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PubMed2005

Sulindac activates nuclear translocation of AIF, DFF40 and endonuclease G but not induces oligonucleosomal DNA fragmentation in HT-29 cells.

Sulindac is one of the most widely studied nonsteroidal anti-inflammatory drugs in the prevention of colon cancer. Thus, from the viewpoint of colon cancer chemotherapy it is important to reveal the mechanism of sulindac-induced cell death. This study was undertaken to dissect the molecular mechanism underlying sulindac-induced apoptosis in human colon cancer cell line HT-29 (mutant p53), focusing on nuclear translocation of AIF, DFF and endonuclease G. On induction of apoptosis by sulindac, it was associated with decreased mitochondrial membrane potential, nuclear expression of active caspase-3, cleavage of poly(ADP-ribose) polymerase, translocation of mitochondrial proteins to the nucleus, and morphological evidence of nuclear condensation. However, sulindac led to only disintegration of nuclear DNA into high molecular weight DNA fragments of about 100-300 kbp as determined by a pulse-field gel electrophoresis, suggesting a predominantly AIF-mediated cell death process. In summary, our findings indicate that sulindac induces large-scale DNA fragmentation without oligonucleosomal DNA fragmentation. This result suggests that nuclear translocation of DFF and endonuclease G are not sufficient for the induction of oligonucleosomal DNA fragmentation in HT-29 cells.

Active Transport, Cell Nucleus↗
PubMed1994

Structural analyses of DNA fragments integrated by illegitimate recombination in Schizosaccharomyces pombe.

In order to elucidate the mechanisms of illegitimate recombination in eukaryotes, we have studied the structure of DNA fragments integrated by illegitimate recombination into the genome of fission yeast. Nonhomologous recombination was rarely identified when a long region of homology with the chromosomal leu1+ gene was present in the introduced leu1::ura4+ DNA fragment; but a decrease in length of homology leads to an increase in the ratio of non-homologous to homologous recombination events. The introduced DNA fragments were integrated into different sites in the chromosomes by nonhomologous recombination. The results suggested that there are multiple modes of integration; most events simply involve both ends of the fragments, while in other cases, fragments were integrated in a more complicated manner, probably via circularization or multimerization. To analyze the mechanism of the major type of integration, DNA fragments containing the recombination junctions of three recombinants were amplified by inverted polymerase chain reaction (IPCR) and their nucleotide sequences were determined. There was no obvious homology between introduced DNA and chromosomal DNA at these recombination sites. Furthermore it was found that each terminal region of the introduced DNA was deleted, but that there were no or very small deletions in the target sites of chromosomal DNA. Two models are proposed to explain the mechanism of nonhomologous integration.

Base Sequence↗
PubMed1989

Cloning and sequence analysis of the genomic DNA fragment encoding oryzacystatin.

A genomic DNA clone encoding oryzacystatin (Oc), a cysteine proteinase inhibitor (cystatin) of rice, was isolated from a lambda EMBL3 phage library constructed with Sau3AI partial digests of rice chromosomal DNA, by screening with an oc cDNA as a probe. The restriction map of the isolated DNA fragment was consistent with the pattern of the genomic Southern-blot analysis using a cDNA probe, and consequently, the gene is considered to be a single-copy gene. The oc gene is about 1.4 kb long and composed of three exons and two introns. The first intron (336 bp) intervenes between Ala-38 and Asp-39. The second intron (372 bp) exists in the 3'-noncoding region at the G residue next to the stop codon. S1 nuclease mapping showed the major transcription start point (tsp) at A, 104 bp upstream from the start codon (ATG). Typical CAT and TATA box sequences were found in the 5'-upstream region of the tsp. The nucleotide sequences around the TATA box, the tsp, the start codon, and the stop codon essentially matched the consensus sequences of other higher plant genes. The intron boundaries of the oc gene were quite different from those of the human kininogen-encoding gene and the human salivary cystatin (cystatin S)-encoding gene.

Amino Acid Sequence↗
PubMed1983

The influence of agarose--DNA affinity on the electrophoretic separation of DNA fragments in agarose gels.

The effects of DNA concentration, buffer composition, added "carrier" DNA, and chemical modification of agarose on the electrophoretic separation of DNA restriction fragments in agarose gels were tested. Electrophoretic zones of migrating DNA were found to broaden by trailing as sample load was decreased, and this effect was found to be more pronounced for species of higher molecular weight. As DNA sample load was increased, DNA fragments were found to move faster in the direction of electrophoresis (front forward). Sharp, well-resolved electrophoretic zones were obtained at very low DNA loads only when a high-salt, high-pH, high-EDTA buffer was employed or when "carrier DNA" having a broad and uniform molecular weight distribution was included in the sample. Moreover, DNA in high concentration was found to displace DNA in low concentration from a given gel region. Unmodified agaroses were found to differ only slightly in their effectiveness in retarding DNA fragments at a given agarose concentration. However, hydroxyethylated agarose was much more effective in retarding DNA, at a given gel concentration, than the unmodified agaroses tested. These results show that it is useful to consider the agarose gel matrix as possessing the properties of both a molecular sieve and a chromatographic adsorbent when designing electrophoretic separation techniques for DNA. A model for these separations which includes the effects of DNA-agarose interaction and molecular sieving is discussed.

Buffers↗
PubMed1994

[Complementary-addressed photomodification of DNA-targets by arylazide and perfluoroarylazide oligonucleotide derivatives. IV. Photomodification of ss- and ds-DNA-fragments].

A highly efficient sequence-specific photomodification of single stranded (ss) and double stranded (ds) DNA fragments was carried out with hexadecathymidilate derivative, R-p(T)16(R--p-azidotetrafluorobenzamide) and 27-meric DNA fragments as a targets. [formula: see text] The main points of the modification were G7 and G24 for the ss target and G7 and G22 of purine- and pyrimidine-rich strands, respectively, for the ds DNA fragment. The photomodification extent was 60-77% for ss DNA and 10-53% for ds DNA depending on the reaction conditions: it increased in a buffer with a high ionic strength (1.0 M) and at a low temperature (4 degrees C) when the triplexes are more stable.

Azides↗
PubMed1993

Coinduction of c-jun gene expression and internucleosomal DNA fragmentation by ionizing radiation.

Previous work has demonstrated that the cellular response to ionizing radiation includes transcriptional activation of the c-jun early response gene. The present studies demonstrate that this induction of c-jun expression is temporally related to the appearance of internucleosomal DNA fragmentation. These events were maximal at 6 h and transient after exposure to lethal doses (20 Gy) of ionizing radiation. We also demonstrate that N-acetyl-L-cysteine (NAC), an antioxidant, inhibits X-ray-induced c-jun expression and endonucleolytic DNA cleavage. These findings suggested that both events are mediated at least in part through the formation of reactive oxygen intermediates (ROIs). Since ROIs damage DNA and X-ray-induced DNA damage is associated with activation of poly(ADP-ribose) polymerase (ADPRP), we studied the effects of the ADPRP inhibitors 3-aminobenzamide (3-AB), nicotinamide, and theophylline. 3-AB blocked both X-ray-induced c-jun expression and internucleosomal DNA fragmentation. Similar findings were obtained with nicotinamide and theophylline. In contrast, 3-AB had little if any effect on induction of c-jun transcripts or DNA fragmentation induced by the alkylating agent mitomycin C. While c-jun expression is restricted to cells in G1 and G1/S phases, we have found that X-ray-induced c-jun transcripts are detectable throughout all phases of the cell cycle. The induction of internucleosomal DNA fragmentation by X-rays was also detectable throughout the cell cycle. Taken together, these results support the coinduction of c-jun transcription and internucleosomal DNA fragmentation by ionizing radiation. Similar studies were performed with H2O2 since this agent also results in the production of ROIs.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Line↗
PubMed2000

Intermediate filaments reconstituted from vimentin, desmin, and glial fibrillary acidic protein selectively bind repetitive and mobile DNA sequences from a mixture of mouse genomic DNA fragments.

Employing the whole-genome PCR technique, intermediate filaments (IFs) reconstituted from vimentin, desmin, and glial fibrillary acidic protein were shown to select repetitive and mobile DNA sequence elements from a mixture of mouse genomic DNA fragments. The bound fragments included major and minor satellite DNA, telomere DNA, minisatellites, microsatellites, short and long interspersed nucleotide elements (SINEs and LINEs), A-type particle elements, members of the mammalian retrotransposon-like (MaLR) family, and a series of repeats not assignable to major repetitive DNA families. The latter sequences were either similar to flanking regions of genes; possessed recombinogenic elements such as polypurine/polypyrimidine stretches, GT-rich arrays, or GGNNGG signals; or were characterized by the distribution of oligopurine and pyrimidine motifs whose sequential and vertical alignment resulted in patterns indicative of high recombination potentials of the respective sequences. The different IF species exhibited distinct quantitative differences in DNA selectivities. Complexes consisting of vimentin IFs and DNA fragments containing LINE, (GT)(n) microsatellite, and major satellite DNA sequences were saturable and dynamic and were formed with high efficiency only when the DNAs were partially denatured. The major-groove binder methyl green exerted a stronger inhibitory effect on the binding reaction than did the minor-groove binder distamycin A; the effects of the two compounds were additive. In addition, DNA footprinting studies revealed significant configurational changes in the DNA fragments on interaction with vimentin IFs. In the case of major satellite DNA, vimentin IFs provided protection of the T-rich strand from cleavage by DNase I, whereas the A-rich strand was totally degraded. Taken together, these observations suggest that IF protein(s) bind to double-stranded DNAs at existing single-stranded sites and, taking advantage of their helix-destabilizing potential, further unwind them via a cooperative effort of their N-terminal DNA-binding regions. A comparison of the present results with literature data, as well as a search in the NCBI database, showed that IF proteins are related to nuclear matrix attachment region (MAR)-binding proteins, and the DNA sequences they interact with are very similar or even identical to those involved in a plethora of DNA recombination and related repair events. On the basis of these comparisons, IF proteins are proposed to contribute in a global fashion, not only to genetic diversity, but also to genomic integrity, in addition to their role in gene expression.

Animals↗
PubMed1996

DNA fragmentation induced by protease activation in p53-null human leukemia HL60 cells undergoing apoptosis following treatment with the topoisomerase I inhibitor camptothecin: cell-free system studies.

We studied the role of proteases in apoptosis using a cell-free system prepared from a human leukemia cell line. HL60 cells are p53 null and extremely sensitive to a variety of apoptotic stimuli including DNA damage induced by the topoisomerase I inhibitor, camptothecin. We measured DNA fragmentation induced in isolated nuclei by cytosolic extracts using a filter elution assay. Cytosol from camptothecin-treated HL60 cells induced internucleosomal DNA fragmentation in nuclei from untreated cells. This fragmentation was suppressed by serine protease inhibitors. Serine proteases (trypsin, endoproteinase Glu-C, chymotrypsin A, and proteinase K) and papain by themselves induced DNA fragmentation in naive nuclei. This effect was enhanced in the presence of cytosol from untreated cells. Cysteine protease inhibitors (E-64, leupeptin, Ac-YVAD-CHO [ICE inhibitor]) did not affect camptothecin-induced DNA fragmentation. The apopain/Yama inhibitor, Ac-DEVD-CHO, and the proteasome inhibitor, MG-132, were also inactive both in the cell-free system and in whole cells. Interleukin-1 beta converting enzyme (ICE) or human immunodeficiency virus protease failed to induce DNA fragmentation in naive nuclei. Together, these results suggest that DNA damage activates serine protease(s) which in turn activate(s) nuclear endonuclease(s) during apoptosis in HL60 cells.

Apoptosis↗
PubMed1996

Apparent inhibition of apoptosis by polyamines and aminothiols in DNA fragmentation assays is artifactual.

We report that, in commonly used DNA fragmentation assays, polyamines and the radioprotective aminothiol WR1065 artifactually depress the degree of spontaneous or induced cellular apoptosis in two distinct ways. Firstly, in assays utilizing Hoechst 33258 dye to measure apoptotic DNA, both amines quench the fluorescence of low affinity dye/DNA binding resulting in preferential underestimation of DNA in the apoptotic DNA fraction and a resultant underestimation of the extent of DNA fragmentation. Secondly, these amines can cause aggregation and condensation of apoptotic DNA, causing anomalous sedimentation under conditions universally employed to separate apoptotic from intact DNA in cell lysates. This anomalous sedimentation of apoptotic DNA leads to underestimation of fragmentation in fluorescence assays as well as in agarose gel assays. We demonstrate that manipulation of the ionic strength of the lysis buffer or lowering the dye concentration ameliorates the effects of dye quenching in the Hoechst assay. Alternatively, this effect is alleviated by substituting DAPI for Hoechst in this assay. Inclusion of a polyanion to the lysis buffer antagonizes the condensation and anomalous sedimentation of apoptotic DNA observed regardless of which dye is used in the assay. These studies call into question the validity of previously reported studies suggesting that polyamines and the radioprotective aminothiol, WR1065, inherently suppress the apoptotic process and underline the need to consider alternative endpoints of apoptosis such as morphology in order to assess effects on cellular apoptosis of exogenously added agents, particularly di- or polycations.

Journal Article↗
PubMed1997

[Isolation and identification of DNA fragments frequently deleted from tumor cell lines of human esophageal cancer].

OBJECTIVE: To clone tumor suppressor genes associated with human esophageal cancer (EC). METHODS: DNA fragments deleted from cell lines of esophageal cancer EC8712 and EC8733 were isolated by using modified genomic substractive hybridization. Twenty EC tissues paired with adjacent normal mucosa were analysed by Southern blot and PCR with these fragments as probes. RESULTS: Seven DNA fragments deleted from EC cell lines obtained were named as 12H1, 12H2, 33H3, 33H4, 12B1, 33B2 and 33B3. Sequencing revealed their size of 512bp, 428bp, 509bp, 355bp, 680bp (partially sequencing), 519bp and 425bp, respectively. 12H1 and 33H3 showed high homology (98%) to a new repeated sequence in human genome. 33H4 displayed 87% nucleotide identity with human alphoid-like repetitive sequence located on chromosome 17. 12B1 was homologous (90%) to LINE 1 transoposon containing two open reading frames. 12H2, 33B2 and 33B3 were novel sequences. These DNA fragments were deleted in 20%-60% of EC tissues and in 10%-20% of adjacent mucosa. CONCLUSIONS: Loss of 33H4 may be accompanied by deletion of an adjacent gene. 12B1 as transposon may suppress the tumorigenesis by activating suppressor genes or/and by inactivating oncogenes. 12H1/33H3, 12H2, 33B2 and 33B3 may be candidates of tumor suppressor genes associated with the development and the progression of esophageal cancer.

Cloning, Molecular↗
PubMed1988

Correlation between the anticellular and DNA fragmenting activities of tumor necrosis factor.

The treatment of cells sensitive to the anticellular effect of tumor necrosis factor (TNF) with TNF results in a degradation of their cellular DNA into DNA fragments that are multiples of 200 base pairs. TNF treatment of cells resistant to the anticellular effect of TNF, but bearing receptors for TNF, fails to result in any DNA fragmentation. Incubation conditions, such as temperature, the presence of metabolic inhibitors or amino acid deprivation, that modulate the effectiveness of TNF or affect the rate at which TNF exerts its anticellular effect have a similar effect on the ability of the TNF to generate DNA fragments. Thus the TNF-mediated DNA fragmentation and the rate at which it occurs correlates with the rate at which cells respond to the anticellular effect of TNF and, as such, might serve as a marker for the responsiveness of cells to TNF.

Antineoplastic Agents↗
PubMed2002

Assessment of DNA fragmentation of spermatozoa that were surgically retrieved from men with obstructive azoospermia.

OBJECTIVE: To determine the degree of DNA fragmentation in spermatozoa of men with obstructive azoospermia or anejaculation compared with that of ejaculated spermatozoa from fertile donors. DESIGN: Observational study. SETTING; University Medical Center St. Radboud, Nijmegen. The Netherlands. PATIENT(S): Forty-one patients with obstructive azoospermia or anejaculation and 10 fertile donors. MAIN OUTCOME MEASURE(S): Sperm samples were obtained surgically from the epididymis or testis of men with azoospermia or anejeculation and by ejaculation in fertile patients. DNA fragmentation was analyzed in the total sample and in a motile fraction that was isolated as in routine ICSI procedures. DNA breaks were measured by using the TdT-mediated dUTP nick-end labeling assay. RESULT(S): A higher percentage of cells with DNA breaks was found in men with obstructive azoospermia or anejaculation compared with donors (mean, 18.9% vs. 6.2%). A significant lower degree of DNA fragmentation was observed in the motile fraction from patients compared with donors (0.4% vs. 0.6%). CONCLUSION(S): High percentages of cells with DNA damage were found in sperm samples from men with obstructive azoospermia or anejaculation, but a very low frequency of damage to the DNA was observed in the motile fraction. In an ICSI setting, the use of motile sperm retrieved from epididymis or testis of men with obstructive azoospermia does not seem to pose a higher genetic risk to the progeny than does use of motile ejaculated sperm.

DNA Fragmentation↗
PubMed1995

High molecular weight DNA fragmentation: a critical event in nucleoside analogue-induced apoptosis in leukemia cells.

Cleavage of DNA into internucleosomal fragments is one of the characteristics of apoptosis. However, searches for in vivo evidence of nucleosomal DNA fragmentation in leukemia cells freshly obtained from patients during chemotherapy frequently failed to reveal nucleosomal multimers (DNA ladders). It is not clear whether this type of DNA cleavage is an essential event in drug-induced apoptosis and thus a denominator of cell killing, or whether the internucleosomal DNA fragments are merely the by-products of the apoptotic process. Here, we report our investigation into the role of DNA fragmentation in apoptotic cell death induced by anticancer nucleoside analogues, both in cell culture and in leukemia patients undergoing chemotherapy. Using a 5'-end DNA-labeling technique and pulsed field gel electrophoresis, we detected fragmentation of DNA in two distinct size classes, internucleosomal and high molecular weight (predominantly 50 kb) DNA fragments, in a human leukemia cell line exposed to the nucleoside analogues fludarabine and gemcitabine. We further demonstrated that the two types of DNA fragmentation were separate events, distinguishable by their requirements for Ca2+ and responses to phorbol ester treatment. The drug-treated cells underwent morphological changes of apoptosis even after internucleosomal DNA fragmentation was selectively inhibited by intracellular Ca2+ chelation, or by treatment with phorbol ester. In contrast, neither apoptotic morphology nor internucleosomal DNA fragmentation was observed when the high molecular weight DNA fragmentation was blocked by inhibition of nucleoside analogue incorporation into DNA. These results suggest that cleavage of DNA into large fragments may be an initial event that is critical for drug-induced apoptosis, whereas activation of a Ca2+-dependent endonuclease to cleave DNA at internucleosomal sites is not an absolute requirement for the execution of the apoptotic cell death program. Further studies of leukemic lymphocytes obtained from 9 patients with chronic lymphocytic leukemia during therapy with fludarabine revealed high molecular weight DNA fragmentation, which was correlated with a decrease of peripheral lymphocytes in 6 patients, whereas only 1 of the 15 patients evaluated for nucleosomal DNA fragments showed the DNA ladders. These results indicate that high molecular weight DNA fragmentation occurs in vivo, and may be correlated with the cytotoxic action of the anticancer drugs. Further study of the association of high molecular weight DNA fragmentation with clinical response to chemotherapy is warranted.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2001

Increased cytochrome c-mediated DNA fragmentation and cell death in manganese-superoxide dismutase-deficient mice after exposure to subarachnoid hemolysate.

BACKGROUND AND PURPOSE: We sought to investigate the mechanisms for oxidative injury caused by subarachnoid hemolysate, a pro-oxidant. METHODS: Injection of 50 microL of subarachnoid hemolysate or saline was performed in CD1 mice (n=75), mutant mice deficient in Mn-superoxide dismutase (Sod2+/-; n=23), and their wild-type littermates (n=23). Subcellular location of cytochrome c was studied by immunocytochemistry, immunofluorescence, and immunoblotting of cellular fractions. DNA fragmentation was assessed though DNA laddering and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL). Cell death was examined through basic histology. RESULTS: Cytochrome c immunoreactivity was present in the cytosol of neurons at 2 hours after hemolysate injection and increased by 4 hours compared with saline-injected animals (P:<0.02). Cytosolic cytochrome c was more abundant in Sod2+/- mutants. DNA fragmentation was evident at 24 hours, but not 4 hours, after hemolysate injection as determined by DNA laddering and TUNEL staining (P:<0.02). DNA fragmentation colocalized to cells with cytosolic cytochrome c and iron. In Sod2+/- mutants, the extent of fragmentation was increased as determined by TUNEL staining (52% increase; P:<0.02) and DNA laddering (optical density=0.819 versus 0.391; P:<0.01). Cell death was evident on basic histology as early as 4 hours after hemolysate injection. No cell death was evident in controls. In Sod2+/- mutants, cell death was increased by 51% compared with wild-type littermates (P:<0.05). CONCLUSIONS: These results demonstrate that subarachnoid blood products are associated with the presence of cytochrome c in the cytosol and subsequent cell death in neurons. It appears that Mn-superoxide dismutase plays a role in preventing cell death after exposure to subarachnoid blood products.

Animals↗
PubMed2001

Extensive DNA fragmentation in oxyphilic cell lesions of the thyroid.

The in situ end-labeling (ISEL) method demonstrates DNA fragmentation, commonly regarded as a marker of apoptosis. We investigated by the ISEL procedure a series of 52 thyroid lesions, including 24 lesions of mitochondrion-rich oxyphilic cells, both benign and malignant, and 28 non-oxyphilic control tumors. A high percentage of nuclear ISEL staining (approximating to 100% in most cases) was observed in the vast majority of oxyphilic cells from both adenomas and carcinomas, in the absence of morphological apoptotic changes and with no immunocytochemical evidence of caspase activation. This pattern of DNA fragmentation was not observed in non-oxyphilic lesions and was confirmed in total extracted DNA. Moreover, a peculiar cytoplasmic staining was also observed in oxyphilic cells from both benign and malignant lesions, probably related to abnormal fragmentation of mitochondrial DNA. Similar staining patterns were detected in oxyphilic cell tumors of other organs (parathyroids, salivary glands, and kidneys). These findings are consistent with an extensive DNA fragmentation peculiar to oxyphilic cells, which is not directly related to apoptosis and whose origin and biological significance are presently unknown.

Caspases↗
PubMed1997

Methylprednisolone and 2-chloroadenosine induce DNA fragmentation at different stages of human T-lymphocyte development.

Both methylprednisolone (MPS) and 2-chloroadenosine (2-CA) were shown previously to induce DNA fragmentation and cell death in human thymocytes at an optimum concentration of 1 and 40 microM, respectively. Though both compounds affected the CD4+CD8+ population, 2-CA depleted primarily thymocytes expressing medium or high levels of CD3-T-cell receptor molecule, while the glucocorticoid treatment affected cells expressing a lower level of CD3-T-cell receptor. Their effect on thymocyte viability and DNA fragmentation was observed already at day 1 of culture and involved the bcl-2 negative thymocytes. Incubation of peripheral T-lymphocytes (which express bcl-2) with the same concentration of MPS did not affect the viability for up to 5 days, while 2-CA induced 100% cell death and DNA fragmentation by day 5. If T-cells were stimulated with concanavalin A in the presence of MPS or 2-CA the cell proliferation was inhibited and a decrease in cell viability with a concomittant increase in DNA fragmentation was observed. If MPS was added at 24 h or later after mitogenic stimulation, it was not able to induce apoptosis and the inhibition of proliferation was less pronounced. 2-CA, on the other hand, inhibited proliferation and induced cell death whenever it was added to the culture. The decreased sensitivity towards the apoptosis induction effects of glucocorticoids at later phases of mitogenic stimulation can not be explained by an increased bcl-2 expression, since its expression level remained constant up to 48 h after mitogenic stimulation. Our data presented in this paper suggest: (1) that T-cells may show different sensitivity towards the same apoptosis inducer signals at different stages of the T-cell development; (2) the apoptotic sensitivity towards various signals may be different at the same stage of T-cell differentiation; and (3) their apoptotic sensitivity does not always correlate with the bcl-2 expression alone.

2-Chloroadenosine↗
PubMed2006

Caspase-activated DNase (CAD)-independent oligonucleosomal DNA fragmentation in chronic myeloid leukaemia cells; a requirement for serine protease and Mn2+-dependent acidic endonuclease activity.

We have previously reported that the pro-apoptotic pyrrolobenzoxazepine, PBOX-6, induces apoptosis in chronic myelogenous leukaemia (CML) cells which is accompanied by oligonucleosomal DNA fragmentation. In this study we show that PBOX-6-induced oligonucleosomal DNA fragmentation occurs in the absence of caspase and CAD activation in CML cells. Dissection of the signalling pathway has revealed that induction of apoptosis requires the upstream activation of a trypsin-like serine protease that promotes the phosphorylation and inactivation of anti-apoptotic Bcl-2. In addition, in this system chymotrypsin-like serine proteases are dispensable for high molecular weight DNA fragmentation, however are required for the activation of a relatively small manganese-dependent acidic endonuclease that is responsible for oligonucleosomal fragmentation of DNA. Furthermore, we demonstrate mitochondrial involvement during PBOX-6-induced apoptosis and suggest the existence of unidentified mitochondrial effectors of apoptosis.

Apoptosis↗
PubMed2001

Biliary dysplasia, cell proliferation and nuclear DNA-fragmentation in primary sclerosing cholangitis with and without cholangiocarcinoma.

OBJECTIVES: To study the extent of biliary dysplasia, and the degree of cell proliferation and apoptosis in bile duct cells (BDC) from patients with primary sclerosing cholangitis (PSC), with and without cholangiocarcinoma (CC). METHODS: Specimens of liver tissue from 16 patients suffering from PSC and CC, and 16 patients with end-stage PSC without cancer, were investigated. Histological evaluation of presence of biliary dysplasia and bile duct proliferation was made. Immunohistochemistry, applying antibodies against Ki-67, p53 and bcl-2, was used. Nuclear DNA fragmentation was assessed by in situ DNA labelling (ApopTag). The numbers of positive cells expressed as a percentage of the total number of BDCs constituted the labelling index (LI). RESULTS: Bile duct dysplasia was significantly more frequent in nontumorous liver tissue from patients with PSC and CC than from patients having end-stage PSC without cancer (P < 0.05). Patients with biliary dysplasia had a higher frequency of marked bile duct proliferation (P < 0.01) than patients without dysplasia. In tumour tissue, the LI for Ki-67 positive nuclei was more than four times the LI of nuclear DNA fragmentation (P < 0.01). Ki-67, bcl-2, p53 or DNA fragmentation were not significantly different in nontumorous liver tissue from patients with and without CC. Immunohistochemical staining for p53 was positive in 75% of the tumours, whilst in nontumorous tissue no such overexpression was found. CONCLUSION: PSC patients with CC more often display biliary dysplasia than those with end-stage PSC, indicating that biliary dysplasia may be a precancerous stage in PSC. Additionally, p53 mutation seems to be a late event in tumour development, since no p53 expression was found in the premalignant areas with nontumorous BDC.

Adult↗