Identification of Age-associated Proteins and Functional Alterations in Human Retinal Pigment Epithelium

Retinal pigment epithelium(RPE) has essential functions, such as nourishing and supporting the neural retina, and is of vital importance in the pathogenesis of age-related retinal degeneration. However, the exact molecular changes of RPE during aging remain poorly understood.Here, we isolated human primary RPE(h RPE) cells from 18 eye donors distributed over a wide age range(10–67 years old). A quantitative proteomic analysis was performed to analyze changes in their intracellular and secreted p...     

Apoptosis and aging in mitochondrial morphology mutants of<Emphasis Type="Italic">S. cerevisiae</Emphasis>

Cell viability during chronological aging and after apoptotic stimuli in some yeast mutants with altered mitochondrial morphology was followed; a function for the corresponding genes in the apoptotic process was assessed. MDM30 and DNM1, the genes encoding an F-box protein and the dynamin-related GTPase, respectively, are involved in triggering aging and apoptosis. In contrast, YME1, encoding a subunit of the mitochondrial inner membrane i-AAA proteinase complex, has a protective role in these processes. FIS1, the mitochondrial fission gene, might play a protective role after an apoptotic insult while it seems to promote cell death in aging cells.     

Involvement of DNA mismatch repair in folate deficiency-induced apoptosis small star,filled

Folate is a critical factor for DNA metabolism and its deficiency is associated with a number of human diseases and cancers. Although it has been shown that folate deficiency induces genomic instability and apoptotic cell death, the underlying mechanism is largely unknown. Given the role of mismatch repair in maintaining genomic integrity, mismatch repair was tested for its involvement in folate deficiency-induced genomic instability and cell death. Cells proficient in mismatch repair were highly sensitive to folate deficiency compared with cells defective in either hMutSalpha or hMutLalpha. Since wild-type cells but not mutant cells underwent apoptosis upon extensive folate depletion, the apoptotic response is dependent on a functional mismatch repair system. Our data also indicate that p53 is required for the folate depletion-induced apoptosis. In vitro biochemical studies demonstrated that hMutSalpha specifically recognized DNA damage induced by folate deficiency, suggesting a direct participation of mismatch repair proteins in mediating the apoptotic response. We conclude that while the mismatch repair-dependent apoptosis is necessary to protect damaged cells from tumorigenesis, it may damage a whole tissue or organ, as seen in patients with megaloblastic anemia, during extensive folate deficiency.     

Cancer and aging: symposium of the 27th annual meeting of the Japanese society for biomedical gerontology, Tokyo

Although many hypotheses have been proposed to explain the strong link between aging and cancer, the exact mechanisms responsible for the increased frequency of occurrence of cancer with advancing age have not been fully defined. Recent evidence indicates that malregulation of the apoptotic process may be involved in some aging process as well as in the development of cancer. Although it is still under debate how apoptosis is expressed during aging in vivo, this phenomenon is an important factor in unwinding the complicated mechanisms that link cancer and aging. In this review, we report on the discussion at the symposium of the 27th annual meeting of the Japanese society for biomedical gerontology, regarding recent findings from aging and carcinogenesis studies using animal models, the characteristics of cancer in patients with Werners syndrome, the epigenetic changes in human cancers and aging, and the characteristics of human cancers in the elderly. It was concluded that apoptosis plays a role in the aging process and carcinogenesis in vivo, likely as an inherent protective mechanism against various kinds of damages to genes/chromosomes.     

Apoptosis in ventricular myocytes: the role of tumor suppressor proteins

Apoptosis or programmed cell death is an important physiologic event crucial for the selective removal of damaged or unwanted cells from body tissues. In the cardiovascular system, apoptosis has been observed in the vasculature and myocardium. Untimely or inappropriate myocardial cell loss through an apoptotic process may contribute to ventricular remodeling and the ultimate demise of ventricular function following injury. Therapeutic interventions designed to modulate or prevent myocardial apoptotic cell loss may therefore prove beneficial in maintaining cardiac function. Incite into the molecular mechanisms that govern apoptosis in mammalian cells has led to the identification of several key factors that promote or prevent the apoptotic process. In this report, we discuss putative regulators of cardiac cell apoptosis with specific reference to the tumor suppressor proteins, p53 and Rb. The interplay between these factors, as well as the anti-apoptotic molecules related to the Bcl-2 the family are discussed in the context of the heart under normal and disease conditions.     

Early detection of staurosporine-induced apoptosis by comet and annexin V assays

Comet, TUNEL, and annexin V assays were used to identify DNA fragmentation and plasma membrane alterations occurring during staurosporine-induced apoptosis in Chinese hamster ovary cells. TUNEL assay detected apoptotic cells after 6 h treatment. The occurrence of annexin V immunofluorescence staining after 1 h treatment confirms that exposure of phosphatidylserine (PS) residues is an early biochemical feature of apoptosis. According to intensity, three annexin staining patterns were distinguished, related to different steps in the apoptotic process. The detection of highly damaged cells by the comet assay after 3 h treatment occurred earlier than the detection of DNA modifications by the TUNEL assay, but later than the exposure of PS residues. However, late apoptotic cells, otherwise characterized by plasma membrane disruption and high annexin V staining, were not detected by the comet assay. In this case, comet assay modified by omitting electrophoresis (halo assay) was more sensitive for an accurate quantification of the apoptotic fraction. Accepted: 2 June 1999     

Comet assay and DNA flow cytometry analysis of staurosporine-induced apoptosis.

BACKGROUND: The ability of the comet assay to quantify DNA strand breaks and alkali labile sites has been widely demonstrated. In this study, this assay was tested for its ability to identify DNA fragmentation occurring during apoptosis in comparison with standard DNA flow cytometry analysis. METHODS: Staurosporine-induced apoptosis in CHO cells is an adequate model to study a rapid time- and dose-dependent appearance of this process. RESULTS: Nuclear staining with DAPI confirmed the induction of apoptosis with a typical chromatin condensation and fragmentation. Analysis of propidium-iodide- (PI) stained DNA by flow cytometry showed the presence of a pre-G1 peak, characteristic of apoptotic cells, 6 h after drug treatment. The detection of highly damaged cells (HDC) by the comet assay after 3 h treatment occurred earlier than the detection of apoptotic cells by flow cytometry. However, HDC were missed when the DNA fragmentation was too high, preventing accurate quantification of late apoptotic cells. CONCLUSIONS: The comet assay is more sensitive than standard DNA flow cytometry to detect early DNA fragmentation events occurring during apoptosis. However, the comet assay modified by omitting electrophoresis was necessary to quantify apoptotic fraction at later stages.     

Functional mitochondria are required for alpha-synuclein toxicity in aging yeast

alpha-Synuclein is one of the principal toxic triggers of Parkinson disease, an age-associated neurodegeneration. Using old yeast as a model of alpha-synuclein expression in post-mitotic cells, we show that alpha-synuclein toxicity depends on chronological aging and results in apoptosis as well as necrosis. Neither disruption of key components of the unfolded protein response nor deletion of proapoptotic key players (including the yeast caspase YCA1, the apoptosis-inducing factor AIF1, or the serine protease OMI) did prevent alpha-synuclein-induced cell killing. However, abrogation of mitochondrial DNA (rho(0)) inhibited alpha-synuclein-induced reactive oxygen species formation and subsequent apoptotic cell death. Thus, introducing an aging yeast model of alpha-synuclein toxicity, we demonstrate a strict requirement of functional mitochondria.     

Appetizing rancidity of apoptotic cells for macrophages: oxidation,externalization, and recognition of phosphatidylserine

Programmed cell death (apoptosis) functions as a mechanism to eliminate unwanted or irreparably damaged cells ultimately leading to their orderly phagocytosis in the absence of calamitous inflammatory responses. Recent studies have demonstrated that the generation of free radical intermediates and subsequent oxidative stress are implicated as part of the apoptotic execution process. Oxidative stress may simply be an unavoidable yet trivial byproduct of the apoptotic machinery; alternatively, intermediates or products of oxidative stress may act as essential signals for the execution of the apoptotic program. This review is focused on the specific role of oxidative stress in apoptotic signaling, which is realized via phosphatidylserine-dependent pathways leading to recognition of apoptotic cells and their effective clearance. In particular, the mechanisms involved in selective phosphatidylserine oxidation in the plasma membrane during apoptosis and its association with disturbances of phospholipid asymmetry leading to phosphatidylserine externalization and recognition by macrophage receptors are at the center of our discussion. The putative importance of this oxidative phosphatidylserine signaling in lung physiology and disease are also discussed.     

In vivo time course of morphological changes and DNA degradation during the degeneration of castration-induced apoptotic prostate cells

The in vivo time course of the morphological changes and DNA degradation in castration-induced apoptotic prostate cells was studied from the earliest to the latest stage of the degeneration process. To study this problem, we first induced apoptotic prostate cells in rats by castration for 3 days and then promptly and continuously blocked the death of healthy prostatic cells in the castrated rats by in vivo testosterone replacement. Because testosterone replacement could not stop the irreversible lysis of already damaged prostate cells, apoptotic cells at different stages of the degeneration process were eliminated sequentially from the prostate after the healthy prostate cells had been protected. Prostate cells at the earliest stage of apoptosis at the time when the castrated rats received testosterone replacement disappeared last. By tracing the morphological and DNA degradation of apoptotic cells after hormone treatment, we estimated the time course of prostate cell death from the early to the final stage. In the morphological evolution of apoptotic prostate cells, the clumping of nuclear chromatin, the degeneration of cytoplasm and the involution of the cell surface occurred and progressed simultaneously, resulting in the rapid formation of apoptotic bodies that were gradually digested by other cells. The DNA ladders of apoptotic cells were progressively cleaved into a mononucleosomal subunit that was further degraded at an additional site, generating a heterogeneous population of small nucleotides. The final digestion of DNA fragments occurred within the apoptotic bodies. The whole course of prostate cell death after castration took about 44 h.     

Activation of Wnt/β-catenin protein signaling induces mitochondria-mediated apoptosis in hematopoietic progenitor cells

The canonical Wnt/β-catenin signaling is activated during development, tumorigenesis, and in adult homeostasis, yet its role in maintenance of hematopoietic stem/progenitor cells is not firmly established. Here, we demonstrate that conditional expression of an active form of β-catenin in vivo induces a marked increase in the frequency of apoptosis in hematopoietic stem/progenitor cells (HSCs/HPCs). Activation of Wnt/β-catenin signaling in HPCs in vitro elevates the activity of caspases 3 and 9 and leads to a loss of mitochondrial membrane potential (ΔΨ(m)), indicating that it induces the intrinsic mitochondrial apoptotic pathway. In vivo, expression of activated β-catenin in HPCs is associated with down-regulation of Bcl2 and expression of Casp3. Bone marrow transplantation assays reveal that enhanced cell survival by a Bcl2 transgene re-establishes the reconstitution capacity of HSCs/HPCs that express activated β-catenin. In addition, a Bcl2 transgene prevents exhaustion of these HSCs/HPCs in vivo. Our data suggest that activation of the Wnt/β-catenin pathway contributes to the defective function of HPCs in part by deregulating their survival.     

Senescence and apoptosis in yeast mother cell-specific aging and in higher cells: a short review

It is our intention to give the reader a short overview of the relationship between apoptosis and senescence in yeast mother cell-specific aging. We are studying yeast as an aging model because we want to learn something of the basic biology of senescence and apoptosis even from a unicellular eukaryotic model system, using its unrivalled ease of genetic analysis. Consequently, we will discuss also some aspects of apoptosis in metazoa and the relevance of yeast apoptosis and aging research for cellular (Hayflick type) and organismic aging of multicellular higher organisms. In particular, we will discuss the occurrence and relevance of apoptotic phenotypes for the aging process. We want to ask the question whether apoptosis (or parts of the apoptotic process) are a possible cause of aging or vice versa and want to investigate the role of the cellular stress response system in both of these processes. Studying the current literature, it appears that little is known for sure in this field and our review will therefore be, for a large part, more like a memorandum or a program for future research.     

“Licensed to kill”: Tyrosine dephosphorylation and Bak activation

The genomes of multicellular organisms are under constant assault from a host of environmental agents. The efficient elimination of cells harboring damage is essential to avoid the accumulation of deleterious changes that may promote tumorigenesis. Consequently, a complex and elaborate series of damage responses have evolved to either ensure that correct repair of the DNA has been carried out, or alternatively, to initiate programmes that result in the ablation of the damaged cell. Apoptosis is recognized as both a fast an efficient way of disposing of damaged or unwanted cells before they accumulate changes that may result in the acquisition of neoplastic autonomy. The mitochondrial apoptotic pathway relies upon two effector proteins of the Bcl2 family, Bax and Bak, that when activated form pores in the outer mitochondrial membrane that release cytochrome c and other apoptogenic factors. We have recently shown that the initiation of Bak activation is controlled by dephosphorylation. In particular, we found that a specific tyrosine dephosphorylation was required for Bak activation to proceed and that tyrosine phosphatases may serve to integrate apoptotic signals that culminate in Bak dephosphorylation. Here, we discuss these findings and present additional data underlining the importance of dephosphorylation in the Bak activation process and how the modulation of Bak phosphorylation status may be modified to enhance cell killing.Key words: apoptosis, mitochondria, phosphatase, BH3, p53, Bak, signaling, phosphorylation     

Vitamin E analogues as inducers of apoptosis: implications for their potential antineoplastic role

Recent evidence suggests that vitamin E and its analogues, which have been used for many years as antioxidants, may not only protect cells from free radical damage but also induce apoptotic cell death in various cell types. While alpha-tocopherol (alpha-TOH) is mainly known as an anti-apoptotic agent, its redox-silent analogues either have no influence on cell survival (alpha-tocopheryl acetate, alpha-TOA), or induce apoptosis (alpha-tocopheryl succinate, alpha-TOS). Although precise mechanisms of apoptosis induction by alpha-TOS remain to be elucidated, there is evidence that this process involves both the antiproliferative and membrane destabilising activities of the agent. Alpha-TOS has been shown to induce apoptosis in malignant cell lines but not, in general, in normal cells, and to inhibit tumorigenesis in vivo. These features suggest that this semi-synthetic analogue of vitamin E could be a promising antineoplastic agent.     

Cell cycle and apoptosis: Common pathways to life and death

Programmed cell death, or apoptosis, is a highly regulated process used to eliminate unwanted or damaged cells from multicellular organisms. The morphology of cells undergoing apoptosis is similar to cells undergoing both normal mitosis and an aberrant form of mitosis called mitotic catastrophe. During each of these processes, cells release substrate attachments, lose cell volume, condense their chromatin, and disassemble the nuclear lamina. The morphological similarities among cells undergoing these processes suggest that the underlying biochemical changes also may be related. The susceptibility of cells to apoptosis frequently depends on the differentiation state of the cell. Additionally, cell cycle checkpoints appear to link the cell cycle to apoptosis. Deregulation of the cell cycle components has been shown to induce mitotic catastrophe and also may be involved in triggering apoptosis. Some apoptotic cells express abnormal levels of cell cycle proteins and often contain active Cdc2, the primary kinase active during mitosis. Although cell cycle components may not be involved in all forms of apoptosis, in many instances cell proliferation and cell death may share common pathways.     

Atypical antiinflammatory activation of microglia induced by apoptotic neurons

In the central nervous system (CNS), apoptosis plays an important role during development and is a primary pathogenic mechanism in several adult neurodegenerative diseases. A main feature of apoptotic cell death is the efficient and fast removal of dying cells by macrophages and nonprofessional phagocytes, without eliciting inflammation in the surrounding tissue. Apoptotic cells undergo several membrane changes, including the externalization of so-called "eat me" signals whose cognate receptors are present on professional phagocytes. Among these signals, the aminophospholipid phosphatidylserine (PS) appears to have a crucial and unique role in preventing the classical pro-inflammatory activation of macrophages, thus ensuring the silent and safe removal of apoptotic cells. Although extensively studied in the peripheral organs, the process of recognition and removal of apoptotic cells in the brain has only recently begun to be unraveled. Here, we summarize the evidence suggesting that upon interaction with PS-expressing apoptotic neurons, microglia may no longer promote the inflammatory cascade, but rather facilitate the elimination of damaged neurons through antiinflammatory and neuroprotective functions. We propose that the anti-inflammatory microglial phenotype induced through the activation of the specific PS receptor (PtdSerR), expressed by resting and activated microglial cells, could be relevant to the final outcome of neurodegenerative diseases, in which apoptosis seems to play a crucial role.     

Actin depolymerization and polymerization are required during apoptosis in endothelial cells

In order to understand the role of actin microfilaments in the apoptotic process, we followed their evolution during tumor necrosis factor-alpha (TNF)-induced apoptosis in bovine aortic endothelial (BAE) cells. Using Western blotting analysis and immunofluorescence microscopy, we observed that the actin microfilaments network was disrupted in apoptotic cells. Depolymerization of F-actin was concomitant with internucleosomal DNA degradation and with the morphological changes associated with apoptotic cell death. However, using the actin microfilament disrupting agent, cytochalasin, we present evidence that the formation of blebs leading to apoptotic cell fragmentation requires neopolymerization of actin. Indeed, in the presence of cyochalasin, induction of apoptosis (internucleosomal DNA degradation) in BAE cells by TNF and cycloheximide was not associated with these classical morphological markers of apoptosis. Moreover, when added to BAE cells showing incipient apoptotic fragmentation, cytochalasin E reversed this process. We also observed an accumulation of actin at the basis of the apoptotic bodies in formation in these cells. Together, these results suggest that the actin network of flattened cells is disrupted concomitantly to the morphological modifications associated to the apoptotic cell death, and that the cytochalasin-sensitive reorganisation of actin is required to the formation of apoptotic blebs.