Mouse mammary gland involution is associated with cytochrome c release and caspase activation.

At weaning, milk producing mammary epithelial cells undergo apoptosis and are removed by phagocytosis. Here, we show that mouse mammary gland involution is associated with mitochondrial cytochrome c release and processing of numerous caspases, including caspase-1, -3, -7, -8 and -9. Induction of caspase-3-like activity paralleled cleavage of poly-(ADP--ribose) polymerase. Dexamethasone inhibited processing of caspase-3, -7 and -8 and apoptosis, but had no effect on caspase-1 accumulation and cytochrome c release. In Bcl-2 transgenic animals, cytochrome c release, caspase activation and apoptosis were impaired. Thus, the pro-apoptotic signaling pathway in mammary epithelial cells during involution involves the release of cytochrome c and activation of caspases. It is inhibited by Bcl-2 at the mitochondrial level and by dexamethasone at a post-mitochondrial level.

Caspases: decoders of apoptotic signals during mammary involution. Caspase activation during involution.

At weaning most of the alveolar epithelial cells in the mammary gland die by apoptosis and are removed by phagocytosis. Caspases are a family of aspartate specific cysteine proteases. Activation of caspases is generally thought to represent a major and irreversible event in the apoptotic process. We analyzed caspase expression and activation during mammary gland involution. A quantitative RT-PCR based approach revealed that levels of mRNA expression of several caspases are induced during involution. Using an antibody that specifically recognizes activated caspases we measured a transient induction of caspase activity in situ and found a maximal activation at days two and three of involution. These data were corroborated by monitoring caspase-3 like activity in mammary extracts with a synthetic DEVD-afc peptide as caspase-3 substrate. Using Fas-deficient mice we present evidence that the Fas signaling pathway is not essential for caspase activation and apoptosis during mammary gland involution. In summary, signaling pathways during involution seem to involve activation of caspases as intraepithelial triggers of mammary epithelial cell apoptosis.

Nuclear localization of procaspase-9 and processing by a caspase-3-like activity in mammary epithelial cells.

Caspases are aspartate-specific proteases that are specifically activated by numerous death stimuli. Caspase activation is thought to play a major role for the execution of apoptosis. Inactive caspase-9 zymogen is known to be localized within the mitochondrial intermembrane space where it is involved in monitoring mitochondrial damage-associated cytochrome c release and subsequent activation of procaspase-3. Here we show that in mammary epithelial cell lines a significant fraction of caspase-9 proform is associated with discrete structures in the nucleus. Stimulation of cells with chemotherapeutic agents leads to the processing of nuclear procaspase-9 and to the accumulation of nuclear and cytoplasmic caspase activity. Using cell-free extracts from caspase-3-deficient MCF-7 cells we show that caspase-8-mediated processing of nuclear procaspase-9 requires caspase-3. In caspase-3-expressing breast cancer cells, cytochrome c-induced processing of nuclear procaspase-9 is blocked by the caspase inhibitors z-VAD and DEVD but not by YVAD. Purified active caspase-3 is sufficient to cleave nuclear caspase-9 zymogen. These results suggest that, in addition to the mitochondrial localization, caspase-9 proform is found within the nucleus and its processing can be regulated by caspase-3.

Physiological apoptosis in hormone-dependent tissues: involvement of caspases.

Physiological apoptosis in mammals is a type of programmed cell death, an important element in the developmental repertoire ensuring tissue homeostasis and proper disposal of cells that are no longer needed, such as milk-producing epithelial cells in the mammary gland after lactation, luteal cells in the post partum Corpus luteum or secretory cells in the prostate after castration. Although incompletely described, apoptosis in hormone-dependent tissues is apparently initiated and executed using common biochemical strategies. These include survival pathways governed by local and systemic factors and hormones, diverse regulatory pathways and caspase-dependent execution pathways. Using an antibody that recognizes processed effector caspases or a fluorogenic caspase substrate, we present for the first time evidence that caspases are activated in the mammary gland, in the prostate and in the ovary at the time when apoptosis occurs. Most likely phagocytosis of apoptotic cells by neighboring cells may represent an important step, since only a modest involvement of professional phagocytes is apparent. Here, we will summarize and discuss recent data and will attempt to draw a generalized picture of how physiological apoptosis may occur in these organs.