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Query: EC:3.1.30.2 (endonuclease)
 18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The nature of the endonucleases responsible for DNA fragmentation in apoptosis has not yet been clearly defined. The intracellular acidity has been known to greatly affect apoptosis probably by affecting the activity of the endonucleases. In this study, the implication of pH in the apoptosis was investigated through the use of human HL-60 leukemia cells. The cells were incubated in media with different pH ranging from 3.5 to 7.5 for 4 hrs and the mode of cell death was investigated. The trypan blue exclusion assay showed that close to 25% and 90% of the cells were dead when incubated in pH 6.4 and pH 5.0 media, respectively. The agarose gel electrophoresis of DNA demonstrated that significant DNA fragmentation occurred in the HL-60 cells incubated in the pH 6.2-6.4 media for 4 hr indicating cell death by apoptosis. The electron microscopy study also demonstrated that many of the cells incubated in the pH 6.4 medium were in the process of apoptosis while the cells maintained in the pH 5.0 medium were dying by necrosis. The intracellular pH (pHi) of HL-60 cells was 6.6-6.9 when the extracellular pH (pHe) was 6.2-6.4. These results demonstrated that DNase I which has a maximal endonuclease activity near pH 7.0 may be responsible for the apoptosis accompanied by DNA fragmentation in HL-60 cells in the pH 6.4 medium. This observation is at variance with the previous reports that DNase II mediate the DNA fragmentation in apoptosis. The cell death at extremely low pH (pH 5.0) appeared to be due mainly to necrosis.
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PMID:Effects of intracellular pH on apoptosis in HL-60 human leukemia cells. 859 48

Transcription of the osteocalcin gene, which encodes a 10 kDa bone-specific protein, is controlled by modularly organized basal regulatory sequences and hormone-responsive enhancer elements. We have previously shown that in the ROS 17/2.8 rat osteosarcoma cell line, which continuously expresses the osteocalcin gene, key regulatory elements reside in two DNase I hypersensitive sites that are fucntionally correlated with transcriptional activity. We now report that a specific nucleosomal organization supports this constitutive expression in ROS 17/2.8 cells, and that chromatin remodeling directly correlates with the developmentally regulated transcriptional activation of the osteocalcin gene during differentiation of normal diploid rat osteoblasts. By combining DNase I, micrococcal nuclease, and specific restriction endonuclease digestion analysis, we observed that the presence of DNAse I hypersensitive sites (-170 to -70 and -600 to -400) and a selective nucleosome positioning over the OC gene promoter are directly associated with developmental stage-specific transcriptional activation in bone-derived cells.
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PMID:Changes in chromatin structure support constitutive and developmentally regulated transcription of the bone-specific osteocalcin gene in osteoblastic cells. 866 2

The relationship between chromatin structure and endonuclease sensitivity was investigated. The cells used in this study were a) human myelogenous leukemic cell lines (HL-60, ML-I, U-937, THP-I) (Group I), which produced internucleosomal DNA cleavage, and b) human T-cell leukemia (MOLT-4), erythroleukemia (K562), glioblastoma (T98G, U87MG) and glioma (KG-1-C) cell lines (Group II), which produced no internucleosomal DNA cleavage, upon treatment with various apoptosis-inducing agents. When the nuclei, isolated from these cells were digested with micrococcal nuclease, chromatin DNA was cleaved into oligonucleosomal units. Although sensitivity to micrococcal nuclease considerably differed from cell to cell, Group I cells were generally more sensitive to micrococcal nuclease digestion than Group II cells. Similar sensitivity to DNase I was observed in both groups of cells. Acid-urea polyacrylamide gel electrophoresis of histone fractions from control and apoptosing HL-60 cells (induced either by hydrogen peroxide or UV irradiation) revealed no significant change in the relative composition of five major histones, indicating the absence of selective degradation of histone HI, but rather the nonspecific degradation of many nuclear proteins. These data suggest a difference in a chromatin structure between Group I and II cells, which might result in the selective production of internucleosomal DNA cleavage only in Group I cells.
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PMID:Chromatin structure and endonuclease sensitivity in human leukemic cell lines. 870 41

The induction of cell death along with cell-cycle arrest is one of the foremost mechanisms regulating cell growth. In the human breast carcinoma cell line MCF-7 we investigated two chemotherapeutic agents, the antiestrogen tamoxifen and the DNA-damaging drug cisplatin, for the relative contribution of these mechanisms to growth inhibition in culture. Growth kinetics and flow cytometry confirmed that tamoxifen at 1 microM acts mainly by arresting cells in the G0/G1 phase of the cell cycle. Compared to untreated controls, only a few more cells were detached from the monolayer and dead after a 5-day incubation. On the other hand, cisplatin at 1 microM did not induce the well-defined G2/M-arrest reported for other cell types, but resulted in a marked increase in the rate of cell death. A morphological feature observed, especially with cisplatin-treated MCF-7 cells, was the formation of numerous micronuclei (in up to 30% of the cells) and an increase in the number of binucleate cells (up to 20%). In both tamoxifen- and cisplatin- treated cultures, cell death appeared to occur by apoptosis, as indicated morphologically by cellular and nuclear shrinkage accompanied by DNA-condensation and ultimately the formation of DNA containing apoptotic bodies. However, no internucleosomal DNA degradation or endogenous endonuclease activity could be detected in the cells of the monolayer or in the mainly dead and detached cells of the culture supernatant. DNA fragmentation was only observed when isolated MCF-7 nuclei were incubated with exogenous endonucleases. However, as determined by reverse transcriptase/polymerase chain reaction amplification, MCF-7 cells do express the mRNA for DNase I, an endonuclease known to be involved in apoptosis. Thus, apoptosis is part of the growth-inhibitory process and occurs without apparent internucleosomal DNA fragmentation in MCF-7 cell cultures.
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PMID:Cell-cycle arrest, micronucleus formation, and cell death in growth inhibition of MCF-7 breast cancer cells by tamoxifen and cisplatin. 887 58

DNA fragmentation is a common biochemical hallmark of apoptosis. It is catalyzed by endogenous Ca2+, Mg(2+)-dependent endonuclease(s). Although the exact identity of the apoptotic endonuclease is still a matter of debate, a number of candidate nucleases have been proposed like NUC18, DNase II and DNase I. Relatively large amounts of nucleases are also expressed by mycoplasmas, cell wall-less bacteria of the class Mollicutes, which are found as contaminants in up to 45% of the continuous cell lines in current use. In order to clarify the effect of these pathogens on the investigation of apoptosis in cell culture systems, we looked for biochemical markers (DNA fragmentation, nuclease expression) and morphological changes characteristic of apoptosis (cell shrinkage, chromatin condensation, apoptotic bodies) in Mycoplasma hyorhinis-free and -infected cultures of the human pancreatic adenocarcinoma cell line PaTu 8902 and of mouse NIH 3T3 fibroblasts. For that purpose we employed cells cultured under standard conditions and cells exposed to the protein synthesis inhibitor cycloheximide, which is known to induce apoptosis in various cell systems. After exposure to cycloheximide only the mycoplasma-positive cells exhibited internucleosomal DNA degradation. In contrast, nuclease activities in the molecular range of 47 to 54 kDa were detected in cell homogenates and culture supernatants of infected cultures of both control and cycloheximide-treated cells, whereas mycoplasma-free cultures were nuclease-negative. The expression of the nucleases and the cycloheximide-induced DNA fragmentation were suppressed by the prokaryote-specific protein synthesis inhibitor chloramphenicol. Moreover, partially purified nucleases from supernatants of infected cells were able to cleave the DNA of isolated substrate nuclei at internucleosomal sites. These data indicate that DNA ladder formation in cell culture systems can also be caused by mycoplasmal nucleases which apparently penetrate the host cells after cycloheximide treatment or more generally after cellular stress. Therefore, internucleosomal DNA fragmentation in established cell lines has to be regarded with care, unless mycoplasmal infection can be excluded, or the existence of endogenous endonucleases can be proven. The presence of endonucleolytic activities of about 47 to 54 kDa molecular mass has now to be regarded as highly indicative of contaminations with M. hyorhinis. In contrast, the expression of an apoptotic morphology was not restricted to infected cells; in both mycoplasma-free and -contaminated cultures, cells with condensed chromatin were observed after staining with the DNA binding dye Hoechst 33342. Electron microscopic studies revealed that most of the cells containing compacted DNA were phagocytosed by unaffected fellow cells. Presumably because of the relatively long exposure (72 h) to cycloheximide we also observed secondary necrosis as indicated by the parallel occurrence of morphological characteristics of apoptosis (chromatin condensation) and necrosis (loss of membrane integrity and organelle swelling).
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PMID:Internucleosomal DNA fragmentation in cultured cells under conditions reported to induce apoptosis may be caused by mycoplasma endonucleases. 888 84

The testis is a tissue of high proliferative activity. In this organ, sperm cells (spermatozoa) are produced from stem cells (spermatogonia) by two consecutive steps of cell multiplication and spermatid cytodifferentiation. Mitotic proliferation of spermatogonia generates primary spermatocytes which enter meiosis, leading to the generation of spermatids. The number of cells entering meiosis is held constant, since outnumbering spermatogonia or premeiotic spermatocytes are eliminated by apoptosis (programmed cell death). During apoptosis, the nuclear chromatin is internucleosomally degraded by the activity of a Ca2+, Mg2+-dependent endonuclease. Recent data indicate that deoxyribonuclease I (DNase I) is identical to the apoptotic endonuclease responsible for the internucleosomal DNA degradation. Previous results using primers specific for rat parotid DNase I in a polymerase chain reaction have demonstrated the presence of DNase I-specific gene transcripts in rat testis. We have therefore analysed the presence of DNase I in rat testis by immunohistochemistry and biochemical procedures. The presence of DNase I-like endonucleolytic activity was verified enzymatically. DNase I immunoreactivity was detected in the nuclei of a few spermatogonia and premeiotic spermatocytes, but within the acrosomic vesicle of all spermatids and spermatozoa. In situ hybridisation revealed the accumulation of DNase I-specific gene transcripts in a small number of spermatogonia and/or premeiotic spermatocytes, but in a large number of spermatids. The occurrence of apoptotic DNA fragmentation was investigated by in situ end-labelling (ISEL) of free 3'-OH DNA ends and gave positive nuclear staining of only very few spermatogonia. No positive ISEL staining was observed in maturing spermatids and/or spermatozoa. These data support the notion that, within the seminiferous epithelium, the number of primary spermatocytes entering meiosis is controlled by apoptosis. In addition, they demonstrated that mature sperm cells are equipped with an endonuclease that might be used for DNA degradation during their elimination at later stages of their life span. The expression and distribution of the tumour suppressor gene product, p53, was analysed by immunostaining. Strong p53 immunoreactivity was observed in the nuclei of a number of spermatogonia, of some premeiotic spermatocytes and probably in all spermatids. Thus, p53 expression appeared to parallel that of DNase I. In contrast, p53 immunoreactivity was absent in mature spermatozoa present in the lumen of the testicular tubules or the ductus epididymidis. It is therefore proposed that at later stages of spermatid maturation most probably before their release as mature spermatozoa-the p53 gene product was either degraded or retained in residual bodies, since p53 immunoreactivity was found to be concentrated within these organelles.
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PMID:Distribution of deoxyribonuclease I (DNase I) and p53 in rat testis and their correlation with apoptosis. 891 66

Locus control region (LCR) is known to occur 5'-upstream of the globin gene clusters in humans and a number of other animals. It comprises four DNase I hypersensitive sites, HS 1-4, and has been considered to play a key role in regulating the globin gene expression in tissue- and developmental stage-specific manners. The occurrence of LCR in the rat genome, however, has not been documented so far. In the present study, the author intended to identify and analyze the rat beta-LCR HS 1 and HS 2, in order to further facilitate studies on the regulatory mechanism involved in globin gene expression. The results obtained in this study are summerized as follows: 1. A DNA region of about 700 bp on the rat genome was amplified by polymerase chain reaction (PCR) using synthetic primers derived from portions of the mouse beta-LCR HS 2. The nucleotide sequence of the PCR product (R 700) shows 67% and 83% homologies with those of the human and mouse HS 2, respectively, indicating that R 700 represents beta-LCR HS 2 of rats. 2. In order to locate beta-LCR HS 2 on the rat genome, a 7 kb DNA fragment (R 7,000) harboring a region between beta-LCR HS 2 and the epsilon 1-globin gene was obtained by PCR. Restriction endonuclease mapping of R 7,000 revealed that the rat beta-LCR HS 2 is located 6.0 kb 5'-upstream relative to the cap site of the epsilon 1-globin gene. 3. The rat beta-LCR HS 1 was then located 4.2 kb 5'-upstream of the epsilon 1-globin gene by Southern blot hybridization of R 7,000 using a human HS 1 probe. Nucleotide sequencing revealed that the rat HS 1 has 83% homology to the mouse HS 1. 4. Comparisons of the structures of the rat beta-LCR HS 1 and HS 2 with those of other animal species indicate that several motifs and consensus sequences for binding of transcription factors, such as NF-E 2/AP-1 and GATA-1, are well conserved during evolutional periods, indicating an indispensable role of LCR in globin gene expression.
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PMID:[Identification and characterization of the rat beta-globin locus control region (LCR) site 1 and site 2]. 893 14

Apoptotic cells in rat thymus were labeled in situ in paraffin-embedded and frozen tissue sections by ligation of double-stranded DNA fragments containing digoxigenin or Texas red. Two forms of double-stranded DNA fragments were prepared using the polymerase chain reaction: one was synthesized using Taq polymerase, which yields products with single-base 3' overhangs, and one using Pfu polymerase, which produces blunt-ended products. Both types of fragment could be ligated to apoptotic nuclei in thymus, indicating the presence in such nuclei of DNA double-strand breaks with single-base 3' overhangs as well as blunt ends. However, in nuclei with DNA damage resulting from a variety of nonapoptotic processes (necrosis, in vitro autolysis, peroxide damage, and heating) single-base 3' overhangs were either nondetectable or present at much lower concentrations than in apoptotic cells. Blunt DNA ends were present in such tissues, but at lower concentrations than in apoptotic cells. In contrast, in all of these forms of DNA damage, nuclei contained abundant 3'-hydroxyls accessible to labeling with terminal deoxynucleotidyl transferase. Thus, although single-base 3' overhangs and blunt ends are present in apoptotic nuclei, the specificity of the in situ ligation of 3'-overhang fragments to apoptotic nuclei indicates that apoptotic cells labeled in this way can readily be distinguished from cells with nonapoptotic DNA damage. These data are consistent with the involvement of an endonuclease similar to DNase I in apoptosis, which is predicted to leave short 3' overhangs as well as blunt ends in digestion of chromatin.
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PMID:Presence of double-strand breaks with single-base 3' overhangs in cells undergoing apoptosis but not necrosis. 894 57

Apoptosis has received increased attention over the past decade, and it is established as an essential process in physiological and disease states. Much effort has been devoted to understanding the intracellular mechanisms culminating in apoptosis; intense investigation has also focused on its role during inflammation. Despite these efforts, these events remain incompletely understood. It has been suggested that the Ca(2+)- and Mg2+-dependent endonuclease that mediates DNA fragmentation is DNase I; however, the precise role of DNase I during apoptosis has been debated. Recent observations using anti-DNA antibodies derived from autoimmune mice (MRL-lpr/lpr) provided both the means and the reagents to approach these issues in a more direct manner. We previously discovered that many anti-DNA antibodies cross-react with DNase I, and a subset of these Ig inhibited DNase I enzymatic activity in vitro. Serendipitously, in separate studies, a subset of these antibodies were observed to enter and localize within the nuclei of living cells. The aim of the present investigation was to determine whether these nuclear-localizing anti-DNA antibodies could interact with DNase I in living cells. We found that, once internalized, these autoantibodies bound DNase I and inhibited activity of the enzyme. Furthermore, living cells containing the intracellular antibodies appeared resistant to apoptotic stimuli; both morphological features of nuclear apoptosis and DNA fragmentation were inhibited. These results support a pivotal role for DNase I in apoptosis, and they provide a novel paradigm for autoantibody-mediated inflammatory disease.
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PMID:Spontaneously produced anti-DNA/DNase I autoantibodies modulate nuclear apoptosis in living cells. 897 1

Most developing lymphocytes spontaneously die in the thymus during positive and negative selection of the T cell repertoire. By evaluating the expression of the proliferation antigens Ki-67 and PCNA, we demonstrated here that more than 95% of thymocytes are potentially proliferating. The coincidence within the same cell population of death and proliferation is thus apparent in developing thymocytes. Using dual-parameter cytometric techniques to evaluate in single cells the amount of DNA versus light-scattering values, we found that spontaneous thymocyte apoptosis occurs with similar frequency in all the cycle phases, whereas apoptosis induced by the anti-topoisomerase-II, etoposide (which is the consequence of irreversible DNA damage), takes place with higher frequency in S and G2 phases (i.e., in those cycle phases in which DNA is subjected to torsional constraints). The capability of thymocytes to enter apoptosis was also monitored by digesting DNA in situ with DNase I (a nuclease that cleaves DNA mimicking the nuclear damage common to most apoptotic suicides). We also show that endonuclease-mediated DNA digestion occurs to a similar extent in cells with different DNA contents, i.e., in cycle phases in which the superstructural organization of chromatin is markedly different.
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PMID:Spontaneous apoptosis of thymocytes is uncoupled with progression through the cell cycle. 898 20


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