Abstract |
The human mammary gland in vivo is made up of branching epithelial ducts
embedded in a supporting stroma. One of the earliest changes seen in the
development of breast cancers is the disruption of normal tissue
architecture, thereby leading to the loss of the organisation of the
branching ducts. The objective of this study was to set up an in vitro
system whereby the molecules involved in branching morphogenesis of a non
tumorigenic human mammary epithelial cell line could be analysed. These
molecules would be potential targets for alteration in tumorigenesis, and
also have a potential role in mammary gland development in vivo. This
thesis describes the in vitro system used, analysis of the roles of
fibroblast secreted factors and integrins in the branching morphogenesis
process, and the effect of overexpression of oncogenes on the ability of
cells to form organised morphogenetic structures. The in vitro system
involves culturing a human mammary epithelial cell line (HB2) in 3-
dimensions within a collagen type I (or fibrin) matrix. The cells grow to
form compact, organised, spherical cysts, or can be induced to perform
branching morphogenesis when cultured with conditioned medium from
fibroblasts. We have found that there are at least two distinct soluble
factors that can induce branching morphogenesis; hepatocyte growth factor
(HGF), and an unidentified and potentially novel factor secreted by human
foreskin fibroblasts (HFF). Preliminary characterisation of this factor
has shown that it is a heat stable protein with a size of between 35 and
45 kDa. In the analysis of the role of integrins in branching
morphogenesis, we have analysed both the expression of integrins at the
RNA level throughout the morphogenetic time course, and the effect of
specific monoclonal antibodies (mAbs) to integrin subunits. Use of
specific integrin mAbs that inhibit cell-matrix interaction, has shown
that integrin mediated maintenance of cell-matrix interaction is vital
both for the growth of HB2 cells and for maintaining cell-cell
interactions in the branching structures. In both cases the integrins
involved were found to differ depending on the matrix in which the
branching morphogenesis occurred, with the a2?1 important in the collagen
system and the [alpha]v[beta]1 in fibrin. Both HGF and HFF conditioned
medium are shown to stimulate the motility of HB2 cells on collagen, and
it is hypothesised that the stimulation of cell motility is a necessary
event in the branching morphogenesis process, and that the [alpha]2[beta]1
integrin has a specific role in branching morphogenesis by way of
regulating this motility. Evidence for this is provided by showing that
mAbs to the [alpha]2 or [beta]1 integrin subunits that reduce HGF induced
motility also inhibit branching morphogenesis induced by HGF.
Overexpression of the v-Ha-ras oncogene in HB2 cells also led to the
disruption of cell-cell interactions in 3-dimensional morphogenetic
structures. Overexpression of ras induced a reduced level of expression of
the [alpha]2 integrin subunit and a concurrent reduction in adhesion of
the cells to collagen type I. Further evidence that the [alpha]2[beta]1
integrin-collagen interaction is involved in regulating cell-cell
interaction in 3-dimensional structures was provided by showing that the
HB2 ras colony phenotype could be significantly reverted with a mAb to the
[beta]1 integrin subunit which increases the integrin mediated adhesion of
cells to collagen.
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