al., 2010). C-Myc is a proto-oncogene that acts as a multifunctional transcription factor regulating a myriad of cellular processes including cell cycle, growth, metabolism and genomic stability (Campaner and Amati, 2012). CDKN2A/ p16 induces cell cycle arrest in G1 and G2 phase, and the homozygous loss has been postulated to initiate the process of immortalisation of MCF10A cells (Cowell et al., 2005). Karyotypic analysis of late passage cells demonstrated the genetic stability of these cells in culture with relatively minimal rearrangements (Soule et al., 1990; Vitolo et al., 2009). Another group that used CGH arrays showed that MCF10A cells overexpress SLD5 (DNA replication complex GINS protein SLD5), THAP1 (THAP domain containing apoptosis associated protein 1) and TMEPAI (transforming growth factor-β (TGF-β)–induced transmembrane protein) (Neve et al., 2006). SLD5 in the GINS complex has been characterised as a novel factor to initiate and elongate DNA during DNA replication (MacNeill, 2010). THAP1 is a DNA binding transcription factor that regulates endothelial cell proliferation and G1/S cell-cycle progression (Cayrol et al., 2007). TMEPAI overexpression has been linked to growth of breast cancer cells (Singha et al., 2010). Despite identification of these genetic aberrations, none have been reported to confer malignancy or transform MCF10A cells without exposure to external carcinogen.
Chapter 1: Introduction
Experimentally, MCF10A cells are characterised as ‘near normal mammary epithelial cells’ as growth in culture requires hormones and growth factors. In 2D cell culture, they organise into ‘cobblestone’ monolayers (Soule et al., 1990). They also grow and form three-dimensional structures in collagen and Matrigel (Debnath et al., 2003; Soule et al., 1990). Crucially these cells lack anchorage-independent growth and tumorigenicity in nude mice (Soule et al., 1990).
1.5.2 MCF10A cells in three-dimensional (3D) culture
The mammary gland is a unique organ named after an entire class of animal and demonstrates remarkable plasticity throughout a mammalian’s lifetime from development to puberty and finally during pregnancy and lactation (Gjorevski and Nelson, 2011). It consists of a branching network of ducts lodged within an adipocyte rich environment (Campbell and Watson, 2009). The bilayer of luminal and myoepithelial cells maintains the ductal epithelium with the latter in contact with the basement membrane (Figure 1.7). Knowledge of the signals and mechanisms
regulating gland remodelling in vivo remain fragmented due to the difficulty in
mimicking the exact processes in vitro (Campbell and Watson, 2009).
MCF10A cells have been cultured in 3D environments using different substrates such as collagen and reconstituted basement membrane, Matrigel. Culture in collagen allows formation of duct-like structures whereas in Matrigel, growth suppressed cyst-like acini spheroids form from MCF10A cells (Debnath et al., 2003; Soule et al., 1990; Chapter 3: Figure 3.10).
The development of MCF10A cells into complex structures in vitro allows extensive
research to interrogate the molecular mechanisms involved in mammary gland morphogenesis during normal processes and the subsequent dysregulation in cancers. Brugge and colleagues have dissected in detail the morphogenetic processes involved in the formation and polarisation of non-malignant MCF10A cells within acini (Debnath et al., 2003; Debnath et al., 2005). One of the important non- malignant characteristics preserved in MCF10A acini is the suppression of proliferation and lumen clearance observed in the final stages of acini development. This parameter allows researchers to elucidate the effects of introducing oncogenic mutations in this aspect of mammary alveolar morphogenesis (Debnath et al., 2003; Isakoff et al., 2005).