It is well established that lysosomes play an active role during the execution of cell death. A range of stimuli can lead to lysosomal membrane permeabilization (LMP), thus inducing programmed cell death without involvement of the classical apoptotic programme. However, these lysosomal pathways of cell death have mostly been described in vitro or under pathological conditions. Here we show that the physiological process of post-lactational regression of the mammary gland is accomplished through a non-classical, lysosomal-mediated pathway of cell death. We found that, during involution, lysosomes in the mammary epithelium undergo widespread LMP. Furthermore, although cell death through LMP is independent of executioner caspases 3, 6 and 7, it requires Stat3, which upregulates the expression of lysosomal proteases cathepsin B and L, while downregulating their endogenous inhibitor Spi2A (ref. 8). Our findings report a previously unknown, Stat3-regulated lysosomal-mediated pathway of cell death under physiological circumstances. We anticipate that these findings will be of major importance in the design of treatments for cancers such as breast, colon and liver, where cathepsins and Stat3 are commonly overexpressed and/or hyperactivated respectively.

STAT3 is the key mediator of apoptosis in mammary gland. We demonstrate here that LIF is the physiological activator of STAT3, because in involuting mammary glands of Lif -/-mice, pSTAT3 is absent and the STAT3 target, C/EBPδ, is not upregulated. Similar to Stat3 knockouts, Lif -/-mammary glands exhibit delayed involution, reduced apoptosis and elevated levels of p53. Significantly, Lif -/-glands display precocious development during pregnancy, when pSTAT3 is not normally detected. We show that pERK1/2 is significantly reduced in Lif -/-glands at this time, suggesting that at this stage LIF mediates its effects through pERK1/2. Inhibition of LIFmediated ERK1/2 phosphorylation potentiates the proapoptotic effects of STAT3. LIF therefore signals alternately through ERK1/2, then STAT3, to regulate mammary growth and apoptosis.

IntroductionMammary gland development during the pregnancy cycle is characterized by successive phases of cell growth, differentiation, high metabolic activity and apoptosis. At the ultrastructural level this includes dramatic changes in tissue architecture, involving ductal epithelial branching and morphogenesis, invasion of tissue compartments, vascularization and subsequent organized remodelling. These events are highly reproducible and strictly controlled at the transcriptional level by circulating hormones and locally derived factors [1]. Thus, many transcription factors have been shown either to directly affect this developmental program or to exhibit altered activity at specific stages in the pregnancy cycle [1]. CAM = cell adhesion molecule; C/ebp = CAAT-enhancer binding protein; IGF = insulin-like growth factor; IL = interleukin; LIF = leukaemia inhibitory factor; LPS = lipopolysaccharide; NF-κB = nuclear factor-κB; Stat = signal transducer and activator of transcription. Breast Cancer ResearchVol 6 No 2 Clarkson et al. Research articleGene expression profiling of mammary gland development reveals putative roles for death receptors and immune mediators in post-lactational regression Richard AbstractIntroduction In order to gain a better understanding of the molecular processes that underlie apoptosis and tissue regression in mammary gland, we undertook a large-scale analysis of transcriptional changes during the mouse mammary pregnancy cycle, with emphasis on the transition from lactation to involution.

Purpose:To assess image quality and image-guidance capabilities of a cone-beam CT based smallanimal image-guided irradiation unit ͑micro-IGRT͒. Methods: A micro-IGRT system has been developed in collaboration with the authors' laboratory as a means to study the radiobiological effects of conformal radiation dose distributions in small animals. The system, the X-Rad 225Cx, consists of a 225 kVp x-ray tube and a flat-panel amorphous silicon detector mounted on a rotational C-arm gantry and is capable of both fluoroscopic x-ray and cone-beam CT imaging, as well as image-guided placement of the radiation beams. Image quality ͑voxel noise, modulation transfer, CT number accuracy, and geometric accuracy characteristics͒ was assessed using water cylinder and micro-CT test phantoms. Image guidance was tested by analyzing the dose delivered to radiochromic films fixed to BB's through the endto-end process of imaging, targeting the center of the BB, and irradiation of the film/BB in order to compare the offset between the center of the field and the center of the BB. Image quality and geometric studies were repeated over a 5-7 month period to assess stability. Results: CT numbers reported were found to be linear ͑R 2 Ն 0.998͒ and the noise for images of homogeneous water phantom was 30 HU at imaging doses of approximately 1 cGy ͑to water͒. The presampled MTF at 50% and 10% reached 0.64 and 1.35 mm −1 , respectively. Targeting accuracy by means of film irradiations was shown to have a mean displacement error of ͓⌬x,⌬y,⌬z͔ = ͓−0.12, −0.05, −0.02͔ mm, with standard deviations of ͓0.02, 0.20, 0.17͔ mm. The system has proven to be stable over time, with both the image quality and image-guidance performance being reproducible for the duration of the studies. Conclusions:The micro-IGRT unit provides soft-tissue imaging of small-animal anatomy at acceptable imaging doses ͑Յ1 cGy͒. The geometric accuracy and targeting systems permit dose placement with submillimeter accuracy and precision. The system has proven itself to be stable over 2 yr of routine laboratory use ͑Ͼ1800 irradiations͒ and provides a platform for the exploration of targeted radiation effects in small-animal models.

Prolactin and leukemia inhibitory factor (LIF) have different roles in the adult mammary gland, which are mediated in part by the signal transducers and activators of transcription (STAT)5 and STAT3. In vivo studies have shown that STAT5 contributes to prolactin-dependent lobuloalveolar development and lactation whereas STAT3 mediates LIF-dependent epithelial apoptosis during postlactational involution. To understand the molecular basis of these STAT-dependent pathways, we demonstrate the ligand-independent effects of STAT5 and STAT3 in mammary epithelial cells in vitro and also identify the genes regulated by these related transcription factors. Thus, using conditionally active STAT3- or STAT5a-GyraseB fusion proteins, we observed that enforced and specific dimerization of STAT3 induced apoptosis whereas STAT5 induced differentiation of mammary epithelial cells. Furthermore, STAT5 attenuated apoptosis mediated by LIF, the physiological inducer of STAT3. Microarray analysis of STAT3- and STAT5-induced genes using this system demonstrated a marked specificity, which reflected their different physiological effects in vitro and in vivo. STAT5-specific gene targets included the milk protein genes alpha-casein and kallikrein-8 and the survival factors prosaposin and Grb10. STAT3-specific genes included the apoptosis regulators CCAAT enhancer binding protein-delta, phosphatidylinositol 3-kinase-regulatory subunits, purine nucleoside phosphorylase, and c-fos. These data illustrate that specific activation of STAT3 and STAT5 alone is sufficient to induce and suppress apoptosis, respectively, and that these transcription factors elicit their actions by inducing distinct subsets of target genes in mammary epithelial cells.