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.     

Higher order structure in metaphase chromosomes

The morphology of metaphase chromosome-derived chromatin fibers released from cells by non-ionic detergent cell lysis in the presence of divalent cations has been studied by electron microscopy. In these preparations the euchromatic arms appear as a series of loops, 200–300 Å in diameter, which are composed of closely-apposed nucleosome arrays. The higher order fiber in chromosomes derived from detergent-lysed cells appears to be less stable than chromatin fibers obtained by mechanical cell lysis. The fiber breaks down into a series of non-uniform nucleosome aggregates (superbeads) and finally to chromatin in a beads-on-a-string morphology upon incubation at 31° for 20 min. These observations allow us to suggest a relationship between uniform thick fibers, superbead-containing fibers, and beads-on-a-string chromatin within metaphase chromosomes.     

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.     

Programmed cell death of secretory cavity cells in fruits of Citrus grandis cv. Tomentosa is associated with activation of caspase 3-like protease

Previously, we found that secretory cell degradation typically occurred through programmed cell death during secretory cavity development in Citrus sinensis L. (Osbeck). This finding indicated that secretory cavities could be utilized as a new cell biology model for investigating the regulatory mechanisms of plant programmed cell death. To study further the programmed cell death during secretory cavity development in Citrus fruit, we studied the morphogenetic characteristics of secretory cavities during their development in Citrus grandis cv. Tomentosa. Using light microscope- and electron microscope-TUNEL assays, immunohistochemistry and immunocytochemistry, we described the precise spatial and temporal alterations in caspase 3-like distribution, chromatin condensation and DNA fragmentation during the programmed cell death of secretory cavity cells. Caspase 3-like was found to be significantly located in both the cytoplasm and the nucleus of secretory cavity cells undergoing programmed cell death, and caspase 3-like is closely associated with chromatin condensation and DNA fragmentation. Interestingly, both caspase 3-like and DNA fragmentation were detected in the nucleoli. Our findings suggest that caspase 3-like may be involved in the programmed cell death of secretory cavity cells, especially in chromatin condensation, DNA fragmentation, nuclear degradation and the degradation of certain organelles.     

Apoptosis and Its Modulation in Human Promyelocytic HL-60 Cells Treated with DNA Topoisomerase I and II Inhibitors

Electron microscopy studies demonstrate unequivocally that the observed oligonucleosome-sized secondary DNA fragmentation in human promyelocytic HL-60 cells treated with the topoisomerase inhibitors camptothecin and teniposide is correlated with the morphological changes in cell structure typical of programmed cell death (apoptosis). Since apoptosis has been associated with potential involvement of intracellular signaling linked to the Ca²⁺/calmodulin and protein kinase C transduction pathways, we also investigated the effects of signaling modulators on camptothecin- and teniposide-induced secondary DNA fragmentation in HL-60 cells. Neither calcium chelators, calcium/calmodulin inhibitors (calmidazolium or cyclosporine A), protein kinase C stimulation by TPA, protein phosphatase inhibition by okadaic acid, protein kinase inhibition by staurosporine, calphostin C, genistein or H7, nor cell cycle alterations by caffeine had any detectable effect. Interestingly, most of these intracellular signaling modulators were able to induce DNA fragmentation in HL-60 cells by themselves. These results may suggest that even though modulation of these signaling pathways was unable to prevent topoisomerase inhibitor-induced apoptosis, their sole deregulations could induce apoptosis in HL-60 cells. In contrast, aphidicolin blocked camptothecin-induced secondary DNA fragmentation, indicating that replication-induced DNA damage is required for camptothecin- but not teniposide-induced secondary DNA fragmentation. Zinc, 3-aminobenzamide, and spermine also modulated both camptothecin- and teniposide-induced secondary DNA fragmentation without significant alteration of topoisomerase-mediated primary DNA strand breaks. Hence, poly(ADP-ribosyl)ation and chromatin structure may be important in modulating oligonucleosomesized DNA fragmentation associated with apoptosis in HL-60 cells treated with topoisomerase inhibitors.     

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.     

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.     

Distinction of apoptotic and necrotic cell death by in situ labelling of fragmented DNA

The occurrence and spatial distribution of intracellular DNA fragmentation was investigated by in situ 3 end labelling of DNA breaks in K562 cells treated in such a way to cause either apoptotic or necrotic cell death. The localisation of DNA breaks was examined by confocal laser microscopy and compared with the electron-microscopic appearance of the cells. In addition, the number of cells with fragmented DNA was counted and compared with the number of dead cells, as determined by the nigrosin dye exclusion test. Apoptosis was induced by cultivation of the cells in the presence of actinomycin D. Cells undergoing apoptosis were characterised by massive intracellular DNA fragmentation that was highly ordered into successive steps. Cells in early stages of the apoptotic process had DNA breaks diffusely distributed in the entire nucleus, except the nucleolus, with crescent-like accumulations beyond the nuclear membrane. In the more advanced stages, the nucleus was transformed into many round bodies with intense labelling. Intracellular accumulations of fragmented DNA corresponded exactly to electron-dense chromatin seen in the electron microscope, whereas diffuse DNA breaks had no morphological correlate at the ultrastructural level. In necrosis induced by ionomycin, NaN₃, or rapid freezing combined with thawing, no DNA fragmentation occurred at the onset of cell death, but appeared 24 h later. This fragmentation was not characterised by a unique morphology, but represented the breakdown of the chromatin in the configuration remaining after cell death. Therefore, apoptosis is characterised by DNA fragmentation that proceeds in a regular orderly sequence at the beginning of cell death, and can be detected by in situ 3end labelling of DNA breaks.     

Evidence of apoptosis in some cell types due to pentachlorophenol (PCP) in Heteropneustes fossilis

The study aimed to clarify the role of apoptosis in pentachlorophenol (PCP) induced testicular, ovarian and renal cell genotoxicity of Heteropneustes fossilis. It was further intended to find the target germ cell type and assess the cellular and nuclear damage. Treatment of PCP was used for multiduration on the germinal tissues and they were processed to detect structural changes by light and electron microscopic evaluation and kidney cells for subsequent detection of DNA fragmentation by agarose gel electrophoresis. Findings suggest functional and morphological changes in the tissues are due to apoptosis, as evidenced by some biochemical and cytological signs. Histological observation on germinal epithelium reveals cell suicidal symptoms such as vacuolization, liquefied regions in the cytoplasm of oocytes, margination of nuclei, clumping of chromatin, and compaction of cytoplasmic organelle. Biochemical manifestation concurrent to this, is; cleavage of kidney cell DNA into low molecular weight fragments confirming apoptosis. Subsequently, it is further cleaved into nucleosome size fragments or its multiples. Ultra-structural histopathology and DNA studies conclusively lead to the PCP induced apoptosis in the exposed cell types. Results further support the usefulness of this assay in the related studies and its feasibility in generating a base line data.     

Distinct mechanisms underlay DNA disintegration during apoptosis induced by genotoxic and nongenotoxic agents in neuroblastoma cells

Exposure of mouse NB-2a neuroblastoma cells to genotoxic (etoposide or cytosine arabinoside) or nongenotoxic challenges (serum deprivation or okadaic acid) resulted in progressive cell death with biochemical and morphological characteristics typical of apoptosis. Apoptotic cell death induced by nongenotoxic agents was associated with the disintegration of nuclear DNA into high molecular weight (HMW) and oligonucleosomal-DNA fragments, while the formation of HMW-DNA fragments, but not oligonucleosomal-DNA ladder accompanied apoptosis induced by genotoxic agents. Combination of genotoxic and nongenotoxic insults, i.e. incubation of etoposide-treated cells in the serum-free medium, resulted in an additive effect on the profile of DNA disintegration, which involved both HMW fragmentation pattern as in etoposide alone treated cells and the oligonucleosomal-DNA ladder observed with serum-deprived cells. On the other hand, incubation of serum-deprived cells in the presence of Zn²⁺-ions led to the abrogation of internucleosomal DNA fragmentation but accumulation of HMW-DNA fragments. Differences in the pattern of DNA fragmentation were reproducible in a cell free apoptotic system after treatment of isolated normal nuclei with cytosolic extracts prepared from the cells treated with genotoxic or nogenotoxic apoptotic inducers. Cell free experiments also revealed that activities responsible for the formation of HMW- and oligonucleosomal-DNA fragments are separable in cytosolic extract prepared from the serum-deprived cells. Finally, DNA fragmentation induced by nongenotoxic apoptotic inducers was effectively prevented by cycloheximide and suramin, while both cycloheximide and suramin had only a slight inhibitory effect on DNA fragmentation induced by genotoxic agents. The results presented suggest that distinct pathways underlay disintegration of nuclear DNA during apoptosis induced by genotoxic and nongenotoxic inducers, and that the formation of HMW- and oligonucleosomal-DNA fragments proceeds via separate mechanisms in NB-2a neuroblastoma cells.     

Mutation of the cohesin related gene PDS5 causes cell death with predominant apoptotic features in Saccharomyces cerevisiae during early meiosis

Pds5p is a cohesin related protein. It is required for maintenance of sister chromatid cohesion in mitosis and meiosis. Here we report that pds5-1 causes cell death in yeast Saccharomyces cerevisiae during early meiosis. The pds5-1 caused cell death possesses characteristics of apoptosis and necrosis, including externalization of phosphatidylserine at cytoplasmic membrane, accumulation of DNA breaks, chromatin condensation and fragmentation, nuclei fragmentation, membrane degeneration and cell size enlargement. Our results also suggest that (1) The defect of DNA repair; (2) The production of reactive oxygen species, in pds5-1 mutant are involved in pds5-1 induced cell death.     

DNA loop organization and DNA fragmentation during radiation-induced apoptosis in human lymphocytes

Apoptotic DNA fragmentation induced by gamma-rays has been compared with the DNA loop sizes in G0-human lymphocytes using pulsed field gel electrophoresis (PFGE). Genomic DNA was cleaved into the DNA loops at the topoisomerase II mediated attachment points using short treatment of cells with etoposide. The apoptotic fragmentation, with a distinct cut-off around 50 kb for a maximum length of fragments, appeared 5 h after irradiation when the most part of radiation-induced DNA double strand breaks (DSBs) have been repaired. The data indicate that apoptotic fragmentation of DNA in the G0-human lymphocytes begins when repair of radiation-induced DSBs has been completed. Similar apoptotic DNA fragmentation was also observed following the treatment of cells with etoposide. All genomic DNA was fragmented into 50-kb fragments during the final stages of apoptosis. Most of the DNA in resting lymphocytes is organized into Mb-size loops but loops of sizes down to 50 kb were also observed. A sharp border between the size distributions of DNA loops and apoptotic fragments was found. The data suggest that 50 kb apoptotic fragmentation is not based on excision of the DNA loops. No apoptotic fragments with the sizes more than 5.7 Mb were seen during the whole course of apoptosis. This observation indicates that despite intensive apoptotic fragmentation into the 50-kb fragments the chromosomes maintain integrity during radiation-induced apoptosis in human lymphocytes. We propose a model for radiation-induced apoptotic fragmentation in human lymphocytes that involves four stages: induction of DNA breaks and relaxation of DNA loops; DNA repair followed by reorganization of the DNA loops into the 50-kb units of condensed chromatin; co-operative fragmentation of the reorganized DNA loops into the distinct 50-kb fragments and resealing of the chromosome ends at the sites of this fragmentation; cleavage of the 50-kb fragments at the internucleosomal spacers.     

维甲酸诱导的人大肠癌细胞凋亡

本研究应用光镜、电镜技术、DNA凝胶电泳、流式细胞术及末端脱氧核苷酰转移酶原位标记(TUNEL法),观察全反式维甲酸ATRA诱导的人大肠癌CCL229细胞凋亡特征。RA诱导CCL229细胞凋亡,光、电镜下观察到凋亡小体形成等典型的形态学改变,琼脂糖凝胶电泳上呈现特征性的DNA ladder,DNA直方图上显示亚二倍体峰。10-8mol/L-105mol/L范围内,RA诱导CCL229细胞凋亡表现出时间和剂量依赖性。     

Nick-forming sequences may be involved in the organization of eukaryotic chromatin into ∼50 kbp loops

Phenomena involving the disassembly of chromosomes to ∼50 kbp double-stranded fragments upon protein denaturing treatments of normal and apoptotic mammalian nuclei as well as yeast protoplasts may be an indication of special, hypersensitive regions positioned regularly at loop-size intervals in the eukaryotic chromatin. Here we show evidence in yeast cell systems that loop-size fragmentation can occur in any phase of the cell cycle and that the plating efficiency of these cells is ∼100%. The possibility of sequence specificity was investigated within the breakpoint cluster region (bcr) of the human MLL gene, frequently rearranged in certain leukemias. Our data suggest that DNA isolated from yeast cultures or mammalian cell lines carry nicks or secondary structures predisposing DNA for a specific nicking activity, at non-random positions. Furthermore, exposure of MLL bcr-carrying plasmid DNA to S1 nuclease or nuclear extracts or purified topoisomerase II elicited cleavages at the nucleotide positions of nick formation on human genomic DNA. These data support the possibility that certain sequence elements are preferentially involved in the cleavage processes responsible for the en masse disassembly of chromatin to loop-size fragments upon isolation of DNA from live eukaryotic cells.     

Nucleosome positioning as a critical determinant for the DNA cleavage sites of mammalian DNA topoisomerase II in reconstituted simian virus 40 chromatin.

We have assessed the ability of nucleosomes to influence the formation of mammalian topoisomerase II-DNA complexes by mapping the sites of cleavage induced by four unrelated topoisomerase II inhibitors in naked versus nucleosome-reconstituted SV40 DNA. DNA fragments were reconstituted with histone octamers from HeLa cells by the histone exchange method. Nucleosome positions were determined by comparing micrococcal nuclease cleavage patterns of nucleosome-reconstituted and naked DNA. Three types of DNA regions were defined: 1) regions with fixed nucleosome positioning; 2) regions lacking regular nucleosome phasing; and 3) a region around the replication origin (from position 5100 to 600) with no detectable nucleosomes. Topoisomerase II cleavage sites were suppressed in nucleosomes and persisted or were enhanced in linker DNA and in the nucleosome-free region around the replication origin. Incubation of reconstituted chromatin with topoisomerase II protected nucleosome-free regions from micrococcal nuclease cleavage without changing the overall micrococcal nuclease cleavage pattern. Thus, the present results indicate that topoisomerase II binds preferentially to nucleosome-free DNA and that the presence of nucleosomes at preferred DNA sequences influences drug-induced DNA breaks by topoisomerase II inhibitors.     

Chromatin condensation of ovarian nurse and follicle cells is regulated independently from DNA fragmentation during Drosophila late oogenesis

Programmed cell death constitutes a common fundamental incident occurring during oogenesis in a variety of different organisms. In Drosophila melanogaster, it plays a significant role in the maturation process of the egg chamber. In the present study, we have used an in vitro development system for studying the effects of inducers and inhibitors of programmed cell death during the late stages of oogenesis. Treatment of the developing egg chambers with two widely used inducers of cell death, etoposide and staurosporine, blocks further development and induces chromatin condensation but not DNA fragmentation in nurse and follicle cells, as revealed by propidium iodide staining and terminal transferase-mediated dUTP nick-end labeling assay. Moreover, incubation of the developing egg chambers with the caspase-3 inhibitor Z-DEVD-FMK significantly delays development, prevents DNA fragmentation, but does not affect chromatin condensation. The above results demonstrate, for the first time, that chromatin condensation in Drosophila ovarian nurse and follicle cells is a caspase-3-like independent process and is regulated independently from DNA fragmentation.     

Large-scale fragmentation of dna and the death of tumor cells by the action of the binary system ascorbic acid-metallocomplexes of cobalt in vitro

High-molecular-weight DNA fragments are the markers of the early stage of apoptosis induced in eukaryotic cells by cytotoxins of different nature. The dynamics of the development of large-scale DNA fragmentation in K-562 leukemia cells by the action of the antitumor drug, the binary system ascorbic acid--cobalt phthalocyanine within 48 h of incubation, which correspond to two periods of the doubling of cell number in growing control cultures, have been studied. It was shown that, within the first hours of incubation, hydrogen peroxide generated by the system induces the formation of DNA fragments from 2200 to 50 kbp long. Later on the cell death accompanied by a decrease in the content of fragmented DNA is observed. Within 24 h of incubation, part of fragmented DNA remains unrepaired; after 48 h of incubation, a delay or a slowed down proliferation of K-562 cells, which differ from control cells also by a high level of death and a higher content of high-molecular-weight DNA fragments, is observed.     

Accumulation of DNA strand breaks during thymineless death in thymidylate synthase-negative mutants of mouse FM3A cells

Thymidylate synthase-negative mutants of cultured mouse cells were immediately committed to cell death upon thymidine deprivation, especially when the cells were synchronized in the S phase. Thymidylate deprivation induced single strand breaks in chromosome-size DNA strands, as measured by alkaline sucrose gradient sedimentation, giving rise to two peaks, one with large and the other with small fragments, the latter about the size of T4 DNA. An increase in the small DNA fragments paralleled that of thymineless death. Thymidine deprivation also produced double strand DNA fragments as determined by a method of neutral filter elution, and their extent paralleled that of cell death. Double-stranded DNA eluted through the filter sedimented as a single peak both in a neutral and in an alkaline sucrose gradient that coincided with that of the above small DNA fragments. Therefore, the strand breaks seemed to occur in some defined portions of the genome and in a specific manner compared to breaks induced by x-rays, which occurred rather randomly. Cycloheximide blocked both thymineless death and the production of the small DNA fragments. The strand breaks induced by thymidine starvation were not repaired but instead advanced on subsequent incubation of the cells in growth medium containing thymidine.     

UV-B Irradiated Cell Lines Execute Programmed Cell Death in Various Forms

We investigated changes typical for apoptosis in various cell lines after UV-B irradiation. Using established methods for detection of apoptosis we demonstrate changes of cellular morphology, phosphatidylserine (PS) exposure, ollgonucleosomal DNA fragmentation and generation of hypochrome nuclei. To isolated high-molecular-weight (hmwt) DNA fragments we engaged a new method avoiding pulse field gel electrophoresis. Most UV-B irradiated cell lines showed oligonucleosomal DNA fragmentation, hypochrome nuclei, morphological changes, annexin-V binding and positive TUNEL reaction. However, no oligonucleosomal DNA fragmentation could be detected in Raji and HaCaT cells. Whereas HaCaT cells displayed all other changes typical for apoptosis, Raji cells were TUNEL negative, formed low amounts of hmwt DNA and showed an 'atypically' low hypochrome shift. Nevertheless, UV-B irradiated Raji cells excluded propidium iodide (PI), bound annexin-V and stopped proliferation. This suggests that Raji cells underwent growth arrest with exposure of PS being the only feature of apoptosis. However, in the presence of phagocytes expressing the phosphatidylserine receptor these cells would share the removal pathway with apoptotic cells. Since UV-B induced programmed cell death differs in dependence of cells under investigation, the failure to detect oligonucleosomal DNA fragmentation or chromatin condensation is not suitable to exclude programmed (apoptotic?) cell death.