Apoptosis of nurse cells at the late stages of oogenesis of Drosophila melanogaster

 In Drosophila a remarkable feature of oogenesis is the regression of the nurse cells after dumping their cytoplasmic contents into the oocyte. We have studied the nature of this process at the late stages of egg chamber development. In egg chambers DAPI staining shows highly condensed chromatin from stage 12 and TUNEL labelling shows DNA fragmentation up to stage 14. Gel electrophoresis of the end-labelled DNA, extracted from isolated egg chambers at the same stages of development, shows a ladder typical of apoptotic nuclei. This provides evidence that, during Drosophila oogenesis, the nurse cells undergo apoptosis. Apoptotic nuclei have also been detected in dumping-defective egg chambers, indicating that the cytoplasmic depletion of nurse cells is concurrent with but apparently not the cause of the process. Received: 12 December 1997 / Accepted: 6 January 1998     

Analysis of atrial natriuretic factor biosynthesis and secretion in adult and neonatal rat atrial cardiocytes

Atrial natriuretic factor (ANF) is stored in atrial cardiocytes as the 126 amino acid polypeptide, proANF, which is later cleaved to the 24-28 amino acid carboxyterminal peptides, the major circulating forms. Earlier studies have demonstrated that isolated, cultured neonatal rat cardiocytes both store and secrete proANF, which can be cleaved to the smaller circulating form(s) by a serum protease. Since differences may exist between neonatal and adult cardiocytes with respect to ANF synthesis and processing, we compared the forms of ANF stored and secreted by neonatal rat cardiocytes with those of adult cells. Using four to five day cultures of isolated atrial cardiocytes prepared from the hearts of neonatal and adult rats, pulse-chase studies were performed with 35S-cysteine and 35S-methionine. Analysis of ANF stored and secreted by these cells was performed by immunoprecipitation of cell extracts and culture media using antibodies directed to either the carboxyterminus or aminoterminus of proANF followed by SDS-PAGE and autoradiography. Cell extracts from both adult and neonatal cultures were found to contain only a 17-kDa polypeptide, previously identified as proANF. The predominant form found in the culture media was also the 17-kDa peptide, with smaller quantities of its 3-kDa carboxyterminal and 14-kDa aminoterminal cleavage products. We conclude from these studies that proANF is the major form stored and secreted by both adult and neonatal cardiocytes in culture; the activity of the protease that cleaves proANF to the smaller forms found in the circulation is either attenuated or is overwhelmed by high ANF-secretory rates in these cultures. Alternatively, the ANF processing and secretory pathways may be somehow altered in culture such that proANF escapes protease cleavage. Further studies will elucidate the nature and location of this protease.     

紫外辐射诱发NIH3T3细胞凋亡时DNA断裂的特性

用常规琼脂糖凝胶电泳UVB照射后培养不同时间的NIH3T3细胞DNA,两个样本均未出现凋亡梯形带,而用相同条件处理的昆明小鼠胸腺细胞DNA出现了典型的梯形带.再用反转电场琼脂糖凝胶电泳(FIGE)UVB照射后培养不同时间的NIH3T3细胞DNA,发现DNA先断裂成低于23 kb的大分子片段,然后进一步断裂成小分子片段,但始终未出现梯形带,说明DNA并不总是从核小体之间断裂的.     

Paraspermatogenesis in Ceratostoma foliatum (Neogastropoda): confirmation of programmed nuclear death

Sperm dimorphism, where ejaculates contain both fertile "eusperm" and sterile "parasperm", has been implicated in sperm competition in animals. Most parasperm lack an acrosome, eliminate the nucleus and develop a complex cytoplasm, swollen with secretory vesicles. This study of parasperm in the foliate whelk Ceratostoma foliatum, focuses on characterizing the process of nuclear breakdown and the nature of cytoplasmic secretions. Testis squash preparations were treated with a Promega apoptosis detection [terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling-TUNEL] assay (Cedar Lane) to detect nuclear apoptosis; acridine orange was used to localize lysosomes. Parasperm were isolated on Percoll gradients and their proteins characterized by running electrophoresis of lysed, purified fractions on polyacrylamide gels. Results show that the process of nuclear breakdown follows the hallmarks of apoptosis in ultrastructure, which is confirmed by the presence of 3'-OH overhangs of DNA fragments. This research implicates snail parasperm as models for elucidating the nuclear events of apoptosis.     

Sequence of events leading to apoptosis in long term cultured HeLa cells

We have maintained HeLa cells in culture in the original medium for increasing times to induce growth arrest. Cell viability was evaluated by trypan blue dye exclusion assay. We observed that when cells are maintained in culture for several days, morphological hallmarks of apoptosis become evident. DNA synthesis rate, followed by (3)H-thymidine incorporation slowed down in long term cultured cells. This evidence was supported by the analysis of cell cycle progression determined by proliferating cell nuclear antigen (PCNA) immunostaining. Apoptotic cells have been characterized with respect to the sequential appearance of high molecular weight and internucleosomal DNA fragmentation. We have provided evidence that in this experimental model the first step in DNA degradation is represented by the formation of high molecular weight fragments, whereas nucleosomal DNA ladder is visible later on. The activation of the enzyme poly(ADP-ribose)polymerase, considered a marker of apoptotic death, has been observed. The results suggest that long term culture conditions activate the apoptotic programme.     

An ELISA for detection of apoptosis.

We describe a simple and convenient enzyme-linked immunosorbent assay (ELISA) for the detection of apoptosis in tissue culture. An early event in apoptosis is DNA fragmentation followed by release of nucleosomes into the cytoplasm. Our sandwich assay uses a pair of monoclonal antibodies specific for two nucleosomal epitopes to capture and detect cytoplasmic nucleosomes onto the ELISA plate. Our assay is about 500 times more sensitive than the detection of apoptotic DNA ladder by agarose electrophoresis and is especially suited for the testing of large numbers of samples.     

Detection of DNA fragmentation and endonucleases in apoptosis

DNA degradation during apoptosis is endonuclease mediated and proceeds through an ordered series of stages commencing with the production of large DNA pieces of 300 kb which are then degraded to fragments of 50 kb. The 50-kb fragments are further degraded, in some but not all cells, to smaller pieces (10-40 kb) releasing the small oligonucleosome fragments that are detected as a characteristic DNA ladder on conventional agarose gels. Methodology is presented for the detection of both DNA ladders and the initial stages of DNA fragmentation using pulsed-field gel electrophoresis. We have developed electrophoresis conditions that resolve large fragments of DNA and also retain the smaller fragments on the same gel. Methods for the detection of endonuclease activities responsible for the cleavage of DNA during apoptosis are also presented.     

DNA fragmentation during apoptosis is caused by frequent single-strand cuts.

One of the hallmarks of apoptosis is the digestion of genomic DNA by an endonuclease, generating a ladder of small fragments of double-stranded DNA. We have examined the nature of the DNA breaks produced in mouse thymocytes triggered to undergo apoptosis by steroids or by stimulation of the T cell receptor. Whereas the typical ladder pattern of oligonucleosomal fragments was observed after agarose gel electrophoresis, numerous single-strand cuts were detected after electrophoresis under denaturing conditions. Single-strand nicks were found to be very frequent in the internucleosomal regions, but also to occur in the core particle-associated DNA. An identical pattern of single-strand nicks was obtained when chromatin DNA was exposed to the single-strand cleaving deoxyribonuclease I. The nicked DNA fragments, extracted from apoptotic thymocytes, were sensitive to the action of S1-nuclease. We propose that DNA fragmentation induced during apoptosis is not due to a double-strand cutting enzyme as previously postulated, but rather is the result of single-strand breaks. This ensures the dissociation of the DNA molecule at sites where cuts are found within close proximity.     

Role of radical oxygen species in rat testicular germ cell apoptosis induced by heat stress.

The present study was designed to clarify the role of radical oxygen species in testicular germ cell apoptosis induced by heat stress. Testicular cells isolated from immature rats were cultured with or without elevated temperature, and occurrence of apoptosis in these cells was defined by the appearance of DNA fragmentation following agarose gel electrophoresis and by flow cytometric quantification of apoptotic cells. At 32.5 degrees C, < 1% of cells showed signs of apoptosis throughout the culture period, whereas under heat stress, the proportion of apoptotic cells increased to 5% at 37 degrees C after 24 h of culture, or to 14% after 1-h exposure at 43 degrees C followed by 23-h culture at 32.5 degrees C. Similar to the effect of heat stress, exogenously supplied oxygen free radicals also induced apoptosis. In contrast, treatment with catalase significantly attenuated heat stress-induced apoptosis. Furthermore, heat stress of testicular cells was associated with an increased intracellular peroxide level as measured by a fluorescent probe, 2', 7'-dichlorofluorescin diacetate. In conclusion, our data indicate the involvement of radical oxygen species during testicular germ cell apoptosis induced by heat stress. This study provides a useful in vitro model for the study of testicular germ cell apoptosis.     

Characterization of DNA damage in yeast apoptosis induced by hydrogen peroxide, acetic acid, and hyperosmotic shock

Saccharomyces cerevisiae has been reported to die, under certain conditions, from programmed cell death with apoptotic markers. One of the most important markers is chromosomal DNA fragmentation as indicated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. We found TUNEL staining in S. cerevisiae to be a consequence of both single- and double-strand DNA breaks, whereas in situ ligation specifically stained double-strand DNA breaks. Cells treated with hydrogen peroxide or acetic acid staining positively for TUNEL assay stained negatively for in situ ligation, indicating that DNA damage in both cases mainly consists of single-strand DNA breaks. Pulsed field gel electrophoresis of chromosomal DNA from cells dying from hydrogen peroxide, acetic acid, or hyperosmotic shock revealed DNA breakdown into fragments of several hundred kilobases, consistent with the higher order chromatin degradation preceding DNA laddering in apoptotic mammalian cells. DNA fragmentation was associated with death by treatment with 10 mM hydrogen peroxide but not 150 mM and was absent if cells were fixed with formaldehyde to eliminate enzyme activity before hydrogen peroxide treatment. These observations are consistent with a process that, like mammalian apoptosis, is enzyme dependent, degrades chromosomal DNA, and is activated only at low intensity of death stimuli.     

Chick embryo cardiocytes in culture synthesize proteins not present in embryonic heart

Proteins synthesized by cardiocytes isolated from 11d embryonic hearts and cultured $\text{in vitro}$ have been compared with proteins present in the 11d embryonic heart. It is shown that cultured cardiocytes synthesize two new proteins, C₁ and C₂ with a molecular weight of 27 500 and pI of 6.35 and 6.05 respectively. The synthesis of these proteins is associated with the appearance of new RNA species. This induction is not related to the conditions of culture since it occurs with either aggregated cardioacytes grown in suspension or cardiocytes grown in monolayer. Finally no other embryonic cell types seem to synthesize these proteins.     

Apoptosis induction in HCT116 cells by cytochalasins isolated from the fungus Daldinia vernicosa.

We examined the induction of apoptosis by cytochalasin (cc) derivatives (1-14) isolated from the Japanese fungus Daldinia vernicosa to HCT116 human colon cancer cell line based on their cytotoxicity, DNA ladder and DNA fragmentation ratio in agarose gel electrophoresis, and morphological changes. Most cc derivatives tested here induced apoptosis. Particularly cytochalasin 1 (cc1), monoacetate of 1 (cc1Ac), and cc14 were the most potent apoptosis inducers. These apoptotic activities were stronger than that of cytochalasin D as a known apoptosis inducer in HCT116 cell. However, cc4 and cc12 induced necrosis. The structure-activity relationship including their cytotoxicity will be discussed.     

Dynamic sieving capillary electrophoresis analysis of xylitol selenite-induced apoptosis in SMMC-7221 cells

DNA ladder fragments, regarded as a biochemical hallmark of apoptosis, have been separated quickly and successfully by capillary electrophoresis. Inter-nucleosomal DNA fragmentations induced by xylitol selenite were determined for the first time, while hydroxypropylmethylcellulose (HPMC) was served as the sieving matrix in dynamic sieving capillary electrophoresis. The calibration curve (r(2)?=?0.991) was established and multiples of two different nucleosomes (140 and 180?bp) were formed in the presence of xylitol selenite. Selenium compounds inhibited carcinogenesis in animal models, SMMC-7221 cells and several other cells by increasing apoptosis. The described method was useful in elucidating the anticancer activities of xylitol selenite and other selenium compounds, which was more effective to detect small fragments than slab gel electrophoresis.     

Apoptosis-like programmed cell death occurs in procambium and ground meristem of pea (Pisum sativum) root tips exposed to sudden flooding

BACKGROUND AND AIMS: Pea (Pisum sativum) primary roots form long vascular cavities when grown under wet or flooded conditions at 25 degrees C. It is thought that the cavities are a form of aerenchyma. At 25 degrees C short roots continue to grow after flooding. After roots reach 10 cm long flooding causes rapid cessation of growth, and root tips often become curled. In longer roots the cavities do not extend into the base of the roots, perhaps rendering them ineffective as aerenchyma. It was hypothesized that the resulting growth arrest was due to programmed cell death (PCD) rather than necrosis. METHODS AND KEY RESULTS: Histological examination by light microscope showed that some cells in the primary meristem (elongation) zone of the primary root tips had morphological abnormalities, including misshapen and fragmented nuclei, and cytoplasmic shrinking and fragmentation. Transmission electron microscopy revealed lobing, invagination and chromatin aggregation in nuclei. The affected cells were positive for terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling. Extracted DNA formed a "DNA ladder" during electrophoresis. Cell death usually began in procambium at one or two protoxylem poles and seemed to spread out to nearby tissues, which asymmetrically inhibited growth and resulted in tip curling. CONCLUSIONS: The above are symptoms of apoptosis-like PCD. Programmed root tip death may rapidly reduce oxygen demand and sink strength, allowing more rapid diversion of resources to lateral roots growing in more permissive conditions.     

Apoptosis of mouse liver nuclei induced in the cytosol of carrot cells

We report here the apoptosis of mouse liver nuclei induced in the cytosol of carrot cells by cytochrome c. Several typical characteristics of apoptosis, such as chromatin condensation, margination and apoptotic bodies, were detected. The result of DNA gel electrophoresis showed that DNA was degraded into nucleosomal fragments. The terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labelling procedure was also performed to detect the breakage of 3'-OH ends of a DNA strand. Furthermore, we found that nuclear lamins were degraded from 88 kDa and 66 kDa to 37 kDa and 47 kDa fragments. The DNA fragmentation could be inhibited by AC-DEVD-CHO and AC-YVAD-CHO. The results indicate that the apoptosis in plant cells may share some similar pathways to apoptosis in animal cells.     

天花粉收白诱发白血病细胞K562凋亡的研究

Trichosanthin (TCS), an eukaryotic ribosome-inactivating protein isolated from the root tuber of Trichosanthes plant, has various biological activities including abortion induction, antitumor, and anti-HIV. In this study, cultured human leukemia K562 cells treated with trichosanthin were examined. Analysis of the cells by single laser flow cytometry showed the sub-G1 peak. DNA extracted from these cells formed a characteristic "ladder" on agarose gel electrophoresis. Under electromicroscope, typical morphological changes of apoptosis were also observed. From all of these findings, we concluded that trichosanthin was able to induce apoptosis in K562 cells.     

天花粉蛋白诱发白血病细胞K562凋亡的研究

天花粉蛋白(Trichosanthin,TCS),是一种从栝楼块根内提取的核糖体失活蛋白,具有流产、抗肿瘤和抗HIV等多种生物活性。本文利用FACS检测到天花粉蛋白可使K562白血病细胞产生明显的凋亡小峰、DNA区带电泳成典型的“梯状”条带,电镜检测可观察到明显的细胞凋亡形态。这些结果表明天花粉蛋白可以诱发K562白血病细胞产生凋亡。     

In vitro apoptotic cell death during erythroid differentiation

Erythropoiesis occurs in bone marrow and it has been shown that during in vivo erythroid differentiation some immature erythroblasts undergo apoptosis. In this regard, it is known that immature erythroblasts are FasL- and TRAIL-sensitive and can be killed by cells expressing these ligand molecules. In the present study, we have investigated the cell death phenomenon that occurs during a common unilineage model of erythroid development. Purified CD34+ human haemopoietic progenitors were cultured in vitro in the presence of SCF, IL-3 and erythropoietin. Their differentiation stages and apoptosis were followed by multiple technical approaches. Flow cytometric evaluation of surface and intracellular molecules revealed that glycophorin A appeared at day 3-4 of incubation and about 75% of viable cells co-expressed high density glycophorin A (Gly(bright)) and adult haemoglobin at day 14 of culture, indicating that this system reasonably recapitulates in vivo normal erythropoiesis. Interestingly, when mature (Gly(bright)) erythroid cells reached their higher percentages (day 14) almost half of cultured cells were apoptotic. Morphological studies indicated that the majority of dead cells contained cytoplasmic granular material typical of basophilic stage, and DNA analysis by flow cytometry and TUNEL reaction revealed nuclear fragmentation. These observations indicate that in vitro unilineage erythroid differentiation, as in vivo, is associated with apoptotic cell death of cells with characteristics of basophilic erythroblasts. We suggest that the interactions between different death receptors on immature basophilic erythroblasts with their ligands on more mature erythroblasts may contribute to induce apoptosis in vitro.     

Cytosine Arabinoside Induces Apoptosis in Cerebellar Neurons in Culture

Abstract: Cytosine arabinoside (AraC) is a pyrimidine antimetabolite that prevents cell proliferation by inhibiting DNA synthesis. We report that AraC kills cultured cerebellar neurons in a concentration-dependent fashion with an EC₅₀ of ∼60 µ M when added shortly after seeding. This cell death has apoptotic features because we observed (1) morphology of apoptotic nuclei as judged by DNA staining with Hoechst 33258, (2) DNA fragmentation with typical ladder pattern on agarose gel, (3) positive nuclear labeling with a specific in situ DNA fragmentation staining, (4) prevention by deoxycytidine (IC₅₀ = 1 µ M ), protein, and RNA synthesis inhibitors, and (5) release of DNA fragments in the incubating medium. We have also observed that several proteins were overexpressed in AraC-treated neurons by two-dimensional polyacrylamide gel electrophoresis. We conclude that AraC induces a signal that triggers a cascade of new mRNA and protein synthesis, leading to apoptotic cell death in cultured cerebellar granule cells.     

Dose-dependent zinc inhibition of DNA ladder in apoptotic HeLa cells regulates the activity of poly(ADP-ribose) polymerase and does not protect from death induced by VP-16

Zinc ions exert an inhibitory effect on Ca(2+)Mg(2+)-dependent endonuclease which is supposed to be responsible for the fragmentation of DNA during apoptosis. In the experimental system we used, that is HeLa cells treated with VP-16, the protection from internucleosomal DNA degradation is modulated by Zn concentration and appears to be dependent on the time after treatment. This effect does not prevent cell death or occurrence of apoptotic parameters, suggesting that DNA ladder appearance is not a crucial event in apoptosis. The activation of poly(ADP-ribose)polymerase following the administration of VP-16, is not observed in cells in which DNA fragmentation has been abolished by zinc, supporting the hypothesis that this event is regulated by the appearance of small-sized DNA fragments.