Programmed cell death (PCD) plays a key role in developmental biology and in maintenance of the steady state in continuously renewing tissues. Currently, its existence is inferred mainly from gel electrophoresis of a pooled DNA extract as PCD was shown to be associated with DNA fragmentation. Based on this observation, we describe here the development of a method for the in situ visualization of PCD at the single-cell level, while preserving tissue architecture. Conventional histological sections, pretreated with protease, were nick end labeled with biotinylated poly dU, introduced by terminal deoxy-transferase, and then stained using avidin-conjugated peroxidase. The reaction is specific, only nuclei located at positions where PCD is expected are stained. The initial screening includes: small and large intestine, epidermis, lymphoid tissues, ovary, and other organs. A detailed analysis revealed that the process is initiated at the nuclear periphery, it is relatively short (1-3 h from initiation to cell elimination) and that PCD appears in tissues in clusters. The extent of tissue-PCD revealed by this method is considerably greater than apoptosis detected by nuclear morphology, and thus opens the way for a variety of studies.

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Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation.

Evidence from clinical and experimental studies indicates that macrophages promote solid-tumour progression and metastasis. Macrophages are educated by the tumour microenvironment, so that they adopt a trophic role that facilitates angiogenesis, matrix breakdown and tumour-cell motility — all of which are elements of the metastatic process. During an inflammatory response, macrophages also produce many compounds — ranging from mutagenic oxygen and nitrogen radicals to angiogenic factors — that can contribute to cancer initiation and promotion. Macrophages therefore represent an important drug target for cancer prevention and cure.

Tumour-educated macrophages promote tumour progression and metastasis

https://www.cell.com/cell/pdf/0092-8674(92)90123-T.pdf

Induction of apoptosis in fibroblasts by c-myc protein

■ Abstract Apoptosis is a genetically programmed, morphologically distinct form of cell death that can be triggered by a variety of physiological and pathological stimuli. Studies performed over the past 10 years have demonstrated that proteases play critical roles in initiation and execution of this process. The caspases, a family of cysteine-dependent aspartate-directed proteases, are prominent among the death proteases. Caspases are synthesized as relatively inactive zymogens that become activated by scaffold-mediated transactivation or by cleavage via upstream proteases in an intracellular cascade. Regulation of caspase activation and activity occurs at several different levels: ( a) Zymogen gene transcription is regulated; ( b) antiapoptotic members of the Bcl-2 family and other cellular polypeptides block proximity-induced activation of certain procaspases; and ( c) certain cellular inhibitor of apoptosis proteins (cIAPs) can bind to and inhibit active caspases. Once activated, caspases cleave a variety of intracellular polypeptides, including major structural elements of the cytoplasm and nucleus, components of the DNA repair machinery, and a number of protein kinases. Collectively, these scissions disrupt survival pathways and disassemble important architectural components of the cell, contributing to the stereotypic morphological and biochemical changes that characterize apoptotic cell death.

Mammalian caspases: structure ,a ctivation ,s ubstrates, and functions during apoptosis

Vulnerable periods during the development of the nervous system are sensitive to environmental insults because they are dependent on the temporal and regional emergence of critical developmental processes (i.e., proliferation, migration, differentiation, synaptogenesis, myelination, and apoptosis). Evidence from numerous sources demonstrates that neural development extends from the embryonic period through adolescence. In general, the sequence of events is comparable among species, although the time scales are considerably different. Developmental exposure of animals or humans to numerous agents (e.g., X-ray irradiation, methylazoxymethanol, ethanol, lead, methyl mercury, or chlorpyrifos) demonstrates that interference with one or more of these developmental processes can lead to developmental neurotoxicity. Different behavioral domains (e.g., sensory, motor, and various cognitive functions) are subserved by different brain areas. Although there are important differences between the rodent and human brain, analogous structures can be identified. Moreover, the ontogeny of specific behaviors can be used to draw inferences regarding the maturation of specific brain structures or neural circuits in rodents and primates, including humans. Furthermore, various clinical disorders in humans (e.g., schizophrenia, dyslexia, epilepsy, and autism) may also be the result of interference with normal ontogeny of developmental processes in the nervous system. Of critical concern is the possibility that developmental exposure to neurotoxicants may result in an acceleration of age-related decline in function. This concern is compounded by the fact that developmental neurotoxicity that results in small effects can have a profound societal impact when amortized across the entire population and across the life span of humans.

Critical periods of vulnerability for the developing nervous system: evidence from humans and animal models.

Mammary epithelial cells are embedded in a unique extracellular environment to which adipocytes and other stromal cells contribute. Mammary epithelial cells are critically dependent on this milieu for survival. However, it remains unknown which adipocyte-secreted factors are required for the survival of the mammary epithelia and what role these adipokines play in the process of ductal carcinoma tumorigenesis. Here, we take a systematic molecular approach to investigate the multiple ways adipocytes and adipokines can uniquely influence the characteristics and phenotypic behavior of malignant breast ductal epithelial cells. Microarray analysis and luciferase reporter assays indicate that adipokines specifically induce several transcriptional programs involved in promoting tumorigenesis, including increased cell proliferation (IGF2, FOS, JUN, cyclin D1), invasive potential (MMP1, ATF3), survival (A20, NFkappaB), and angiogenesis. One of the key changes in the transformed ductal epithelial cells associated with the cell cycle involves the induction of NFkappaB (five-fold) and cyclin D1 (three-fold). We show that by regulating the transcription of these molecules, the synergistic activity of adipocyte-derived factors can potentiate MCF-7 cell proliferation. Furthermore, compared to other stromal cell-secreted factors, the full complement of adipokines shows an unparalleled ability to promote increased cell motility, migration, and the capacity for angiogenesis. Adipocyte-secreted factors can affect tumorigenesis by increasing the stabilization of pro-oncogenic factors such as beta-catenin and CDK6 as a result of a reduction in the gene expression of their inhibitors (i.e. p18). An in vivo coinjection system using 3T3-L1 adipocytes and SUM159PT cells effectively recapitulates the host-tumor interactions in primary tumors. Type VI collagen, a soluble extracellular matrix protein abundantly expressed in adipocytes, is further upregulated in adipocytes during tumorigenesis. It promotes GSK3beta phosphorylation, beta-catenin stabilization, and increased beta-catenin activity in breast cancer cells and may critically contribute towards tumorigenesis when not counterbalanced by other factors.

Adipocyte-secreted factors synergistically promote mammary tumorigenesis through induction of anti-apoptotic transcriptional programs and proto-oncogene stabilization

In higher organisms homeostatic control of cell number is thought to be the result of the dynamic balance between cell proliferation and cell death. While the process of proliferation has received a great deal of attention over the past 20 years, much less emphasis has been placed on the biochemical events that occur before and during cell death. In higher organisms cell death can be classified into one of three different categories: necrosis, which occurs as a result of massive tissue damage; terminal differentiation of specialized tissues such as the skin, intestine, and red blood cells; and apoptosis, which is a process of active cellular selfdestruction that requires the expression of a number of genes. The latter process was originally recognized and described by Wyllie who coined the term "apoptosis" to describe the sequence of events that lead to cell death in a variety of different systems (Kerr et al., Br. J . Cancer, 26, 239-257, 1972), specifically to distinguish this form of cell death from necrosis. Apoptosis is induced in the terminally differentiated cells of hormone-dependent tissues, such as the prostate and mammary gland in the absence of the appropriate trophic hormones, resulting in the regression of the tissue. In other tissues apoptosis can be induced by positive modulators, such as Mullerian duct inhibiting substance (MIS) in the Mullerian ducts, tumor necrosis factor (TNF) in adipocytes, and a variety of cell lines and glucocorticoids in lymphocytes and thymocytes. It can also be induced in cells that retain their proliferative potential (such as hepatocytes) to maintain homeostasis of cell numbers in organs such as the liver. In both proliferating and nonproliferating systems, apoptosis requires specific gene transcription and protein synthesis in the affected cell prior to death, although the biochemical sequence of events may vary slightly from one tissue to another.

The biochemistry of cell death by apoptosis.

Active cell death (ACD) in hormone-dependent tissues such as the prostate and mammary gland is readily induced by hormone ablation and by treatment with anti-androgens or anti-estrogens, calcium channel agonists and TGF beta. These agents induce a variety of genes within the hormone-dependent epithelial cells including TRPM-2, transglutaminase, poly(ADP-ribose) polymerase, Hsp27 and several other unidentified genes. Not all epithelial cells in the glands are equally sensitive to the induction of ACD. In the prostate, the secretory epithelial cells that are sensitive to hormone ablation are localized in the distal region of the prostatic ducts, and are in direct contact with the neighboring stroma. In contrast, the epithelial cells in the proximal regions of the ducts are more resistant to hormone ablation, probably because the permissive effects of the stroma are attenuated by the presence of the basal epithelial cells, which are intercalated between the epithelium and stroma. The underlying biology of ACD in prostate and mammary glands, and its relevance to hormone resistance, is discussed in this review.

Active cell death in hormone-dependent tissues

Poly (A)+ RNA from the prostates of both intact and castrated rats was translated in a message-dependent reticulocyte lysate, and the translation products were electrophoresed on SDS/polyacrylamide gels. Fluorography of these gels showed the expected disappearance, after castration, of the prostate steroid-binding proteins as well as a number of other androgen-dependent proteins. Two major (Mr 40,000 and 45,000) and several minor proteins appeared in the translation products of the castrated rat prostate RNA. Criss-cross liquid hybridization analysis between prostate poly (A+) RNA from intact and castrated rats also showed the disappearance of the abundant prostate steroid-binding protein sequences after castration and the synthesis of several new low to medium abundance sequences. Northern hybridization experiments demonstrated the presence of at least two, and possibly four androgen-repressed poly (A)+ RNA sequences. The most prominent of these, an RNA of 2,000 nucleotides, appeared within 2 days of castration, reaching a maximum on day 4 at a level approximately 400 times greater than the normal level. The other major sequence (a sequence of 1,000 nucleotides) appears after 4 days, reaching a peak between days 8 and 11. Sequences similar to these new RNAs could play an important role in the long-term resistance of prostatic cancer to hormone therapy in humans.

Martin Tenniswood

Papers

Adipocyte-secreted factors synergistically promote mammary tumorigenesis through induction of anti-apoptotic transcriptional programs and proto-oncogene stabilization

The biochemistry of cell death by apoptosis.

Active cell death in hormone-dependent tissues

Androgen-repressed messages in the rat ventral prostate.

Characterization and cloning of androgen-repressed mRNAs from rat ventral prostate.