Fine mapping of 28S rRNA sites specifically cleaved in cells undergoing apoptosis.

Bona fide apoptosis in rat and human leukemia cells, rat thymocytes, and bovine endothelial cells was accompanied by limited and specific cleavage of polysome-associated and monosome-associated 28S rRNA, with 18S rRNA being spared. Specific 28S rRNA cleavage was observed in all instances of apoptotic death accompanied by internucleosomal DNA fragmentation, with cleavage of 28S rRNA and of DNA being linked temporally. This indicates that 28S rRNA fragmentation may be as general a feature of apoptosis as internucleosomal DNA fragmentation and that concerted specific cleavage of intra- and extranuclear polynucleotides occurs in apoptosis. Apoptosis-associated cleavage sites were mapped to the 28S rRNA divergent domains D2, D6 (endothelial cells), and D8. The D2 cuts occurred in hairpin loop junctions considered to be buried in the intact ribosome, suggesting that this rRNA region becomes a target for RNase attack in apoptotic cells. D8 was cleaved in two exposed UU(U) sequences in bulge loops. Treatment with agents causing necrotic cell death or aging of cell lysates failed to produce any detectable limited D2 cleavage but did produce a more generalized cleavage in the D8 region. Of potential functional interest was the finding that the primary cuts in D2 exactly flanked a 0.3-kb hypervariable subdomain (D2c), allowing excision of the latter. The implication of hypervariable rRNA domains in apoptosis represents the first association of any functional process with these enigmatic parts of the ribosomes.     

RNase L-independent specific 28S rRNA cleavage in murine coronavirus-infected cells

We characterized a novel 28S rRNA cleavage in cells infected with the murine coronavirus mouse hepatitis virus (MHV). The 28S rRNA cleavage occurred as early as 4 h postinfection (p.i.) in MHV-infected DBT cells, with the appearance of subsequent cleavage products and a decrease in the amount of intact 28S rRNA with increasing times of infection; almost all of the intact 28S rRNA disappeared by 24 h p.i. In contrast, no specific 18S rRNA cleavage was detected in infected cells. MHV-induced 28S rRNA cleavage was detected in all MHV-susceptible cell lines and all MHV strains tested. MHV replication was required for the 28S rRNA cleavage, and mature cytoplasmic 28S rRNA underwent cleavage. In certain combination of cells and viruses, pretreatment of virus-infected cells with interferon activates a cellular endoribonuclease, RNase L, that causes rRNA degradation. No interferon was detected in the inoculum used for MHV infection. Addition of anti-interferon antibody to MHV-infected cells did not inhibit 28S rRNA cleavage. Furthermore, 28S rRNA cleavage occurred in an MHV-infected mouse embryonic fibroblast cell line derived from RNase L knockout mice. Thus, MHV-induced 28S rRNA cleavage was independent of the activation of RNase L. MHV-induced 28S rRNA cleavage was also different from apoptosis-related rRNA degradation, which usually occurs concomitantly with DNA fragmentation. In MHV-infected 17Cl-1 cells, 28S rRNA cleavage preceded DNA fragmentation by at least 18 h. Blockage of apoptosis in MHV-infected 17Cl-1 cells by treatment with a caspase inhibitor did not block 28S rRNA cleavage. Furthermore, MHV-induced 28S rRNA cleavage occurred in MHV-infected DBT cells that do not show apoptotic signs, including activation of caspase-3 and DNA fragmentation. Thus, MHV-induced 28S rRNA cleavage appeared to differ from any rRNA degradation mechanism described previously.     

7-Hydroxystaurosporine (UCN-01) Induces Apoptosis in Human Colon Carcinoma and Leukemia Cells Independently of p53

7-hydroxystaurosporine (UCN-01) is a more selective protein kinase C inhibitor than staurosporine. UCN-01 exhibits antitumor activity in experimental tumor models and is presently in clinical trials. Our study reveals that human myeloblastic leukemia HL60 and K562 and colon carcinoma HT29 cells undergo internucleosomal DNA fragmentation and morphological changes characteristic of apoptosis after UCN-01 treatment. These three cell lines lack functional p53, and K562 and HT29 cells are usually resistant to apoptosis. DNA fragmentation in HT29 and K562 cells occurred after 1 day of treatment while it took less than 4 h in HL60 cells. Cycloheximide prevented UCN-01-induced DNA fragmentation in HT-29 cells, but not in HL60 and K562 cells, suggesting that macromolecular synthesis is selectively required for apoptotic DNA fragmentation in HT29 cells. UCN-01-induced DNA fragmentation was preceded by activation of cyclin B1/cdc2 kinase. Further studies in HL60 cells showed that UCN-01-induced apoptosis was associated with degradation of CPP32, PARP, and lamin B and that the inhibitor of caspases (ICE/CED-3 cysteine proteases), Z-VAD-FMK, and the serine protease inhibitor, DCI, protected HL60 cells from UCN-01-induced DNA fragmentation. However, only DCI and TPCK, but not Z-VAD-FMK, inhibited DNA fragmentation in the HL60 cell-free system, suggesting that serine protease(s) may play a role in the execution phase of apoptosis in HL60 cells treated with UCN-01. Z-VAD-FMK and DCI also inhibited apoptosis in HT29 cells. These data demonstrate that the protein kinase C inhibitor and antitumor agent, UCN-01 is a potent apoptosis inducer in cell lines that are usually resistant to apoptosis and lack p53 and that caspases and probably serine proteases are activated during UCN-01-induced apoptosis.     

Characterization of apoptotic pathway associated with caspase-independent excision of DNA loop domains

Excision of chromatin loop domains and internucleosomal DNA fragmentation are widely considered as consecutive stages of chromatin disassembly during apoptosis. We report here on apoptosis induced by staurosporine in NB-2a neuroblastoma cells, which was accompanied by excision of chromatin loop domains, but proceeded without internucleosomal DNA cleavage. In contrast to apoptosis associated with internucleosomal DNA fragmentation, the apoptotic pathway associated with excision of chromatin loop domains was largely caspase independent. We identify here MAPK family member, p38/JNK, mitochondria, and topoisomerase II as the components of this caspase-independent apoptotic pathway. While caspase-independent excision of chromatin loop domains was a predominant mechanism of DNA disintegration in staurosporine-treated neuroblastoma, both caspase-dependent internucleosomal DNA fragmentation and caspase-independent excision of chromatin loop domains accompanied staurosporine-induced apoptosis of promyelocytic leukemia cells. Our results suggest that caspase-independent excision of chromatin loop domains represents a separate cell death pathway, which operates either in parallel or independently from caspase-dependent internucleosomal DNA fragmentation.     

Brefeldin A Is a Potent Inducer of Apoptosis in Human Cancer Cells Independently of p53

Brefeldin A (BFA) is a natural product that affects the structure and function of the Golgi apparatus and is in development for cancer chemotherapy. We observed that a wide range of cancer cells could undergo DNA fragmentation associated with apoptosis after BFA treatment. This DNA fragmentation was induced within 15 h in HL60 leukemia cells and after 48 h in K562 leukemia and HT-29 colon carcinoma cells with BFA concentrations as low as 0.1 μM.The DNA fragmentation had the typical internucleosomal pattern in HL60 and HT-29 cells. Apoptotic cells were also detected by microscopy. BFA-induced apoptosis is p53-independent as HL60 and K562 cells are p53 null and HT-29 are p53 mutant cells. BFA could potentiate UCN-01 and staurosporine-induced DNA fragmentation in HL60 cells. Cyclin B1/Cdc2 kinase activity decreased after BFA treatment in HL60 cells, indicating that BFA-induced DNA fragmentation was independent of a cyclin B1/Cdc2 kinase upregulation pathway. Cycloheximide could not prevent BFA-induced DNA fragmentation in HL60 cells, suggesting that protein synthesis is not needed for HL60 cells to undergo apoptosis. On the contrary, cycloheximide blocked BFA-induced DNA fragmentation in HT-29 cells, indicating that apoptosis in HT-29 cells requires macromolecular synthesis. Cell-free system experiments suggested that cytosolic proteins play an important role in triggering DNA fragmentation during apoptosis induced by BFA. Our results show that transduction signaling pathways play central roles in apoptotic regulation.     

Glutathione depletion restores the susceptibility of cisplatin-resistant chronic myelogenous leukemia cell lines to Natural Killer cell-mediated cell death via necrosis rather than apoptosis

We investigated the effect of intracellular glutathione (GSH) levels on Natural Killer-mediated apoptosis in cisplatin-resistant K562 cells. K562/B6 and K562/C9 are cisplatin-resistant K562 cells less susceptible to lysis by natural killer cells. Cisplatin-resistant K562 cells did not present the apoptotic pattern of DNA fragmentation as it was observed for their maternal counterparts. K562/B6 and K562/C9 cell lines produce 1.6- and 1.9-times more GSH than K562 cells. Treatment of both cell lines with D,L-buthionine-(S,R)-sulfoximine (BSO, a gamma-glutamyl cysteine synthetase inhibitor) decreased GSH levels and augmented cell death induced by NK cells via a necrotic rather than an apoptotic process. Proliferating cell nuclear antigen (PCNA) expression was elevated in cisplatin-resistant K562 subclones, and the reduction of GSH levels after treatment with BSO decreased the expression of PCNA. These results suggest that the GSH level affects the NK cell-mediated cell death of cisplatin-resistant K562 cells by inducing necrosis rather than apoptosis.     

Modulation of caspase-3 activity by zinc ions and by the cell redox state

It is known that DNA fragmentation during apoptosis is controlled by a number of factors, a crucial step being the caspase-operated cleavage of ICAD, the DNase inhibitor. We have previously demonstrated that hydrogen peroxide-treated lymphocytes undergo apoptosis without formation of a DNA ladder; however, the use of micromolar amounts of a Zn(2+) chelator allowed DNA cleavage at internucleosomal sites. Such results were extended in the present work, thus allowing their framing into the events related to alterations in the redox state of the cell. Apoptosis in hydrogen peroxide-treated lymphocytes was found to occur with caspase-3 activation, but the enzyme activity was found to be impaired, thus affecting internucleosomal fragmentation as well as nuclear morphology. Caspase-3 activity was found to resume upon mild Zn(2+) chelation. These results provide as well an experimental model from which apoptotic events upstream and downstream of caspase-3 activity can be examined.     

Internucleosomal DNA fragmentation is not obligatory for castration induced rat ventral prostate cell apoptosis in vivo

Castrated male rats were treated with the reversible S1-phase cell cycle blocking drug, mimosine, and the effects of this drug on prostate cell apoptosis was characterized. At a single dose of mimosine (25 mg/kg/day), we found that the internucleosomal DNA fragmentation associated with apoptosis was partially suppressed in the rat ventral prostate at all early time points (24, 48 and 72 h) analyzed post-castration. This suppression was dose-dependent, and treatment with mimosine up to 150 mg/kg/day was sufficient to reduce the internucleosomal DNA fragmentation in the prostate by 90% at 72 h post-castration. Intriguingly, this drug did not suppress the induction of mRNAs for several apoptosis-associated gene products in the ventral prostate gland (bcl-2, p53, TGF-beta and SGP-2/clusterin). Moreover, this treatment did not suppress the histological appearance of apoptotic bodies in the ventral prostate detectable by fast green staining of thin sections of tissue. The apoptotic bodies present in mimosine-treated regressing ventral prostate tissues, however, were refractory to labeling by the in situ gap labeling method, further demonstrating lack of nuclear DNA fragmentation in the condensed nuclei of apoptotic cells. In summary, the cell cycle-blocking drug mimosine does not appear to affect the rate of apoptosis in the regressing rat ventral prostate gland. However, this drug was capable of suppressing the nuclear DNA fragmentation associated with androgen-regulated prostate cell apoptosis. These results support the concept that nuclear DNA fragmentation is not obligatory for apoptosis. Additionally, they imply that cell cycle movement from the G1/S-phase boundary might be important for the terminal DNA degradation associated with androgen-regulated prostate cell apoptosis.     

Activation of caspase-3, proteolytic cleavage of DFF and no oligonucleosomal DNA fragmentation in apoptotic Molt-4 cells

A variety of endonucleases has been implicated in apoptotic DNA fragmentation. DNA fragmentation factor (DFF) is one of the endonucleases responsible for DNA fragmentation. Since an oligonucleosomal DNA ladder is not induced in apoptotic Molt-4 cells, we investigated whether or not the absence of ladder formation is related to an inability of DFF endonuclease in the cells. Semiquantitative RT-PCR analysis showed that the mRNA level of DFF-40 and DFF-45 in Molt-4 cells was approximately the same, compared with in other cells, which exhibit different levels of the fragmentation in apoptosis. When Molt-4 cells were induced to undergo apoptosis by neocarzinostatin (NCS) treatment, both caspase-3 activation and DFF-45 cleavage were observed. Furthermore, DFF immunoprecipitated from Molt-4 cells exhibited DNA degradation activity. These results suggest that functional expression of DFF is not sufficient for the induction of DNA fragmentation in Molt-4 cells.     

Cleavage of Poly(ADP-ribose) polymerase measured in situ in individual cells: relationship to DNA fragmentation and cell cycle position during apoptosis

Poly(ADP-ribose) polymerase (PARP), a nuclear enzyme involved in DNA repair, is a target of caspases during apoptosis: its cleavage onto 89- and 24-kDa fragments is considered to be a hallmark of the apoptotic mode of cell death. Another hallmark is the activation of endonuclease which targets internucleosomal DNA. The aim of the present study was to reveal cell cycle phase specificity as well as the temporal and sequence relationships of PARP cleavage vis-à-vis DNA fragmentation in two model systems of apoptosis, one induced by DNA damage via cell treatment with camptothecin (CPT) (mitochondria-induced pathway) and another by the cytotoxic ligand tumor necrosis factor alpha (TNF-alpha) (cell surface death receptor pathway). PARP cleavage was detected immunocytochemically using antibody which recognizes its 89-kDa fragment (PARP p89) while DNA fragmentation was assayed by in situ labeling of DNA strand breaks. The frequency and extent of PARP cleavage as well as DNA fragmentation were measured by mutiparameter flow and laser scanning cytometry. PARP cleavage, selective to S phase cells, was detected 90 min after administration of CPT. PARP cleavage in the cells treated with TNF-alpha was not selective to any cell cycle phase and was seen already after 30 min. DNA fragmentation trailed PARP cleavage by about 30 min and showed a similar pattern of cell cycle specificity. PARP p89 was present in nuclear chromatin but at least in the early phase of apoptosis it did not colocalize with DNA strand breaks. The rate of cleavage of PARP molecules in individual cells whether induced by CPT or TNF-alpha was rapid as reflected by the paucity of cells with a mixture of cleaved and noncleaved PARP molecules. In contrast, DNA fragmentation proceeded stepwise before reaching the maximal number of DNA strand breaks. Although time windows for PARP cleavage vs DNA fragmentation were different at early stages of apoptosis, a good overall correlation between the cytometric assays of apoptotic cells identification based on these events was observed in both CPT- and TNF-alpha-treated cultures.     

Cytotoxic mechanism of the ribotoxin alpha-sarcin. Induction of cell death via apoptosis.

Alpha-sarcin is a ribosome-inactivating protein that has been well characterized in vitro, but little is known about its toxicity in living cells. We have analyzed the mechanism of internalization of alpha-sarcin into human rhabdomyosarcoma cells and the cellular events that result in the induction of cell death. No specific cell surface receptor for alpha-sarcin has been found. The toxin is internalized via endocytosis involving acidic endosomes and the Golgi, as deduced from the ATP requirement and the effects of NH4Cl, monensin and nigericin on its cytotoxicity. Specific cleavage of 28S rRNA in cultured rhabdomyosarcoma cells, associated with protein biosynthesis inhibition, has been detected. alpha-Sarcin kills rhabdomyosarcoma cells via apoptosis: incubation of cells with alpha-sarcin at a concentration below its IC50 induces internucleosomal genomic DNA fragmentation, reversion of membrane asymmetry, activation of caspase-3-like activity and cleavage of poly(ADP-ribose)polymerase. Apoptosis is not a general direct consequence of protein biosynthesis inhibition, as deduced from the comparative analysis of the effects of alpha-sarcin and cycloheximide; the latter does not induce apoptosis even at concentrations far beyond its IC50, where protein biosynthesis is null. Experiments with a catalytically inactive alpha-sarcin mutant, neither toxic nor apoptotic, reveal that induced apoptosis is directly related to the effects of catalytic activity of the toxin on the ribosomes. The caspase inhibitor z-VAD-fmk does not suppress protein synthesis inhibition by alpha-sarcin. Together, these data suggest that alpha-sarcin-induced caspase activation is a pathway downstream of the 28S rRNA catalytic cleavage and consequent protein biosynthesis inhibition.     

Caspase Activation Is an Early Event in Anthracycline-Induced Apoptosis and Allows Detection of Apoptotic Cells before They Are Ingested by Phagocytes

An increasing number of methods are being described to detect apoptotic cells. However, attempts to detect apoptotic cells in clinical samples are rarely successful. A hypothesis is that apoptotic cells are cleared from the circulation by phagocytosis before they become detectable by conventional morphological or cytometric methods. Using LR73 adhering cells as phagocytes in a model ofin vitrophagocytosis, we found that phagocytosis of daunorubicin (DNR)-treated U937, HL60, or K562 leukemia cell lines occurred prior to phosphatidylserine externalization, DNA hydrolysis, chromatin condensation, nuclear fragmentation, or mitochondrial potential alteration. Moreover DNR-treated K562 cells were eliminated by phagocytes while apoptosis was never observed by any of the above methods. By contrast, using a fluorometric batch analysis assay to detect caspase activity in ceramide- or DNR-treated cells (fluorogenic substrate for caspase), we found that caspase activity increased in apoptosis-committed cells before they were detected by flow cytometry or recognized by phagocytes. Similarly a caspase activity increase was detected in circulating mononuclear cells of leukemic patients 15 h after the beginning of anthracyclin treatment. We suggest that recent findings on enzymatic events (caspase activation) occurring in the early events of apoptosis must now allow the development of new markers for apoptosis, irrespective of the morphological features or internucleosomal fragmentation which are late events in apoptosis.     

Trail-induced apoptosis in Type I leukemic cells is not enhanced by overexpression of bax

We have previously shown that Bax translocation was crucial in TNFalpha or etoposide-induced apoptosis. Overexpression of Bax sensitized chronic myeloid leukemic K562 cells to etoposide-induced apoptosis. Treatment with TNF-related apoptosis-inducing ligand (TRAIL) induces a loss of mitochondrial membrane potential (DeltaPsim), cytochrome c release from mitochondria, activation of caspases-8, -9, and -3, and cleavage of Bid in the K562 cell line. Bax failed to sensitize K562 cells to TRAIL-induced apoptosis. TRAIL did not induce Bax expression and/or translocation from cytosol to mitochondria in the K562 cell line. However, 100 microM Z-VAD.fmk, a pan caspase inhibitor, completely blocked TRAIL-initiated mitochondrial alterations and cleavages of caspases and Bid. We propose that TRAIL-induced apoptosis in K562 cells is via Type I apoptotic signal pathway. Bax translocation is not essential for TRAIL-induced cytochrome c release and DeltaPsim collapse in the Type I cells.     

Rescue of K562 cells from MDM2-modulated p53-dependent apoptosis by growth factor-induced differentiation

The wild-type human MDM2 protooncogene was tested for its ability to modulate apoptotic activity of the de novo expressed p53 tumor suppressor gene in K562 cells. We also studied the role of some cytokines in this phenomenon. K562, a human myeloid leukemia cell line, does not express p53 at the mRNA or protein level. In this study, we stably transfected K562 with eukaryotic vectors containing either normal p53 cDNA (pC53-SN3) or mutated p53 (143Val-->Ala) cDNA (pC53-SCX3). Transfectants expressing WT p53 or those expressing mutant p53 are called K562 SN and K562 SM respectively. Many leukemic cell lines undergo apoptosis when de novo WT p53 is expressed alone. In contrast, while the resulting clones (K562 SN and K562 SM) expressed p53, they did not undergo apoptosis. However, when treated with MDM2 mRNA antisense (MDM2 AS) oligodeoxynucleotides (ODNs), K562 SN demonstrated apoptotic features at both molecular and morphological levels. No change was observed when the other clones (K562 and K562 SM) were treated with MDM2 AS. Apoptosis induced in this manner was associated with a relatively small increase in intracellular calcium [Ca2+]i. Cells cultured in medium previously supplemented with recombinant human (rh) interleukin (IL)-3 and rh-erythropoietin (Epo) did not undergo apoptosis. Moreover, K562 SN cells were induced to differentiate. This differentiation was evaluated by measuring hemoglobin (Hb) level in cellular extracted proteins and by analyzing erythroid colony number and morphology. High Hb synthesis was obtained when K562 SN cells were cultured with cytokines (IL-3 + Epo) combined with MDM2 AS. Our results are consistent with the hypothesis that the function of the proto-oncogene MDM2 is to provide a 'feedback' mechanism for the p53-dependent pathway of apoptosis that could be shunted toward differentiation.     

p53-Independent Apoptosis and p53-Dependent Block of DNA Rereplication Following Mitotic Spindle Inhibition in Human Cells

We have studied the response of human transformed cells to mitotic spindle inhibition. Two paired cell lines, K562 and its parvovirus-resistant KS derivative clone, respectively nonexpressing and expressing p53, were continuously exposed to nocodazole. Apoptotic cells were observed in both lines, indicating that mitotic spindle impairment induced p53-independent apoptosis. After a transient mitotic delay, both cell lines exited mitosis, as revealed by flow-cytometric determination of MPM2 antigen and cyclin B1 expression, coupled to cytogenetic analysis of sister centromere separation. Both cell lines exited mitosis without chromatid segregation. K562 p53-deficient cells further resumed DNA synthesis, giving rise to cells with a DNA content above 4C, and reentered a polyploid cycle. In contrast, KS cells underwent a subsequent G1 arrest in the tetraploid state. Thus, G1 arrest in tetraploid cells requires p53 function in the rereplication checkpoint which prevents the G1/S transition following aberrant mitosis; in contrast, p53 expression is dispensable for triggering the apoptotic response in the absence of mitotic spindle.     

Apicidin, a histone deacetylase inhibitor, induces apoptosis and Fas/Fas ligand expression in human acute promyelocytic leukemia cells.

We previously reported that apicidin arrested human cancer cell growth through selective induction of p21(WAF1/Cip1). In this study, the apoptotic potential of apicidin and its mechanism in HL60 cells was investigated. Treatment of HL60 cells with apicidin caused a decrease in viable cell number in a dose-dependent manner and an increase in DNA fragmentation, nuclear morphological change, and apoptotic body formation, concomitant with progressive accumulation of hyperacetylated histone H4. In addition, apicidin converted the procaspase-3 form to catalytically active effector protease, resulting in subsequent cleavages of poly(ADP-ribose) polymerase and p21(WAF1/Cip1). Incubation of HL60 cells with z-DEVD-fmk, a caspase-3 inhibitor, almost completely abrogated apicidin-induced activation of caspase-3, DNA fragmentation, and cleavages of poly(ADP-ribose) polymerase and p21(WAF1/Cip1). Moreover, these effects were preceded by an increase in translocation of Bax into the mitochondria, resulting in the release of cytochrome c and cleavage of procaspase-9. The addition of cycloheximide greatly inhibited activation of caspase-3 by apicidin by interfering with cleavage of procaspase-3 and DNA fragmentation, suggesting that apicidin-induced apoptosis was dependent on de novo protein synthesis. Consistent with these results, apicidin transiently increased the expressions of both Fas and Fas ligand. Preincubation with NOK-1 monoclonal antibody, which prevents the Fas-Fas ligand interaction and is inhibitory to Fas signaling, interfered with apicidin-induced translocation of Bax, cytochrome c release, cleavage of procaspase-3, and DNA fragmentation. Taken together, the results suggest that apicidin might induce apoptosis through selective induction of Fas/Fas ligand, resulting in the release of cytochrome c from the mitochondria to the cytosol and subsequent activation of caspase-9 and caspase-3.     

16S Mitochondrial Ribosomal RNA Degradation Is Associated with Apoptosis

The use of mitochondrial RNA as an indicator of apoptosis was investigated. Exposure of HA-1 fibroblastic cells to 10 H₂O₂ per 10⁷ cells induced nuclear fragmentation, cell shrinkage, and internucleosomal DNA fragmentation, all characteristics of apoptosis. RNA extracted from control and apoptotic cultures, and analyzed by Northern blot hybridization, revealed a significant increase in the degradation of mitochondrial 16S ribosomal RNA (rRNA) that was associated with apoptosis. Conversely, minimal, if any, degradation of glyceraldehyde-3-phosphate dehydrogenase or actin mRNAs was observed. Similar results were obtained for HA-1 cells treated with the protein kinase inhibitor staurosporine, and for HT-2 T-lymphocytes induced to undergo apoptosis by interleukin-2 withdrawal. In addition, 16S rRNA degradation was an early event that was discernable well before chromatin condensation in hydrogen peroxide-treated HA-1 cells. These observations suggest that degradation of mitochondrial 16S ribosomal RNA is a new marker of mammalian cell apoptosis. © 1997 Elsevier Science Inc.     

Apoptotic DNA fragmentation is not related to the phosphorylation state of histone H1

Changes in chromatin structure, histone phosphorylation and cleavage of DNA into nucleosome-size fragments are characteristic features of apoptosis. Since H1 histones bind to the site of DNA cleavage between nucleosomal cores, the question arises as to whether the state of H1 phosphorylation influences the rate of internucleosomal cleavage. Here, we tested the relation between DNA fragmentation and H1 phosphorylation both in cultured cells and in vitro. In Jurkat cells, hyperosmotic mannitol concentration resulted in apoptosis, including nucleosomal fragmentation, whereas apoptosis induction by increased NaCl concentration was not accompanied by DNA fragmentation. However, both treatments induced dephosphorylation of H1 histones. In contrast, treatment of Raji cells with alkylphosphocholine led to induction of apoptosis with internucleosomal fragmentation, albeit without notable histone H1 dephosphorylation. These results demonstrate that dephosphorylation of H1 histones is neither a prerequisite for nor a consequence of internucleosomal cleavage. Moreover, we observed with an in vitro assay that the known enhancing effect of H1 histones on the activity of the apoptosis-induced endonuclease DFF40 is independent of the subtype or the phosphorylation state of the linker histone.     

Ca<Superscript>2+</Superscript>, Mg<Superscript>2+</Superscript>-dependent DNase Involvement in Apoptotic Effects in Spermatozoa of Sea Urchin <Emphasis Type="Italic">Strongylocentrotus intermedius</Emphasis> Induced by Two-Headed Sphingolipid Rhizochalin

Previously, we have purified three distinct DNases from spermatozoa of sea urchin Strongylocentrotus intermedius and we suppose the role of Ca²⁺, Mg²⁺-dependent DNase (Ca, Mg-DNase) in apoptosis of spermatozoa. Two-headed sphingolipid rhizochalin (Rhz) induced characteristic apoptotic nuclear chromatin changes, internucleosomal DNA cleavage, and activation of caspase-9, caspase-8, and caspase-3 in spermatozoa as was shown by fluorescence Hoechst 33342/PI/FDA analysis, DNA fragmentation assay, and fluorescence caspase inhibitors FAM-LEHD-fmk, FAM-IETD-fmk, and FAM-DEVD-fmk, respectively. Inhibitor of caspase-3 z-DEVD-fmk subdued Rhz-induced internucleosomal ladder formation, which confirmed the major role of caspase-3 in apoptotic DNA cleavage probably through Ca, Mg-DNase activation. Participation of sea urchin Ca, Mg-DNase in apoptosis of spermatozoa was demonstrated by ions Zn²⁺ blocking of Rhz-induced DNA fragmentation due to direct inhibition of the Ca, Mg-DNase and internucleosomal cleavage of HeLa S and Vero E6 cell nuclei chromatin by highly purified Ca, Mg-DNase.