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The use of cell culture models is a principal and fundamental technology used in understanding how mammalian cells work. However, for some cell types such as mammary epithelia, the lines selected for extended culture are often transformed or have chromosomal abnormalities, while primary cultures have such a curtailed lifespan that their use is restricted. For example, mammary luminal epithelial cells (MECs) are used to study mechanisms of breast cancer, but the proliferation of primary cell cultures is highly limited. Here we describe the establishment of a new culture system to allow extended analysis of cultures of primary mouse MECs. In 2D monolayer culture, primary MECs showed a burst of proliferation 2-3 days post isolation, after which cell cycle decreased substantially. Addition of mammary epithelial growth factors, such as Epidermal Growth Factor, Fibroblast Growth Factor-2, Hepatocyte Growth Factor, and Receptor Activator for Nuclear Factor κB Ligand, or extracellular matrix proteins did not maintain their proliferation potential, neither did replating the cells to increase the mitogenic response. However, culturing MECs directly after tissue extraction in a 3D microenvironment consisting of basement membrane proteins, extended the time in culture in which the cells could proliferate. Our data reveal that the cellular microenvironment has profound effects on the proliferative properties of the mammary epithelia and is dominant over growth factors. Moreover, manipulating the cellular environment using this novel method can maintain the proliferative potential of primary MECs, thus enabling cell cycle to be studied as an endpoint after gene transfer or gene deletion experiments.

BACKGROUND: Cellular terminal differentiation (TD) correlates with a permanent exit from the cell cycle and so TD cells become stably post-mitotic. However, TD cells express the molecular machinery necessary for cell proliferation that can be reactivated by experimental manipulation, yet it has not been reported the stable proliferation of any type of reactivated TD cells. Neurons become post-mitotic after leaving the ventricular zone. When neurons are forced to reenter the cell cycle they invariably undergo cell death. Wider evidence indicates that the post-mitotic state cannot solely depend on gene products acting in trans, otherwise mutations in the corresponding genes may lead to reentry and completion of the cell cycle in TD cells, but this has not been observed. In the interphase, nuclear DNA of metazoan cells is organized in supercoiled loops anchored to a nuclear nuclear matrix (NM). The DNA-NM interactions define a higher-order structure in the cell nucleus (NHOS). We have previously compared the NHOS of aged rat hepatocytes with that of early post-mitotic rat neurons and our results indicated that a very stable NHOS is a common feature of both senescent and post-mitotic cells in vivo. PRINCIPAL FINDINGS: In the present work we compared the NHOS in rat neurons from different post-natal ages. Our results show that the trend towards further stabilization of the NHOS in neurons continues throughout post-natal life. This phenomenon occurs in absence of overt changes in the post-mitotic state and transcriptional activity of neurons, suggesting that it is independent of functional constraints. CONCLUSIONS: Apparently the continued stabilization of the NHOS as a function of time is basically determined by thermodynamic and structural constraints. We discuss how the resulting highly stable NHOS of neurons may be the structural, non-genetic basis of their permanent and irreversible post-mitotic state.

The distribution of chromatin within the mammalian nucleus is constrained by its organization into chromosome territories (CTs). However, recent studies have suggested that promiscuous intra- and inter-chromosomal interactions play fundamental roles in regulating chromatin function and so might define the spatial integrity of CTs. In order to test the extent of DNA mixing between CTs, DNA foci of individual CTs were labeled in living cells following incorporation of Alexa-488 and Cy-3 conjugated replication precursor analogues during consecutive cell cycles. Uniquely labeled chromatin domains, resolved following random mitotic segregation, were visualized as discrete structures with defined borders. At the level of resolution analysed, evidence for mixing of chromatin from adjacent domains was only apparent within the surface volumes where neighboring CTs touched. However, while less than 1% of the nuclear volume represented domains of inter-chromosomal mixing, the dynamic plasticity of DNA foci within individual CTs allows continual transformation of CT structure so that different domains of chromatin mixing evolve over time. Notably, chromatin mixing at the boundaries of adjacent CTs had little impact on the innate structural properties of DNA foci. However, when TSA was used to alter the extent of histone acetylation changes in chromatin correlated with increased chromatin mixing. We propose that DNA foci maintain a structural integrity that restricts widespread mixing of DNA and discuss how the potential to dynamically remodel genome organization might alter during cell differentiation.