Genetically programmed cell death (apoptosis)

Extensively and successfully studied problems of programmed cell death are considered. Recent evidence on apoptosis genes is presented, including the bcl-2 family and other genes with similar functions. A scheme of pathways of the main apoptosis mechanism is constructed. Examples of associations of apoptosis and diseases are presented in a special section.     

Genetic control of programmed cell death (apoptosis) in Drosophila

Programmed cell death, or apoptosis, is a highly conserved cellular process that has been intensively investigated in nematodes, flies, and mammals. The genetic conservation, the low redundancy, the feasibility for high-throughput genetic screens and the identification of temporally and spatially regulated apoptotic responses make Drosophila melanogaster a great model for the study of apoptosis. Here, we review the key players of the cell death pathway in Drosophila and discuss their roles in apoptotic and non-apoptotic processes.     

Induction of programmed cell death (apoptosis) in mature lymphocytes

The apoptosis of human peripheral blood lymphocytes was analyzed by the breakdown of DNA into oligonucleosome-sized fragments. The mature lymphocytes were rendered sensitive to apoptosis by either the omission of fetal bovine serum in the culture medium or the addition of polymyxin B. In the first case it was counteracted by phorbol myristate acetate. The possible involvement of protein kinase C in cell survival is pointed out.     

Radiation-induced interphase death of rat thymocytes is internally programmed (apoptosis)

Thymocytes are highly radiosensitive and show 'interphase death' within a few hours after low doses of irradiation. However, the mechanisms responsible for this type of death remain ill-defined. Separation of the dead thymocyte fraction from irradiated thymocyte suspensions by centrifugation on Percoll gradients provided homogeneous populations of dead cells suitable for detailed study. Using this method, radiation-induced interphase death of thymocytes was found to involve a sharp but transient increase in buoyant density, concomitant with the appearance of distinctive morphologic changes which included disappearance of microvilli and blistering of the cell surface. The chromatin in the dead cells had a molecular weight sufficiently low to resist sedimentation, and consisted of short oligonucleosome chains. We were unable to detect populations of cells intermediate between the dead and normal in the above characteristics. Interphase death thus involves a discrete, abrupt transition from the normal state and is not merely the consequence of progressive and degenerative changes. Furthermore, immediate cessation of development of interphase death by cycloheximide suggested a possible involvement of protein synthesis on this transition step.     

Decreased protein kinase C activity is associated with programmed cell death (apoptosis) in freshly isolated rat hepatocytes

Apoptosis of freshly isolated rat hepatocytes was induced by either the omission of fetal bovine serum in the culture medium or addition of the protein kinase C inhibitors polymyxin B or staurosporin. The time-course of DNA breakdown into oligonucleosome-sized fragments and the activity of protein kinase C was determined. Hepatocytes were found to be sensitive to bleomycin which induced a high degree of DNA breakdown even within 30 min incubation. Both staurosporin and polymyxin B induced DNA degradation in hepatocytes after three hours incubation, an effect that was partially prevented by phorbol myristate acetate (PMA). After eight hours incubation, PMA failed to counteract this action and itself produced the apoptosis of rat hepatocytes. The results suggest the involvement of protein kinase C in hepatocyte survival.     

The expression of "tissue" transglutaminase in two human cancer cell lines is related with the programmed cell death (apoptosis).

The expression of "tissue" transglutaminase (tTG) in two human tumor cell lines (the cervix adenocarcinoma line HeLa-TV and the neuroblastoma cells SK-N-BE-2) was found to be in correlation with the rate of physiological cell death (apoptosis) in culture. We investigated the effect of retinoic acid (RA) and alpha-difluoromethylornithine (DFMO) in order to elucidate the relationship between tTG expression and apoptosis. RA led to a 6-fold increase of tTG activity in HeLa-TV cells and to a 12-fold increase in SK-N-BE(2) cells, which was paralleled in both cell lines by a proportional increase in the number of apoptotic bodies recovered from the cultures. On the contrary, DFMO determined a dramatic reduction of tTG expression and of the apoptotic index. Immunohistochemical analysis using an anti-tTG antibody showed that the enzyme was accumulated in both cell lines within typical apoptotic bodies. Immunocytochemistry and cell cloning of SK-N-BE(2) line demonstrated that tTG was absent in cells showing neurite outgrowth, indicating that the enzyme expression is not associated with neural differentiation, even though both phenomena are elicited by retinoic acid. On the whole, these data indicate that also in tumors tTG activation takes place in cells undergoing apoptosis. The enzyme is activated in apoptotic cells to form cross-linked protein envelopes which are insoluble in detergents and chaotropic agents. The number of insoluble protein envelopes as well as the N,N-bis(gamma-glutamyl)polyamine cross-links is related with both tTG expression and apoptotic index, strongly suggesting the participation of the enzyme in the apoptotic program.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantification of apoptosis (programmed cell death) in mammalian testis by DNA-fragmentation ELISA

Apoptosis (programmed cell death) could contribute to fluctuations in sperm production and involution of testis in dependence on seasonal, genetic, environmental or individual factors. Investigations of such factors require a reliable quantitative examination of apoptotic processes. Therefore, a standardized procedure was developed for quantification of apoptosis in samples of testicular parenchyma in bull. This test is based on a highly sensitive DNA-fragmentation ELISA which was used originally for somatic cells in culture. Aliquots of testicular parenchyma were minced and homogenized by freezing/thawing and subsequent sonification at 4 degrees C. In comparison, aliquots were lysed in original buffer from the cell death detection ELISA-kit. Nucleosomes from the cell cytoplasm were obtained in supernatant of homogenate or lysate after centrifugation. The absorbance was linear over the range of sample concentrations from 5 to 20 microg testis equivalent/100 microl solution. Therefore, samples were standardized to a final concentration of 10 microg testis equivalent/100 microl. The recorded values were expressed in units per mg tissue (U/mg). The method was used to study testicular apoptotic processes in the guinea pig and roe deer. The results showed that apoptosis can be detected in testicular homogenate prepared from 0.01 mg testis parenchyma within 24 h after recovery of testes without significant variations. Detectable apoptosis levels showed differences among sexually active guinea pig (7.08 +/- 1.95 U/mg), roe deer (16.32 +/- 3.45 U/mg), and cattle (29.0 +/- 7.1 U/mg). This species-specificity suggests different cross reactivity of the monoclonal antibody used in the ELISA. A significantly higher amount of testicular apoptosis was detected in a population of guinea pigs with increased inbreeding coefficients (f = 0.785 - 0.998) than in outbred animals (11.41 +/- 3.50 U/mg and 7.08 +/- 1.95 U/mg, respectively). The inverse relationship of testicular apoptosis and proliferation in these two populations was significant (r = -0.531; P < 0.05). In conclusion, the relative simplicity and high sensitivity of this nonradioactive method provides a useful approach to investigate spermatogenesis under different conditions. Results in the guinea pig showed that apoptosis plays an important role in the regulation of gonadal efficiency.     

IL-4 enhances programmed cell death (apoptosis) in stimulated human monocytes.

Because IL-4 down-regulates several proinflammatory functions associated with human monocytes/macrophages, we explored the possibility that IL-4 also decreases monocyte survival. IL-4 caused a concentration-dependent decrease in viability of IL-1 or LPS stimulated, but not unstimulated, monocytes. Nonviable cells demonstrated classic features of programmed cell death or apoptosis, in that they were condensed and contained oligonucleosome-sized (200 bp) DNA fragments. When compared with several other cytokines commonly associated with inflammatory lesions, IL-4 was uniquely effective in enhancing cell death. We found that IL-4 enhanced death more quickly in IL-1-stimulated cells than in LPS-stimulated cells, that stimulated monocytes did not become resistant to the effects of IL-4 during culture, and that the effects of IL-4 on viability were antagonized by IFN-gamma. Enhanced cell death was stimulus-specific in that monocyte viability maintained by certain activating agents, such as Con A or CSF, was unaffected by IL-4. These findings represent the first evidence of cytokine-enhanced programmed cell death in monocytes and suggest that the antiinflammatory effects of IL-4 are mediated in part by reducing survival of stimulated monocytes in chronic lesions.     

Heat-induced programmed cell death in Leishmania infantum is reverted by Bcl-XL expression

An increasing number of reports indicate that single-celled organisms are able to die following what seems to be an ordered program of cell death with strong similarities to apoptosis from higher eukaryotes. DNA degradation and several other apoptotic-like processes have also been described in the parasitic protozoa Leishmania. However, the existence of an apoptotic death in this parasite is still a matter of controversy. Our results indicate that most of the processes of macromolecular degradation and organelle dysfunction observed in mammalian cells during apoptosis can also be reproduced in promastigotes of the genus Leishmania when incubated at temperatures above 38°C. These processes can be partially reversed by the expression of the anti-apoptotic mammalian gene Bcl-X_L, which suggests that this family of apoptosis-regulating proteins was present very early in the evolution of eukaryotic cells.     

动物细胞培养过程中的细胞自然凋亡

细胞培养过程中的细胞自然凋亡是细胞受环境压力的影响而发生的现象。随着细胞自然凋亡的分子生物学和生物化学研究的深入,对以动物细胞产品生产为目的的细胞培养产业将产生极有价值的影响。采用DNA重组技术把预防细胞自然凋亡的基因导入细胞和在培基中加入具有抗细胞自然凋亡的化合物等手段已用于预防或减缓细胞培养过程中的细胞自然凋亡。这些技术将大大延长细胞达到饱和密度后的培养时间,从而使细胞培养系统的生产效率得以显著提高。     

Cell cycle kinetics, tissue transglutaminase and programmed cell death (apoptosis).

Studies were undertaken on a highly metastatic hamster fibrosarcoma cell line with a view to assessing whether cells entering into apoptosis, measured by counting the number of transglutaminase mediated detergent insoluble envelopes, has any synchrony with a particular phase of the cell cycle. A double exposure of thymidine was used to block cells in early S-phase. Flow cytometry in combination with [3H]thymidine incorporation into DNA was used to assess the degree of synchrony and progression through the different phases of cell cycle. The apoptotic index was found to be at its maximum in mid-S-phase. Measurement of transglutaminase activity in each phase of the cell cycle indicated that the specific activity was also at its greatest during mid S-phase. The level of enzyme was relatively unchanged throughout the cell cycle indicating that the regulation of transglutaminase activity occurs primarily through effects on catalytic activity rather than enzyme synthesis.     

Analysis and discrimination of necrosis and apoptosis (programmed cell death) by multiparameter flow cytometry.

Necrosis and apoptosis are two distinct modes of cell death which differ in morphology, mechanism and incidence. Membrane disruptants, respiratory poisons and hypoxia cause ATP depletion, metabolic collapse, cell swelling and rupture leading to inflammation. These are typical features of necrosis. Apoptosis plays a crucial role in embryogenesis and development and is also prevalent in tumours. It is characterised by cell shrinkage, chromatin condensation and systematic DNA cleavage. Apoptotic cells are rapidly engulfed by phagocytes, thus preventing inflammatory reaction to degradative cell contents. In vivo, apoptosis is almost impossible to quantify due to problems of heterogeneity and the short half-life of an apoptotic cell. In vitro, mechanistic studies are further complicated by a late phase of apoptosis where the cell membrane becomes permeable to vital dyes and which occurs in the absence of phagocytes. Here we describe a novel and rapid multiparameter flow cytometric assay which discriminates and quantifies viable, apoptotic and necrotic cells via measurement of forward and side light scatter (proportional to cell diameter and internal granularity, respectively) and the DNA-binding fluorophores Hoechst 33342 and propidium. It is anticipated that mechanistic studies of apoptosis in a variety of cell types will greatly benefit from this mode of analysis.     

Clinical and pathological significance of programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) expression in high grade serous ovarian cancer

We investigated programmed cell death 1 (PD-1) / programmed cell death ligand 1 (PD-L1) expression in high grade serous ovarian cancer (HGSOC) and its relationship to tumor infiltrating lymphocytes (TIL) and prognosis. Formalin fixed paraffin embedded (FFPE) samples of 94 HGSOC cases were included in the study. Immunohistochemical analysis (CD3, CD4, CD8, PD-1 and PD-L1) was performed. Samples were analyzed for expression of immune proteins in the peritumoral stromal and intratumoral areas, scored, and expression was correlated with overall survival, stage, and age. PD-L1 staining ratio with a score greater than 0 was found to have lower survival. There were two positive staining patterns, patchy/diffuse and patchy/focal patterns, in 24 (25.5%) cases. Considering the threshold value ≥5%, we demonstrated that the PD-L1 positive cancer cell membrane immunoreactivity rate and patchy/diffuse PD-L1 expression were 9.6% (n = 9). There was statistically significant relationship between high PD-1 scores and PD-L1 cases of ≥ 5%. A statistically significant difference was found between PD-L1 staining and survival in patients with a threshold ≥ 5%. However an appropriate rate for treatment was determined in 9.6% cases. There was a statistically significant correlation between PD-1 positive TIL score and intratumoral CD3, peritumoral stromal CD3, intratumoral CD4 and intratumoral CD8 positive cells. Survival was lower in cases with higher PD-L1 positive stromal TIL score.     

Differential regulation of human monocyte programmed cell death (apoptosis) by chemotactic factors and pro-inflammatory cytokines

In the absence of appropriate stimuli, monocytes undergo programmed cell death (PCD) or apoptosis. IL-1 beta and TNF-alpha prevent monocyte PCD, which suggests that viability may be regulated by biologically active peptides released during inflammation. To explore this possibility, we evaluated several chemotactic factors and pro-inflammatory cytokines for their ability to regulate PCD. The recruitment factors, FMLP, C fragment C5a, monocyte chemotactic protein-1, or transforming growth factor-beta 1, were incapable of rescuing monocytes from PCD nor did they enhance PCD, whereas several inflammatory cytokines in addition to IL-1 beta and TNF-alpha, including granulocyte-monocyte-CSF and IFN-gamma, prevented monocyte PCD provided that sufficient levels of these cytokines were continuously maintained in the cultures. Cytokine-mediated inhibition of PCD could be blocked by specific antisera, ruling out potential effects caused by LPS contamination. When tested at equivalent concentrations, IL-2, IL-4, and IL-6 had no effect on PCD indicating selectivity in cytokine modulation of monocyte PCD. Because monocytes produce IL-1 beta, TNF-alpha, and granulocyte-monocyte CSF when activated, the data suggest autocrine as well as paracrine control of cell survival and accumulation. The results also suggest that monocytes recruited to a site of inflammation will undergo PCD in the absence of specific cytokines and/or other stimuli that block this process.     

Constitutive expression of the machinery for programmed cell death

In the presence of cycloheximide (CHX) to inhibit protein synthesis, a high concentration of staurosporine (STS) induces almost all cells in explant cultures of 8/8 types of newborn mouse organs and 3/3 types of adult mouse organs to die with the characteristic features of apoptosis. Eggs and blastomeres also die in this way when treated with STS and CHX, although they are less sensitive to this treatment than trophectoderm or inner cell mass cells whose sensitivity resembles that of other developing cells. Human red blood cells are exceptional in being completely resistant to treatment with STS and CHX. As (STS plus CHX)-induced cell deaths have been shown to display the characteristic features of programmed cell death (PCD), we conclude that all mammalian nucleated cells are capable of undergoing PCD and constitutively express all the proteins required to do so. It seems that the machinery for PCD is in place and ready to run, even though its activation often depends on new RNA and protein synthesis.