Science Collected Works - Theses
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ItemMolecular mechanisms that regulate apoptosis and involution in the mammary gland(University of Melbourne, 2008)This thesis investigates the hypothesis that milk components play a role in regulating mammary gland function during involution, and explores an in vitro model system to examine the response of mammary tissue in the absence of significant milk accumulation. It was determined that a population of mammary epithelial cells have an intrinsic survival mechanism and that a milk protein can regulate cell fate, which is most likely central to the process of involution in the lactation cycle. Previous studies using eutherian species concluded that mammary explants require addition of exogenous macromolecules to remain hormone responsive in culture. In the absence of added hormones and growth factors, the alveoli and small terminal ducts of mouse explants showed degeneration (Elias, 1957). Furthermore, the withdrawal of hormones from bovine mammary gland explants resulted in DNA laddering (Accorsi et al., 2002). Therefore, the addition of growth factors to the culture media appeared to prevent the onset of apoptosis in mammary explants, which perpetuated the dogma that permissive growth factors are required for mammary epithelial cell survival in vitro. The research presented herein challenges this dogma. Investigation of mammary explants in chemically defined media and in the absence of hormones and growth factors demonstrated definitive evidence for mammary epithelial cell survival without any exogenous hormones. Furthermore, this phenomenon is well conserved, and occurs in the tammar wallaby (Macropus eugenii), the cow (Bos taurus) and the mouse (Mus musculus): three evolutionarily distinct species with markedly different lactation strategies. The specific mechanism that permits a population of mammary epithelial cells to survive in the absence of either hormones or growth factors in mammary explants was not confirmed, however two possibilities were investigated. Although milk proteins are synthesised and secreted by mammary explants incubated with lactogenic hormones, milk constituents do not accumulate to elevated levels such as those at weaning (Nagamatsu and Oka, 1980; Nicholas and Tyndale-Biscoe, 1985). Therefore, milk accumulation that provides the trigger for involution upon milk stasis (Green and Streuli, 2004; Quarrie et al., 1996) is absent in this culture model, which may allow a population of mammary epithelial cells to survive. Alternatively, microarray analysis of wallaby and cow mammary explants showed that a number of antioxidant cytoprotective genes involved in stress-induced cellular defence mechanisms, not yet defined in the mammary gland, are upregulated. The induction of these genes is most likely due to the stress of culture on the mammary tissue, which may be responsible for maintaining a population of mammary epithelial cells in explants. Although it was unclear whether the absence of an accumulated milk factor facilitated mammary epithelial cell survival in mammary explants from the wallaby, cow and mouse, a possible trigger of involution due to milk accumulation was investigated. It is well established that chemically induced forms of the milk protein ?-lactalbumin (LALBA) can cause apoptosis of transformed and immature cells, while sparing healthy cells (Hakansson et al., 1995). More recently, it was demonstrated that some commercial preparations of bovine LALBA (bLALBA) had direct apoptotic effects on an intestinal cell line (Xu et al., 2005) and primary fur seal (Arctocephalus pusillus) mammary epithelial cells (Sharp et al., 2007). Similarly, the results presented in this thesis show that bLALBA had a potent apoptotic effect on a number of mammary cell types, and the effect appears to be a function of a 28kD variant. The naturally occurring 28kD form of bLALBA caused apoptosis of primary mammary epithelial cells, immortal HC11, AGS and Hek293 cell lines, and a number of human breast cancer cell lines. The cells became irreversibly apoptotic within 1-3 hours of exposure to the 28kD bLALBA form, and an equal and potent apoptotic effect occurred when cells were exposed to either the naturally occurring 28kD form of bLALBA or bLALBA treated with EDTA to remove bound calcium. Furthermore, incubation of cells with calcium-depleted bLALBA in calcium free media did not alter the apoptotic effects, which suggests the protein is a stable complex. In contrast to the chemically induced LALBA variants, multimeric LALBA (MAL) and human LALBA made lethal to tumour cells (HAMLET), cell death also occurred after fatty acids were sequestered from bLALBA by pre-incubation with BSA. It therefore appears the apoptotic bLALBA form is a naturally occurring stable structure present in bovine whey with strong and irreversible apoptotic potential. Microarray analysis of HC11 cells exposed to bLALBA showed that important genes involved in the apoptosis pathway were upregulated, and genes previously identified in serum starved apoptotic HC11 cells (Seol et al., 2005) were also differentially expressed. Analysis of the abundance of 28kD bLALBA during lactation showed this form was highest in whey from cows during the drying off period, when cells are undergoing apoptosis due to involution of the mammary gland. We propose that the apoptotic effects of 28kD LALBA may be involved in mammary epithelial cell death during involution, a process initiated by the accumulation of milk. Although the trigger of involution is associated with milk accumulation, a myriad of downstream signaling pathways are required to conduct the apoptosis stimuli (Watson, 2006; Green and Streuli, 2004). This study shows that the conditional deletion of STAT6 delays involution in the mouse mammary gland by 72 h, which highlights that apoptosis regulation in vivo is multifactorial and depends on the critical juxtaposition of several signaling pathways. Using microarray analysis it was demonstrated that the expression of important involution genes involved in apoptosis and immune-responses have been altered in STAT6-/- mice. Involution requires immuno-modulatory signals to suppress the potentially damaging effects of cellular inflammatory responses, which may correlate with a Th2 cytokine environment that operates through the actions of STAT6 within the immune system. The precise mechanisms that regulate these actions remains unclear, however, as the specific roles of the immune system in mammary gland involution have not been clearly defined these results emphasize the importance of considering immune response signaling events in any future studies of involution.