Sugeetha Ramakrishnan, School of Medicine, Deakin University, Victoria, Australia
Sugeetha Ramakrishnan, Julie Sharp, Kevin Nicholas
School of Medicine, Deakin University, Victoria, Australia
Mammary gland involution is a physiological process associated with rapid apoptosis (programmed cell death) of the lactating mammary epithelial cells followed by remodelling of the mammary gland to a pre-pregnant state. The loss of secretory alveolar epithelium is attributed to a number of factors like mild ischemia as a result of milk engorgement and compression of vasculature, falling levels of prolactin upon cessation of suckling, factors in milk that promote cell death, physical distension of the luminal epithelium and increased activity of the basement membrane degrading enzymes (matrix metalloproteinases). Therefore it is clear that the retention of milk plays an essential role in this process.
Mammary gland involution in the mouse is a 2 step process according to its reversibility. Early involution, occurring within the first 24-48 hours, is reversible and induced by local factors. If weaning is re-initiated during this phase, the process of programmed cell death can be reversed. The second phase, a remodelling programme initiated to return the gland to a pre-pregnant state, is irreversible and requires systemic factors.
Alpha Lactalbumin, a major whey protein secreted by the mammary gland epithelium, may undergo partial unfolding, bind to a fatty acid cofactor like oleic acid and become tumoricidal. HAMLET (Human alpha-lactalbumin made lethal to tumor cells) induces apoptosis in tumor and immature cells, but normal differentiated cells are resistant to its effects. Recent studies (Sharp and Nicholas Laboratory) have shown that a different form of the α-lactalbumin dimer complexed with unsaturated fatty acids has the capacity to program apoptosis in a variety of cells. The objective of this study is to re-evaluate the regulation of mammary gland involution, particularly to study the role of specific milk proteins and the extracellular matrix proteins involved in mechanisms of transition between the reversible and irreversible phases.
Analysis of microarray data analysing murine mammary gland during lactation and involution reveals significant changes in the expression of genes coding for secretory proteins that may play a role in phase 2 involution. In order to examine the interplay between the role of milk factors and ECM components on epithelial cell function and fate during involution, an in-vitro model has been established. Mouse mammary epithelial cells have been isolated from day 12 pregnant mice (C57BL/6J) and cultured on extra-cellular matrix extracted from day 15 lactating (day 0 involuting) to day 4 involuting mammary tissue. In contrast to the 3-D acini structures (mammospheres) formed on day 15 lactating matrix, the cells did not polarize and lost the capacity to form these structures when cultured on day 3 and day 4 involuting matrices. When these 3D acini structures were transferred to lactating and early involuting matrices (day 1 and day 2) they depolarized and were programmed into apoptosis. Current studies are examining the relationship of milk proteins, particularly a-lactalbumin and ECM to promote cell death.