The role of topoisomerase II in the excision of DNA loop domains during apoptosis

Disintegration of nuclear DNA into high molecular weight (HMW) and oligonucleosomal DNA fragments represents two major periodicities of DNA fragmentation during apoptosis. These are thought to originate from the excision of DNA loop domains and from the cleavage of nuclear DNA at the internucleosomal positions, respectively. In this report, we demonstrate that different apoptotic insults induced apoptosis in NB-2a neuroblastoma cells that was invariably accompanied by the formation of HMW DNA fragments of about 50-100 kb but proceeded either with or without oligonucleosomal DNA cleavage, depending on the type of apoptotic inducer. We demonstrate that differences in the pattern of DNA fragmentation were reproducible in a cell-free apoptotic system and develop conditions that allow in vitro separation of the HMW and oligonucleosomal DNA fragmentation activities. In contrast to apoptosis associated with oligonucleosomal DNA fragmentation, the HMW DNA cleavage in apoptotic cells was accompanied by down-regulation of caspase-activated DNase (CAD) and was not affected by z-VAD-fmk, suggesting that the caspase/CAD pathway is not involved in the excision of DNA loop domains. We further demonstrate that nonapoptotic NB-2a cells contain a constitutively present nuclease activity located in the nuclear matrix fraction that possessed the properties of topoisomerase (topo) II and was capable of reproducing the pattern of HMW DNA cleavage that occurred in apoptotic cells. We demonstrate that the early stages of apoptosis induced by different stimuli were accompanied by activation of topo II-mediated HMW DNA cleavage that was reversible after removal of apoptotic inducers, and we present evidence of the involvement of topo II in the formation of HMW DNA fragments at the advanced stages of apoptosis. The results suggest that topo II is involved in caspase-independent excision of DNA loop domains during apoptosis, and this represents an alternative pathway of apoptotic DNA disintegration from CAD-driven caspase-dependent oligonucleosomal DNA cleavage.     

Caspase-activated DNase Is Necessary and Sufficient for Oligonucleosomal DNA Breakdown,but Not for Chromatin Disassembly during Caspase-dependent Apoptosis of LN-18 Glioblastoma Cells

Caspase-dependent apoptosis is a controlled type of cell death characterized by oligonucleosomal DNA breakdown and major nuclear morphological alterations. Other kinds of cell death do not share these highly distinctive traits because caspase-activated DNase (DFF40/CAD) remains inactive. Here, we report that human glioblastoma multiforme-derived LN-18 cells do not hydrolyze DNA into oligonucleosomal fragments after apoptotic insult. Furthermore, their chromatin remains packaged into a single mass, with no signs of nuclear fragmentation. However, ultrastructural analysis reveals that nuclear disassembly occurs, although compacted chromatin does not localize into apoptotic nuclear bodies. Caspases become properly activated, and ICAD, the inhibitor of DFF40/CAD, is correctly processed. Using cell-free in vitro assays, we show that chromatin from isolated nuclei of LN-18 cells is suitable for hydrolysis into oligonuclesomal fragments by staurosporine-pretreated SH-SY5Y cytoplasms. However, staurosporine-pretreated LN-18 cytoplasms do not induce DNA laddering in isolated nuclei from either LN-18 or SH-SY5Y cells because LN-18 cells express lower amounts of DFF40/CAD. DFF40/CAD overexpression makes LN-18 cells fully competent to degrade their DNA into oligonucleosome-sized fragments, and yet they remain unable to arrange their chromatin into nuclear clumps after apoptotic insult. Indeed, isolated nuclei from LN-18 cells were resistant to undergoing apoptotic nuclear morphology in vitro. The use of LN-18 cells has uncovered a previously unsuspected cellular model, whereby a caspase-dependent chromatin package is DFF40/CAD-independent, and DFF40/CAD-mediated double-strand DNA fragmentation does not warrant the distribution of the chromatin into apoptotic nuclear bodies. The studies highlight a not-yet reported DFF40/CAD-independent mechanism driving conformational nuclear changes during caspase-dependent cell death.     

Chromatin Collapse during Caspase-dependent Apoptotic Cell Death Requires DNA Fragmentation Factor, 40-kDa Subunit-/Caspase-activated Deoxyribonuclease-mediated 3′-OH Single-strand DNA Breaks

Apoptotic nuclear morphology and oligonucleosomal double-strand DNA fragments (also known as DNA ladder) are considered the hallmarks of apoptotic cell death. From a classic point of view, these two processes occur concomitantly. Once activated, DNA fragmentation factor, 40-kDa subunit (DFF40)/caspase-activated DNase (CAD) endonuclease hydrolyzes the DNA into oligonucleosomal-size pieces, facilitating the chromatin package. However, the dogma that the apoptotic nuclear morphology depends on DNA fragmentation has been questioned. Here, we use different cellular models, including MEF CAD^−/− cells, to unravel the mechanism by which DFF40/CAD influences chromatin condensation and nuclear collapse during apoptosis. Upon apoptotic insult, SK-N-AS cells display caspase-dependent apoptotic nuclear alterations in the absence of internucleosomal DNA degradation. The overexpression of a wild-type form of DFF40/CAD endonuclease, but not of different catalytic-null mutants, restores the cellular ability to degrade the chromatin into oligonucleosomal-length fragments. We show that apoptotic nuclear collapse requires a 3′-OH endonucleolytic activity even though the internucleosomal DNA degradation is impaired. Moreover, alkaline unwinding electrophoresis and In Situ End-Labeling (ISEL)/In Situ Nick Translation (ISNT) assays reveal that the apoptotic DNA damage observed in the DNA ladder-deficient SK-N-AS cells is characterized by the presence of single-strand nicks/breaks. Apoptotic single-strand breaks can be impaired by DFF40/CAD knockdown, abrogating nuclear collapse and disassembly. In conclusion, the highest order of chromatin compaction observed in the later steps of caspase-dependent apoptosis relies on DFF40/CAD-mediated DNA damage by generating 3′-OH ends in single-strand rather than double-strand DNA nicks/breaks.     

Auxin-induced Rapid Degradation of Inhibitor of Caspase-activated DNase (ICAD) Induces Apoptotic DNA Fragmentation,Caspase Activation,and Cell Death: A CELL SUICIDE MODULE*

Caspase-activated DNase (CAD) is a major apoptotic nuclease, responsible for DNA fragmentation and chromatin condensation during apoptosis. CAD is normally activated in apoptosis as a result of caspase cleavage of its inhibitory chaperone ICAD. Other aspects of CAD regulation are poorly understood. In particular, it has been unclear whether direct CAD activation in non-apoptotic living cells can trigger cell death. Taking advantage of the auxin-inducible degron (AID) system, we have developed a suicide system with which ICAD is rapidly degraded in living cells in response to the plant hormone auxin. Our studies demonstrate that rapid ICAD depletion is sufficient to activate CAD and induce cell death in DT40 and yeast cells. In the vertebrate cells, ectopic CAD activation triggered caspase activation and subsequent hallmarks of caspase-dependent apoptotic changes, including phosphatidylserine exposure and nuclear fragmentation. These observations not only suggest that CAD activation drives apoptosis through a positive feedback loop, but also identify a unique suicide system that can be used for controlling gene-modified organisms.     

Loss of Acinus inhibits oligonucleosomal DNA fragmentation but not chromatin condensation during apoptosis

Chromatin condensation and oligonucleosomal DNA fragmentation are the nuclear hallmarks of apoptosis. A proteolytic fragment of the apoptotic chromatin condensation inducer in the nucleus (Acinus), which is generated by caspase cleavage, has been implicated in mediating apoptotic chromatin condensation prior to DNA fragmentation. Acinus is also involved in mRNA splicing and a component of the apoptosis and splicing-associated protein (ASAP) complex. To study the role of Acinus for apoptotic nuclear alterations, we generated stable cell lines in which Acinus isoforms were knocked down by inducible and reversible RNA interference. We show that Acinus is not required for nuclear localization and interaction of the other ASAP subunits SAP18 and RNPS1; however, knockdown of Acinus leads to a reduction in cell growth. Most strikingly, down-regulation of Acinus did not inhibit apoptotic chromatin condensation either in intact cells or in a cell-free system. In contrast, although apoptosis proceeds rapidly, analysis of nuclear DNA from apoptotic Acinus knockdown cells shows inhibition of oligonucleosomal DNA fragmentation. Our results therefore suggest that Acinus is not involved in DNA condensation but rather point to a contribution of Acinus in internucleosomal DNA cleavage during programmed cell death.     

Oligonucleosome DNA fragmentation of caspase 3 deficient MCF-7 cells in palmitate-induced apoptosis

Oligonucleosomal fragmentation of nuclear DNA is the late stage hallmark of the apoptotic process. In mammalian apoptotic cells fragmentation is catalyzed by DFF40/ CAD DNase. DFF40/CAD primary activated through site-specific proteolytic cleavage by caspase 3. The absence of caspase 3 in MCF-7 leads to lack of oligonucleosomal DNA fragmentation under numerous apoptotic stimuli. In this study it was shown that palmitate induces apoptotic changes of nuclei and oligonucleosomal DNA fragmentation in casp3 deficient MCF-7. Activation and accumulation of 40-50 kDa DFF40 like DNases in nuclei and cytoplasm of palmitate-treated MCF-7 were detected by SDS-DNA-PAGE assay. Microsomes of apoptotic MCF-7 activate 40-50 kDa nucleases when incubated with human placental chromatin and induce oligonucleosomal fragmentation of chromatin in cell free system. Both DNases activation and chromatin fragmentation are suppressed in presence of caspase 3/7 inhibitor Ac-DEVD-CHO. Microsome associated caspase 7 is suggested to play the principal role in induction of oligonucleosomal DNA fragmentation of casp3 defitient MCF-7.     

Oligonucleosomal DNA Fragmentation in MCF-7 Cells Undergoing Palmitate-Induced Apoptosis

Oligonucleosomal fragmentation of nuclear DNA is the late-stage apoptosis hallmark. In apoptotic mammalian cells the fragmentation is catalyzed by DFF40/CAD DNase primarily activated by caspase 3 through the site-specific proteolytic cleavage of DFF45/ICAD. A deletion in the casp3 gene of human breast adenocarcinoma MCF-7 results in lack of procaspase 3 in these cells. The absence of caspase 3 in MCF-7 leads to disability to activate oligonucleosomal DNA fragmentation in TNF-alpha induced cell death. In this study, sodium palmitate was used as an apoptotic stimulus for MCF-7. It has been shown that palmitate but not TNF-alpha induces both apoptotic changes in nuclei and oligonucleosomal DNA fragmentation in casp3-mutated MCF-7. Activation and accumulation of 40-50 kD DFF40-like DNases in nuclei of palmitate-treated apoptotic MCF-7 were detected by SDS-DNA-PAGE assay. Microsomal fraction of apoptotic MCF-7 does not contain any detectable DNases, but activates 40-50 kD nucleases when incubated with human placental chromatin. Furthermore, microsomes of apoptotic MCF-7 induce oligonucleosomal fragmentation of chromatin in a cell-free system. Both the activation of DNases and chromatin fragmentation are suppressed in the presence of the caspase 3/7 inhibitor Ac-DEVD-CHO. Microsome-associated caspase 7 is suggested to play an essential role in the induction of oligonucleosomal DNA fragmentation in casp3-deficient MCF-7 cells.     

Modeling apoptotic chromatin condensation in normal cell nuclei. Requirement for intranuclear mobility and actin involvement

Hallmarks of the terminal stages of apoptosis are genomic DNA fragmentation and chromatin condensation. Here, we have studied the mechanism of condensation both in vitro and in vivo. We found that DNA fragmentation per se of isolated nuclei from non-apoptotic cells induced chromatin condensation that closely resembles the morphology seen in apoptotic cells, independent of ATP utilization, at physiological ionic strengths. Interestingly, chromatin condensation was accompanied by release of nuclear actin, and both condensation and actin release could be blocked by reversibly pretreating nuclei with Ca2+, Cu2+, diamide, or low pH, procedures shown to stabilize internal nuclear components. Moreover, specific inhibition of nuclear F-actin depolymerization or promotion of its formation also reduced chromatin condensation. Chromatin condensation could also be inhibited by exposing nuclei to reagents that bind to the DNA minor groove, disrupting native nucleosomal DNA wrapping. In addition, in cultured cells undergoing apoptosis, drugs that inhibit depolymerization of actin or bind to the minor groove also reduced chromatin condensation, but not DNA fragmentation. Therefore, the ability of chromatin fragments with intact nucleosomes to form large clumps of condensed chromatin during apoptosis requires the apparent disassembly of internal nuclear structures that may normally constrain chromosome subdomains in non-apoptotic cells.     

The major apoptotic endonuclease DFF40/CAD is a deoxyribose-specific and double-strand-specific enzyme

DFF40/CAD endonuclease is primarily responsible for internucleosomal DNA cleavage during the terminal stages of apoptosis. The nuclease specifically introduces DNA double strand breaks into chromatin substrates. Here we performed a detailed study on the specificity of the nuclease using synthetic single-stranded and double-stranded ribo- and deoxyribo-oligonucleotides as substrates. We have found that neither single-stranded DNA, single-stranded RNA, double-stranded RNA nor RNA–DNA heteroduplexes are cleaved by the DFF40/CAD nuclease. Noteworthy, all types of oligonucleotides that are not cleaved by the nuclease inhibit cleavage of double-stranded DNA. We have also observed that in cells undergoing apoptosis in vivo neither the activation of DFF40/CAD nor oligonucleosomal chromatin fragmentation was temporally correlated with either total cellular or nuclear RNA degradation. We conclude that DFF40/CAD is exclusively specific for double-stranded DNA. Jakub Hanus and Magdalena Kalinowska-Herok contributed equally to the work.     

LDFF, the large molecular weight DNA fragmentation factor, is responsible for the large molecular weight DNA degradation during apoptosis in Xenopus egg extracts

DNA degradation is a biochemical hallmark in apoptosis. It has been demonstrated in many cell types that there are two stages of DNA fragmentation during the apoptotic execution. In the early stage, chromatin DNA is cut into large molecular weight DNA fragments, although the responsible nuclease(s) has not been recognized. In the late stage, the chromatin DNA is cleaved further into short oligonucleosomal fragments by a well-characterized nuclease in apoptosis,the caspase-activated DNase (CAD/DFF40). In this study, we demonstrate that large molecular weight DNA fragmentation also occurs in Xenopus egg extracts in apoptosis. We show that the large molecular weight DNA fragmentation factor (LDFF) is not the Xenopus CAD homolog XCAD. LDFF is activated by caspase-3. The large molecular weight DNA fragmentation activity of LDFF is Mg^2 -dependent and Ca^2 -independent, can occur in both acidic and neutral pH conditions and can tolerate 45℃ treatment. These results indicate that LDFF in Xenopus egg extracts might be a new DNase (or DNases) responsible for the large DNA fragmentation.     

Correlation between nucleocytoplasmic transport and caspase-3-dependent dismantling of nuclear pores during apoptosis

During apoptosis (also called programmed cell death), the chromatin condenses and the DNA is cleaved into oligonucleosomal fragments. Caspases are believed to play a major role in nuclear apoptosis. However, the relation between dismantling of nuclear pores, disruption of the nucleocytoplasmic barrier, and nuclear entry of caspases is unclear. We have analyzed nuclear import of the green fluorescent protein fused to a nuclear localization signal (GFP-NLS) in tissue culture cells undergoing apoptosis. Decreased nuclear accumulation of GFP-NLS could be detected at the onset of nuclear apoptosis manifested as dramatic condensation and redistribution of chromatin toward the nuclear periphery. At this step, dismantling of nuclear pores was already evident as indicated by proteolysis of the nuclear pore membrane protein POM121. Thus, disruption of nuclear compartmentalization correlated with early signs of nuclear pore damage. Both these events clearly preceded massive DNA fragmentation, detected by TUNEL assay. Furthermore, we show that in apoptotic cells, POM121 is specifically cleaved at aspartate-531 in its large C-terminal portion by a caspase-3-dependent mechanism. Cleavage of the C-terminal portion of POM121, which is adjoining the nuclear pore complex, is likely to disrupt interactions with other nuclear pore proteins affecting the stability of the pore complex. A temporal correlation of apoptotic events supports a model where caspase-dependent disassembly of nuclear pores and disruption of the nucleocytoplasmic barrier paves the way for nuclear entry of caspases and subsequent activation of CAD-mediated DNA fragmentation.     

BAPTA blocks DNA fragmentation and chromatin condensation downstream of caspase-3 and DFF activation in HT-induced apoptosis in HL-60 cells

DFF ((DNA Fragmentation Factor) is a heterodimer composed of 40 kDa (DFF40, CAD) and 45 kDa (DFF45, ICAD) subunits. During apoptosis, activated caspase-3 cleaves DFF45 and activates DFF40, a DNase that targets nucleosomal linker region and cleaves chromatin DNA into nucleosomal fragments. We have previously reported that HT induced apoptosis in HL-60 cells, and intracellular Ca²⁺ chelator BAPTA blocked apoptosis-associated DNA fragmentation induced by HT. We report here that HT also induced activation of caspase-3 and cleavage of DFF45. BAPTA prevented neither the caspase-3 activation nor the cleavage of DFF45. Mitochondrial membrane potential was disrupted in BAPTA-AM treated cells. However, BAPTA did prevent DNA fragmentation and chromatin condensation in HT-treated cells. These data suggest a novel role for intracellular calcium in regulating apoptotic nuclease that causes DNA fragmentation and chromatin condensation.     

Genetic analysis of the short splice variant of the inhibitor of caspase-activated DNase (ICAD-S) in chicken DT40 cells

We have studied the regulation of the caspase-Activated DNase (CAD) by its inhibitor, ICAD. To study the role of ICAD short and long splice forms ICAD-S and ICAD-L, respectively, in vivo, we constructed chicken DT40 cell lines in which the entire coding regions of ICAD alone or ICAD plus CAD were deleted. ICAD and ICAD/CAD double knock-outs lacked both DNA fragmentation and nuclear fragmentation after the induction of apoptosis. We constructed a model humanized system in which human ICAD-L and CAD proteins expressed in DT40 ICAD/CAD double knock-out cells could rescue both DNA fragmentation and stage II chromatin condensation. ICAD-S could not replace ICAD-L as a chaperone for folding active CAD in these cells. However, a modified version of ICAD-S, in which the two caspase-3 cleavage sites were replaced with two tobacco etch virus (TEV) protease cleavage sites (ICAD-S(2TEV)) and which was therefore resistant to caspase cleavage, did inhibit CAD activation upon induction of apoptosis in vivo. Moreover, ICAD-L(2TEV) was functional as a chaperone for the production of active CAD in DT40 cells. In extracts prepared from these cells, we were able to activate CAD by cleavage of ICAD-L(2TEV) with TEV protease under non-apoptotic conditions. Thus, ICAD appears to be the only functional inhibitor of CAD activation in these cell-free extracts. Taken together, these observations indicate that ICAD-S may function together with ICAD-L as a buffer to prevent inappropriate CAD activation, particularly in cells where ICAD-S is the dominant form of ICAD protein.     

Curcumin induces cell death without oligonucleosomal DNA fragmentation in quiescent and proliferating human CD8+ cells

Cytotoxic CD8+ cells play an important role in determining host response to tumor, thus chemotherapy is potentially dangerous as it may lead to T cells depletion. The purpose of this study was to elucidate the propensity of quiescent and proliferating human CD8+ cells to undergo cell death upon treatment with curcumin, a natural dye in Phase I of clinical trials as a prospective chemopreventive agent. Methods: We treated human quiescent or proliferating CD8+ cells with 50 microM curcumin or irradiated them with UVC. Cell death symptoms such as decreased cell viability, chromatin condensation, activation of caspase-3 and specific DFF40/CAD endonuclease and oligonucleosomal DNA fragmentation were analyzed using MTT test, microscopic observation, Western blotting and flow cytometry. Results: Curcumin decreased cell viability, activated caspase-3 and decreased the level of DFF45/ICAD, the inhibitor of the DFF40/CAD endonuclease. However, this did not lead to oligonucleosomal DNA degradation. In contrast, UVC-irradiated proliferating, but not quiescent CD8+ cells revealed molecular and morphological changes characteristic for apoptosis, including oligonucleosomal DNA fragmentation. Curcumin can induce cell death in normal human lymphocytes both quiescent and proliferating, without oligonucleosomal DNA degradation which is considered as a main hallmark of apoptotic cell death. Taking into account the role of CD8+ cells in tumor response, their depletion during chemotherapy could be particularly undesirable.     

Caspase-6 gene disruption reveals a requirement for lamin A cleavage in apoptotic chromatin condensation

To study the role of caspase-6 during nuclear disassembly, we generated a chicken DT40 cell line in which both alleles of the caspase-6 gene were disrupted. No obvious morphological differences were observed in the apoptotic process in caspase-6- deficient cells compared with the wild type. However, examination of apoptosis in a cell-free system revealed a block in chromatin condensation and apoptotic body formation when nuclei from HeLa cells expressing lamin A or lamin A-transfected Jurkat cells were incubated in caspase-6-deficient apoptotic extracts. Transfection of exogenous caspase-6 into the clone reversed this phenotype. Lamins A and C, which are caspase-6-only substrates, were cleaved by the wild-type and heterozygous apoptotic extracts but not by the extracts lacking caspase-6. Furthermore, the caspase-6 inhibitor z-VEID-fmk mimicked the effects of caspase-6 deficiency and prevented the cleavage of lamin A. Taken together, these observations indicate that caspase-6 activity is essential for lamin A cleavage and that when lamin A is present it must be cleaved in order for the chromosomal DNA to undergo complete condensation during apoptotic execution.     

DNA localization in nuclear fragments of apoptotic ameloblasts using anti-DNA immunoelectron microscopy: Programmed cell death of ameloblasts

Ameloblasts responsible for tooth enamel formation are classified into two different phases: secretion and maturation. At the transition between these secretion and maturation stages, a considerable number of cells die. In this study, we examined the morphology of degenerating ameloblasts by conventional electron microscopy, and DNA cleavage in degenerating ameloblast nuclei by the in situ terminal transferase assay. The results suggest that apoptosis (programmed cell death) in ameloblasts, including DNA ligation is induced at the transitional stage. The nuclear fragments, chromatin condensation and DNA relocation in apoptotic nuclei were examined quantitatively by post-embedding anti-DNA immunogold electron microscopy and the in situ terminal transferase assay combined with electron microscopy. Numerical analysis revealed that immunogold labeling density in the condensed chromatin of apoptotic nuclei was comparable on the average to that in the perinuclear heterochromatin of normal nuclei, and that individual apoptotic nuclear fragments exhibited highly variable gold particle density, from fragments with lower density to that of normal heterochromatin, to fragments with densities twice as high as that of normal heterochromatin. The in situ terminal transferase assay combined with electron microscopy detected DNA ends exposed by ultrathin sectioning as well as DNA cleavage by a putative endonuclease. In conclusion, the state of the DNA, including its ligation and degeneration, changes gradually during chromatin condensation and nuclear fragmentation of apoptosis.     

Apoptotic DNA degradation into oligonucleosomal fragments, but not apoptotic nuclear morphology, relies on a cytosolic pool of DFF40/CAD endonuclease

Apoptotic cell death is characterized by nuclear fragmentation and oligonucleosomal DNA degradation, mediated by the caspase-dependent specific activation of DFF40/CAD endonuclease. Here, we describe how, upon apoptotic stimuli, SK-N-AS human neuroblastoma-derived cells show apoptotic nuclear morphology without displaying concomitant internucleosomal DNA fragmentation. Cytotoxicity afforded after staurosporine treatment is comparable with that obtained in SH-SY5Y cells, which exhibit a complete apoptotic phenotype. SK-N-AS cell death is a caspase-dependent process that can be impaired by the pan-caspase inhibitor q-VD-OPh. The endogenous inhibitor of DFF40/CAD, ICAD, is correctly processed, and dff40/cad cDNA sequence does not reveal mutations altering its amino acid composition. Biochemical approaches show that both SH-SY5Y and SK-N-AS resting cells express comparable levels of DFF40/CAD. However, the endonuclease is poorly expressed in the cytosolic fraction of healthy SK-N-AS cells. Despite this differential subcellular distribution of DFF40/CAD, we find no differences in the subcellular localization of both pro-caspase-3 and ICAD between the analyzed cell lines. After staurosporine treatment, the preferential processing of ICAD in the cytosolic fraction allows the translocation of DFF40/CAD from this fraction to a chromatin-enriched one. Therefore, the low levels of cytosolic DFF40/CAD detected in SK-N-AS cells determine the absence of DNA laddering after staurosporine treatment. In these cells DFF40/CAD cytosolic levels can be restored by the overexpression of their own endonuclease, which is sufficient to make them proficient at degrading their chromatin into oligonucleosome-size fragments after staurosporine treatment. Altogether, the cytosolic levels of DFF40/CAD are determinants in achieving a complete apoptotic phenotype, including oligonucleosomal DNA degradation.     

Lack of internucleosomal DNA fragmentation is related to Cl(-) efflux impairment in hematopoietic cell apoptosis.

The heterodimeric DNA fragmentation factor (DFF) is responsible for DNA degradation into nucleosomal units during apoptosis. This process needs the caspase-dependent release of ICAD/DFF-45, the inhibitory subunit of DFF. Here we report that triggering apoptosis via a hyperosmotic shock in hematopoietic cells causes the appearance of mitochondrial and cytosolic alterations, activation of caspases, chromatin condensation, nuclear disruption, and DNA fragmentation. However, oligonucleosomal but not high molecular weight (50-150 kb) DNA cleavage is abolished if Cl(-) efflux is prevented by using NaCl to raise extracellular osmolarity or by Cl(-) channel blockers, even when apoptosis is initiated by other agents (staurosporine, anti-Fas antibody). In these conditions, all the apoptosis hallmarks investigated remain detectable, including the cleavage of ICAD/DFF-45. In vitro assays with lysates of cells in which Cl(-) efflux is blocked confirm the lack of internucleosomal DNA degradation. These findings establish that neither caspase activation nor ICAD/DFF-45 processing per se is sufficient to induce oligonucleosomal DNA fragmentation and that high molecular weight DNA degradation and chromatin condensation appear independently of it. Finally, they suggest that Cl(-) efflux is a necessary cofactor that intervenes specifically in the activation of the DFF endonuclease.     

The DFF40/CAD endonuclease and its role in apoptosis

The sequential generation of large-scale DNA fragments followed by internucleosomal chromatin fragmentation is a biochemical hallmark of apoptosis. One of the nucleases primarily responsible for genomic DNA fragmentation during apoptosis is called DNA Fragmentation Factor 40 (DFF40) or Caspase-activated DNase (CAD). DFF40/CAD is a magnesium-dependent endonuclease specific for double stranded DNA that generates double strand breaks with 3'-hydroxyl ends. DFF40/CAD is activated by caspase-3 that cuts the nuclease's inhibitor DFF45/ICAD. The nuclease preferentially attacks chromatin in the internucleosomal linker DNA. However, the nuclease hypersensitive sites can be detected and DFF40/CAD is potentially involved in large-scale DNA fragmentation as well. DFF40/CAD-mediated DNA fragmentation triggers chromatin condensation that is another hallmark of apoptosis.     

褐飞虱若虫中肠凋亡细胞的检测

为揭示褐飞虱Niloparvata lugens Stl若虫在发育过程中中肠的凋亡细胞,使用末端脱氧核苷酸转移酶介导的dUTP-生物素断端标记法(TUNEL)进行中肠组织切片检测,结果表明,1～5龄若虫中肠分别存在2%～5%的凋亡细胞。利用4′,6-二脒基-2-苯基吲哚二盐酸(DAPI)染色法检测表明,存在Ⅰ,Ⅱa和Ⅱb期凋亡的细胞核,其特征包括染色体浓缩、边缘化及细胞核碎裂。透射电子显微镜检测结果表明,早期凋亡的细胞呈现染色质浓缩、边缘化特征,晚期凋亡的细胞出现细胞核碎裂、形成凋亡小体及细胞质空泡化等。本研究揭示了在正常发育过程中褐飞虱若虫中肠有少量的细胞发生了凋亡。通过人工干预的方式调控中肠细胞的凋亡进程有可能使之成为防治该水稻害虫的新靶标。