Is apoptosis a "programmed cell death"?]

The purpose of this study was to evaluate the results and complications of anterior cruciate ligament surgery in middle-aged patients. Forty-five consecutive patients over 40 years old (average age, 44.6 years) who had arthroscopically assisted anterior cruciate ligament reconstructions with midthird patellar tendon autografts were evaluated. The patients returned for interviews, physical examinations, radiographs, Biodex dynamometer strength testing, and KT-1000 arthrometer testing at an average of 37 months after their surgeries (range, 24 to 96 months). The mean Lysholm and Gillquist score was 91, which corresponds to symptoms only with vigorous activity. The overall scores from the International Knee Documentation Committee form were 29 (64%) normal or nearly normal and 2 (4%) severely abnormal. Side-to-side differences as determined by the KT-1000 arthrometer were < or = 3 mm in 31 of 40 patients (78%), between 3 and 5 mm in 4 patients (10%), and > 5 mm in 5 patients at 30 pounds of anterior displacement. Seventy-six percent of the patients (N = 34) returned to their preoperative activity levels. Three patients required repeat arthroscopic surgery for persistent knee pain and two patients had graft ruptures. This study shows that when middle-aged patients undergo surgery, their results can be successful and satisfying to a degree similar to those of younger patients.     

Mechanisms and control of programmed cell death in invertebrates

Apoptosis is a morphologically distinct form of programmed cell death that plays important roles in development, tissue homeostasis and a wide variety of diseases, including cancer, AIDS, stroke, myopathies and various neurodegenerative disorders (see Thompson (1995) for review). It is now clear that apoptosis occurs by activating an intrinsic cell suicide program which is constitutively expressed in most animal cells, and that key components of this program have been conserved in evolution from worms to insects to man. Genetic studies of programmed cell death in experimentally highly accessible invertebrate model systems have provided important clues about the molecular nature of the death program, and the intracellular mechanisms that control its activation. This review summarizes some of the key findings in this area, but also touches on some of the many unresolved questions and challenges that remain.     

Cell renewal, cell differentiation and programmed cell death (apoptosis) in pilomatrixoma

Pilomatrixoma is a benign tumour of the cutaneous adnexa. Histologically, pilomatrixoma comprises masses of immature basophilic cells, small numbers of polygonal squamoid cells, few transitional cells, and clusters of 'shadow cells'. The mechanism leading to the formation of shadow cells is still unknown. Skin biopsy specimens of pilomatrixoma (n = 15) were studied histologically, immunohistologically, and by applying the in situ end-labelling technique. The basal layer of the basophilic cells induced most of the proliferating cells with high expression of bcl-2 and cytokeratin 19. The overlying basophilic cells showed a negligible mitotic activity, a high significant accumulation of p53 protein, and a heterogeneous, but progressive loss of bcl-2 and cytokeratin 19. They developed either into squamoid cells or into transitional cells. The squamoid cells were characterized as differentiated cells resembling mature keratinocytes of stratified mucosa. The transitional cells could be shown to represent apoptotic cells proceeding to shadow cells. The data suggest that apoptosis is the main mechanism leading to the development of the dead shadow cells and is most probably responsible for the banal biological behaviour of pilomatrixoma. Apart from that, pilomatrixoma represents a suitable biological model to study apoptosis in humans.     

Genetic control of programmed cell death in the Caenorhabditis elegans hermaphrodite germline

Development of the nematode Caenorhabditis elegans is highly reproducible and the fate of every somatic cell has been reported. We describe here a previously uncharacterized cell fate in C. elegans: we show that germ cells, which in hermaphrodites can differentiate into sperm and oocytes, also undergo apoptotic cell death. In adult hermaphrodites, over 300 germ cells die, using the same apoptotic execution machinery (ced-3, ced-4 and ced-9) as the previously described 131 somatic cell deaths. However, this machinery is activated by a distinct pathway, as loss of egl-1 function, which inhibits somatic cell death, does not affect germ cell apoptosis. Germ cell death requires ras/MAPK pathway activation and is used to maintain germline homeostasis. We suggest that apoptosis eliminates excess germ cells that acted as nurse cells to provide cytoplasmic components to maturing oocytes.     

Alternative splicing and programmed cell death

Programmed cell death (PCD) is critical for development and homeostasis of multicellular organisms. Genetic and biochemical studies have revealed that PCD is under complex and delicate regulation. An important level of such regulation may be pre-mRNA splicing as suggested by the observation that a number of PCD regulatory genes are expressed as functionally distinct or even antagonistic isoforms as a result of alternative splicing. Studies on alternative splicing of these genes are reviewed here. Expression and function of a large number of genes involved in PCD are regulated by alternative splicing, including death receptors and intracellular components of the death machinery. Alternative splicing affects not only intracellular distribution but also functional activity of these death regulators, providing a fine-tuning mechanism in modulating a presumably tightly controlled process of cell death.     

Diverse molecular provocation of programmed cell death

The experimental study and manipulation of programmed cell death has been greatly assisted by the identification of genetic and pharmacological tools that can either induce or block cell lethality. This review discusses new insights into the molecular sensing of perturbations induced by such tools, as well as the possible consequences of this detection in determining cell survival.     

Role of Ced-3/ICE-family proteases in staurosporine-induced programmed cell death

In the accompanying paper by Weil et al. (1996) we show that staurosporine (STS), in the presence of cycloheximide (CHX) to inhibit protein synthesis, induces apoptotic cell death in a large variety of nucleated mammalian cell types, suggesting that all nucleated mammalian cells constitutively express all of the proteins required to undergo programmed cell death (PCD). The reliability of that conclusion depends on the evidence that STS-induced, and (STS + CHS)-induced, cell deaths are bona fide examples of PCD. There is rapidly accumulating evidence that some members of the Ced-3/Interleukin-1 beta converting enzyme (ICE) family of cysteine proteases are part of the basic machinery of PCD. Here we show that Z-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), a cell-permeable, irreversible, tripeptide inhibitor of some of these proteases, suppresses STS-induced and (STS + CHX)-induced cell death in a wide variety of mammalian cell types, including anucleate cytoplasts, providing strong evidence that these are all bona fide examples of PCD. We show that the Ced-3/ICE family member CPP32 becomes activated in STS-induced PCD, and that Bcl-2 inhibits this activation. Most important, we show that, in some cells at least, one or more CPP32-family members, but not ICE itself, is required for STS-induced PCD. Finally, we show that zVAD-fmk suppresses PCD in the interdigital webs in developing mouse paws and blocks the removal of web tissue during digit development, suggesting that this inhibition will be a useful tool for investigating the roles of PCD in various developmental processes.     

Induction of programmed death/apoptosis androgen-dependent mouse mammary tumor cell line (Shionogi Carcinoma 115) by androgen withdrawal

Shionogi Carcinoma 115 (SC 115) cells are a cloned cell line derived from androgen-dependent mouse mammary tumor. They can grow in serum-free culture if a physiological level of androgen is present in the medium, but can not proliferate in culture without testosterone. In the present study, the mechanism of cell death in SC 115 cells after androgen withdrawal was examined. Based upon the temporal sequence of DNA fragmentation, morphologic changes and loss of cell viability, androgen withdrawal induces programmed cell death (apoptosis) of SC 115 cells in serum-free culture. Northern blot analysis was used to identify a series of genes whose expression per cell is enhanced during the recruitment of cells from a nonproliferative (i.e. G0) state into G1 (i.e.,cyclins D1 and C), from G1 into the S phase of the cell cycle (i.e., cdk2), and during the programmed cell death pathway (i.e. testosterone repressed prostatic message-2 (TRPM-2), transforming growth factor-beta1 (TGF-beta1) and glucose regulated 78 kilodalton protein (GRP-78). Expression of TRPM-2, TGF-beta1, GRP-78, and calmodulin genes increases, but that of cyclins C and D1, and cdk2 genes decreases during programmed cell death of SC 115 cells. These results demonstrate that androgen-dependent SC 115 cells undergo programmed cell death induced by androgen withdrawal, and that this death does not require proliferation or progression into G1 of the proliferative cell cycle. SC 115 cells should be a good model for investigating programmed death of hormone-dependent cancer.     

Steroid regulated programmed cell death during Drosophila metamorphosis

During insect metamorphosis, pulses of the steroid hormone 20-hydroxyecdysone (ecdysone) direct the destruction of obsolete larval tissues and their replacement by tissues and structures that form the adult fly. We show here that larval midgut and salivary gland histolysis are stage-specific steroid-triggered programmed cell death responses. Dying larval midgut and salivary gland cell nuclei become permeable to the vital dye acridine orange and their DNA undergoes fragmentation, indicative of apoptosis. Furthermore, the histolysis of these tissues can be inhibited by ectopic expression of the baculovirus anti-apoptotic protein p35, implicating a role for caspases in the death response. Coordinate stage-specific induction of the Drosophila death genes reaper (rpr) and head involution defective (hid) immediately precedes the destruction of the larval midgut and salivary gland. In addition, the diap2 anti-cell death gene is repressed in larval salivary glands as rpr and hid are induced, suggesting that the death of this tissue is under both positive and negative regulation. Finally, diap2 is repressed by ecdysone in cultured salivary glands under the same conditions that induce rpr expression and trigger programmed cell death. These studies indicate that ecdysone directs the death of larval tissues via the precise stage- and tissue-specific regulation of key death effector genes.     

A common pathway mediates retinoic acid and PMA-dependent programmed cell death (apoptosis) of neuronal cells

To assess the maternal and neonatal risk associated with high-order cesarean sections, a case-control study was carried out in two university affiliated maternity wards. The outcome of 154 pregnancies of women undergoing cesarean section for the 4th time or more was compared with 148 women sectioned for the 2nd or 3rd time and 132 women of similar age and parity after spontaneous birth. The main outcome measures were maternal operative and postoperative morbidity and neonatal prematurity and its complications, Apgar scores, and the need for intensive care. Women undergoing multiple (> or = 4) cesarean sections had significantly more intra-abdominal adhesions (P < 0.0001) than women sectioned for the 2nd or 3rd time. However, the time interval from incision to delivery and the total duration of operation were similar. The postoperative course was not adversely affected by multiple cesarean sections. A high incidence (16.2%) of preterm cesarean deliveries was noted in the study group. This was due to non-elective repeat cesarean delivery rather than to poor timing of scheduled cesarean sections. The significantly increased (P < 0.05) need for neonatal intensive care was explained by the higher occurrence of prematurity. Low Apgar scores (< or = 7) at 1 and 5 min were significantly (P < 0.01) related to multiple cesarean sections, even after controlling for the effect of gestational age. We conclude that multiple cesarean sections pose little risk for the mother, but may be associated with increased neonatal risk, attributed mainly to preterm non-elective cesarean sections.     

Detection of programmed cell death using fluorescence energy transfer

Fluorescence energy transfer (FRET) can be generated when green fluorescent protein (GFP) and blue fluorescent protein (BFP) are covalently linked together by a short peptide. Cleavage of this linkage by protease completely eliminates FRET effect. Caspase-3 (CPP32) is an important cellular protease activated during programmed cell death. An 18 amino acid peptide containing CPP32 recognition sequence, DEVD, was used to link GFP and BFP together. CPP32 activation can be monitored by FRET assay during the apoptosis process.     

Spontaneous germ cell apoptosis in humans: evidence for ethnic differences in the susceptibility of germ cells to programmed cell death

Since the first clinical studies regarding sealing of arterial puncture sites with collagen with the use of the vascular hemostatic device (VHD) and the hemostatic puncture closing device (HPCD) in the early 1990s were performed, no analysis summarizing the published patients has been reported. Therefore we performed a Medline search of data as far back as 1990 and included abstracts presented at the major scientific meetings in the United States (American Heart Association, American College of Cardiology), Europe (European Society of Cardiology), and Germany (German Society of Cardiology). A total of 6007 patients were found to have been enrolled in studies with VHD (4448 patients) or with HPCD (1559 patients). Parameters analyzed in this review were hemostasis success rates and local complications. To assess the impact of the sealing devices on local complications, studies without control groups were excluded. The hemostasis success rates immediately after deployment seemed to be higher for HPCD, but at 2' to 5' after sheath removal, they were in the same range for VHD and HPCD. In controlled studies minor local complications occurred at a rate of 7.6% in the VHD group and in 6.7% of the HPCD group. Because the control group in the HPCD studies showed a considerably higher rate of minor complications than the VHD group (11.7% vs 5.7%), the reduction in minor complications was statistically significant for HPCD, whereas VHD did not reduce minor local complications. Major local complications were reported in 3.8% of the VHD group but in only 1.8% of the HPCD group. The increase of major local complications was statistically significant with VHD (control, 1.7%) but not with HPCD (control, 1.4%). Our analysis shows that some differences between collagen devices may exist, but neither device has been proven to reduce major local complications.     

Genes, molecules, and mechanisms regulating programmed cell death

Apoptosis is a morphologically distinct form of programmed cellular death that plays a central role during embryogenesis, tissue homeostasis, and to remove not necessary or potentially dangerous cells. Moreover, disregulation of genes mediating or modulating apoptosis contributes to the pathogenesis of a number of human diseases, including cancer, autoimmune diseases, neurodegenerative disorders, viral infections and acquired immunodeficiency syndrome. A number of genes and molecules promoting or protective against cell death is at present-day known and an important information about the external and internal signals involved in stimulation and suppression of apoptosis is also emerging. In the intracellular pathway of the death deregulation of [Ca2+](i) plays a pivotal role. Increased ionized intracellular calcium stimulates both the activation of enzymes (protein kinases, endonucleases, proteases and phospholipases) and plasma membrane K+ channels. This calcium-mediated activation leads to morphostructural changes, such as cell shrinkage, cytoplasmatic blebbing, nuclear chromatin condensation and DNA degradation into oligonucleosomal fragments. At least some genes of the cell death pathway have been conserved throughout animal evolution; ced-3 e ced-9 that regulate the initiation of cellular suicide in the nematode Caenorhabditis elegans are homologous to genes that in mammalian cells are thought to play a similar role (interleukin-1 beta converting enzyme [ICE] family, Bcl-2). It is possible to suppose that these regulators could constitute a target for treatment of disorders related with disregulation of apoptosis.     

Widespread programmed cell death in early developing chick optic tectum

We demonstrate that widespread programmed cell death exists in proliferative regions of chicken optic tectum during early development using a sensitive fluorescent ISEL method (FISEL+) and antibody staining for an antigen in dying cells. Several developmental stages from embryonic day (E) 3 to E18 were examined. FISEL+-positive cells were rare before E7 and between E9 to E12. However, massive labeling was observed in the ventricular zone (VZ) between stages E7.5 and E8. At this time extensive cell migration is underway and many labeled cells were found not only in the VZ (premigratory cells) but also in the intermediate zone and tectal plate (migratory cells). Many labeled cells were also found in upper tectal laminae at late developmental stages (E15 and E18).     

The regulation of reactive oxygen species production during programmed cell death

Reactive oxygen species (ROS) are thought to be involved in many forms of programmed cell death. The role of ROS in cell death caused by oxidative glutamate toxicity was studied in an immortalized mouse hippocampal cell line (HT22). The causal relationship between ROS production and glutathione (GSH) levels, gene expression, caspase activity, and cytosolic Ca2+ concentration was examined. An initial 5-10-fold increase in ROS after glutamate addition is temporally correlated with GSH depletion. This early increase is followed by an explosive burst of ROS production to 200-400-fold above control values. The source of this burst is the mitochondrial electron transport chain, while only 5-10% of the maximum ROS production is caused by GSH depletion. Macromolecular synthesis inhibitors as well as Ac-YVAD-cmk, an interleukin 1beta-converting enzyme protease inhibitor, block the late burst of ROS production and protect HT22 cells from glutamate toxicity when added early in the death program. Inhibition of intracellular Ca2+ cycling and the influx of extracellular Ca2+ also blocks maximum ROS production and protects the cells. The conclusion is that GSH depletion is not sufficient to cause the maximal mitochondrial ROS production, and that there is an early requirement for protease activation, changes in gene expression, and a late requirement for Ca2+ mobilization.     

Nitric oxide induction of neuronal endonuclease activity in programmed cell death

Neuronal survival is intricately linked to the maintenance of intact DNA. In contrast, neuronal degeneration following nitric oxide (NO) exposure is dependent, in part, on the degradation of DNA through programmed cell death (PCD). We therefore investigated in primary rat hippocampal neurons the role of endogenous deoxyribonucleases, enzymes responsible for metabolically derived DNA cleavage, during NO-induced neurodegeneration. Twenty-four hours following exposure to the NO generators sodium nitroprusside (300 microM) and SIN-1 (300 microM), neuronal survival was reduced from approximately 88 to 23%. Treatment with aurintricarboxylic acid (1-100 microM), an endonuclease inhibitor, during NO exposure increased neuronal survival from 23 to 80% and decreased DNA fragmentation from 70 to 30% over a 24-h period. Enhancement of endonuclease activity alone with zinc chelation actively decreased neuronal survival from approximately 80% to approximately 34%. DNA digestion assays identified not only two constitutively active endonucleases, an acidic endonuclease (pH 4.0-7.0) and a calcium/magnesium-dependent endonuclease (pH 7.2-8.0), but also a NO-inducible magnesium-dependent endonuclease (pH 8.0). In the absence of endonuclease activity, DNA degradation did not occur during NO application, suggesting that endonuclease activity was a requisite pathway for NO-induced PCD. In addition, NO independently altered intracellular pH in ranges that were physiologically relevant for the activity of the endonucleases responsible for DNA degradation. Our identification and characterization of specific neuronal endonucleases suggest that the constitutive endonucleases may play a role in the initial stages of NO-induced PCD, but the subsequent "downstream" degradation of DNA may ultimately be dependent upon the NO-inducible endonuclease.