DNA strand scission and ADP-ribosyltransferase activity during murine erythroleukemia cell differentiation

The accumulation of DNA strand breaks and activation of ADP-ribosyltransferase (ADPRT) have recently been associated with cellular differentiation. Murine erythroleukemia (MEL) cells undergo erythropoietic differentiation when exposed to dimethyl sulfoxide (Me2SO) and several studies have suggested that DNA strand scission induced by this agent is a prerequisite for expression of the differentiated phenotype. Me2SO induction of MEL cells has also been associated with increases in ADPRT activity in one study, but not in another. We have monitored the effects of Me2SO on DNA strand breaks in preformed and replicating MEL cell DNA. The results clearly demonstrate that DNA fragmentation is not detectable during Me2SO induction of MEL differentiation, even in the presence of 3-aminobenzamide, an inhibitor of ADPRT. Further, these results are consistent with an absence of detectable changes in both endogenous and total potential ADPRT activity during Me2SO-induced MEL differentiation. These findings would argue against Me2SO induction of DNA strand scission and ADPRT in MEL cells undergoing differentiation.     

Early nuclear events in lymphocyte proliferation : The role of DNA strand break repair and ADP ribosylation

The previously reported extensive DNA strand breakage in resting murine splenic lymphocytes is not an artifact of the extraction or assay procedure. The benzamide inhibitors of poly(ADP ribose) synthetase (pADPRS), such as 5-methoxybenzamide (MBA), had been shown to block the strand break repair occurring within 2 h of activation of splenic lymphocytes by the mitogen concanavalin A (conA); the inhibitors also blocked early events in proliferation, such as blast formation, as well as entry into S phase. Inhibitors of pADPRS blocked lymphocyte proliferation by inhibiting the activity of this enzyme, rather than by non-specific effects. Aphidicolin, an inhibitor of alpha-polymerase, also prevented DNA strand break repair in conA-stimulated cells but, unlike MBA, did not prevent blast formation. DNA strand breaks accumulated in the presence of MBA at the same linear rate (300-400/h) in both resting and conA-treated cells. We and others had hypothesized that this accumulation was due to a continuous production of strand breaks in lymphocytes, leading to their accumulation in presence of repair inhibitors. However, incubation of the cells with aphidicolin at concentrations that inhibited repair did not result in any increase in strand breaks. The hypothesis of continuous cycling of breaks is incorrect; accumulation of breaks was due to some indirect effect of MBA, such as a possible disinhibition of an ADP-ribosylation-sensitive endonuclease described in other cell types. All of the early stages of lymphocyte proliferation, including blast transformation (but not DNA synthesis) require ADP ribosylation. Repair of DNA strand breaks is not a precondition for blast formation, though experiments involving the combined effects of MBA and aphidicolin showed that repair of the breaks is essential in order for the cells to replicate their DNA. Our data are consistent with a model suggesting that DNA strand breaks introduced into differentiated cells act as an additional safety-catch mechanism that restrains them from replicating their genetic material but not from undergoing the early stages of proliferation.     

Rejoining of DNA strand breaks is an early nuclear event during the stimulation of quiescent lymphocytes

Activation of quiescent human peripheral blood lymphocytes or purified T cells by the mitogen, phytohemagglutinin (PHA), involves a rapid rejoining of DNA breaks present in the resting cells as detected by both nucleoid sedimentation analysis and rate of strand unwinding in alkali. Inhibitors of the enzyme ADP-ribosyltransferase (ADPRT) prevent activation of peripheral lymphocytes or T cells by PHA or concanavalin A in a dose-dependent manner, but only if present during the early stages. They do not affect subsequent proliferation if added later, nor do they inhibit the growth of lymphoblastoid cell lines. The inhibitors slow the rejoining of DNA breaks but do not affect the binding of mitogen to the cell surface or the early PHA-stimulated turnover of plasma membrane inositol phospholipids. DNA breaking and rejoining, regulated by ADPRT, may be involved in controlling gene expression during differentiation.     

Mechanism of phorbol myristate acetate-induced lymphotoxin production by a human T cell hybridoma

Various hydroxyl radical scavengers markedly inhibited phorbol myristate acetate (PMA)-induced lymphotoxin (LT) production by a human T cell hybridoma, AC5-8. Among those we tested, tetramethylurea (TMU) was the most potent scavenger, and it was revealed that TMU must be added before 2 h have elapsed after PMA addition in order for LT production to be inhibited. In concordance with this fact, soluble NADPH dependent O2- forming enzyme(s) were activated several fold by PMA. PMA also induced DNA strand breaks, a process markedly inhibited by TMU. As expected, ADP-ribosyl transferase (ADPRT), which is well known to require DNA strand breaks for its enzymatic activity, was activated by PMA treatment. In addition, specific inhibitors for ADPRT, namely 3-amino-benzamide and nicotinamide, inhibited PMA-induced LT production. Taken together, these three successive events, activation of soluble NADPH dependent O2- forming enzyme(s), DNA strand breaks and activation of ADPRT, may be required for PMA-induced LT production by AC5-8.     

Regulation of repair of naturally occurring DNA strand breaks in lymphocytes

Mouse lymphocytes have been shown to contain DNA strand breaks that were repaired within 2h of onset of culture with mitogen. Inhibitors of ADP ribosylation prevented this repair and blocked cell proliferation. The mitogen concanavalin A caused the internal concentration of NAD⁺, the substrate of the ADP ribose polymerase, to rise to about double that of resting cells within 45 min of stimulation. Addition of 300 μm nicotinamide to the culture in absence of mitogen also resulted in a similar increase in internal [NAD⁺], resulting in increased ADP ribosylation activity (measured in permeabilized cells) and in joining of DNA strand breaks; however, none of the subsequent events of lymphocyte activation such as blast transformation and DNA synthesis occurred. These findings indicate that (1) cellular [NAD⁺] is a rate limiting factor in repair of DNA strand breaks in resting lymphocytes and (2) this repair is necessary but not sufficient for lymphocyte proliferation.     

DNA repair in human promyelocytic cell line, HL-60.

The human promyelocytic cell line, HL-60, shows large changes in endogenous poly(ADP-ribose) and in nuclear ADP-ribosyl transferase activity (ADPRT) during its induced myelocytic differentiation. DNA strand-breaks are an essential activator for this enzyme; and transient DNA strand breaks occur during the myelocytic differentiation of HL-60 cells. We have tested the hypothesis that these post-mitotic, terminally differentiating cells are less efficient in DNA repair, and specifically in DNA strand rejoining, than their proliferating precursor cells. We have found that this hypothesis is not tenable. We observe that there is no detectable reduction in the efficiency of DNA excision repair after exposure to either dimethyl sulphate or gamma-irradiation in HL-60 cells induced to differentiate by dimethyl sulphoxide. Moreover, the efficient excision repair of either dimethyl sulphate or gamma-irradiation induced lesions, both in the differentiated and undifferentiated HL-60 cells, is blocked by the inhibition of ADPRT activity.     

Transient formation of DNA strand breaks during the induced differentiation of a human promyelocytic leukaemic cell line, HL-60.

During the induced differentiation of the human promyelocytic leukaemic cell line, HL-60, along the myelocytic lineage, DNA strand-breaks are formed. These breaks which are formed in the face of a proficient DNA repair mechanism, are only transiently maintained and subsequently become religated. The ligation of these breaks requires the activity of the nuclear adenosine diphosphoribosyl transferase (ADPRT). Inhibition of nuclear ADPRT, an enzyme totally dependent on the presence of DNA strand-breaks for its activity and required for efficient DNA repair in eukaryotic cells, blocks the religation of these breaks but not their formation. The inhibition of DNA strand ligation in the differentiating HL-60 cells results in loss of viability and cell death.     

Topoisomerase II is nonfunctional in polyamine-depleted cells.

The polyamines-putrescine, spermidine, and spermine-are essential for normal cell proliferation. Polyamine depletion affects DNA structure and synthesis. Topoisomerase II (topo II) is also necessary for normal cell proliferation, and it has been shown in vitro that polyamines may affect topo II activity. In order to investigate the effect of polyamine depletion on topo II activity, we treated Chinese hamster ovary cells with either alpha-difluoromethylornithine (DFMO) or 4-amidinoindan-1-one-2'-amidinohydrazone (CGP 48664), which are polyamine biosynthesis inhibitors. Treatment with the topo II inhibitor etoposide results in DNA strand breaks only if there is active topo II in the cells. By quantitating DNA strand breaks after etoposide treatment using single cell gel electrophoresis, we were able to estimate intracellular topo II activity. We also quantitated topo II activity in crude nuclear extracts from control and polyamine biosynthesis inhibitor-treated cells. Using single cell gel electrophoresis, we noted a clear decrease in the function of topo II in polyamine biosynthesis inhibitor-treated cells, as compared with untreated control cells. However, the topo II activity in crude nuclear extracts did not differ significantly in control versus polyamine biosynthesis inhibitor-treated cells. Taken together, these results indicate that although the function of topo II in polyamine-depleted cells was impaired, topo II remained functional in an in vitro assay. Using the single cell gel electrophoresis assay, we also found that spermine depletion itself caused DNA strand breaks.     

Unscheduled DNA synthesis induced by N-acetoxy-2-acetylaminofluorene is not sensitive to regulation by ADP-ribosyl transferase

We have directly compared in resting human mononuclear leukocytes the DNA repair effects caused by ADP-ribosyl transferase (ADPRT) activity following DNA damage induction by gamma radiation, UV radiation, ethylene oxide (EO) and N-acetoxy-2-acetylaminofluorene (NA-AAF). The presence of inhibitors of ADPRT during the quantitation of unscheduled DNA synthesis (UDS) resulted in about a 2-fold increase of UDS when induced by gamma radiation, UV radiation or EO. The stimulation of UDS by EO, UV- or gamma-radiation in the presence of an ADPRT inhibitor was equally strong whether 1 mM or 10 mM hydroxyurea was used to suppress scheduled DNA synthesis. The level of NA-AAF induced UDS was not affected by inhibitors of ADPRT. In addition, direct estimation of ADPRT activity revealed that at doses giving maximal UDS, NA-AAF damage did not induce a measurable enzymatic activity whereas gamma-radiation, UV radiation and EO all showed a significant dose response increase. We have interpreted our data to mean that NA-AAF induced UDS estimates DNA repair relating mainly to DNA lesions that are recognized with difficulty, and hence, the rate of endonuclease-induced DNA strand break accumulation is not sufficient to allow a stimulation of ADPRT and affect the quantitation of UDS.     

DNA strand breaks and DNA repair response in lymphocytes after chronic in vivo exposure to very low doses of ionizing radiation in mice

In contrast to the well-documented negative effects of high-dose oxidant exposure, accumulating evidence supports a positive, perhaps essential physiologic role for very low-level oxidant stress. For example, low-level oxidant exposure, within or below the physiologic range, has been reported to stimulate membrane signal transduction, proliferation, antioxidant defense and DNA repair. In the present study, we have examined whether whole-body exposure to low-dose radiation (LDR) results in an alteration in constitutive (steady state) levels of DNA-strand breaks and whether an adaptive increase in DNA-repair response is induced. C57B1/6J mice were exposed to 0.04 Gy (4 cGy) of gamma-radiation as a model of low level oxidant stress. End points measured after chronic in vivo LDR included: (1) constitutive expression of DNA-strand breaks in quiescent spleen cells; (2) sensitivity to DNA damage after high-dose radiation exposure in vitro; (3) repair of constitutive and radiation-induced DNA strand breaks after mitogen stimulation: (4) activity of the DNA-repair associated enzyme, poly(ADP-ribose)transferase (ADPRT) and its substrate, NAD. The results indicated that the constitutive expression of DNA-strand breaks is significantly decreased after chronic LDR; however, DNA-repair capacity after high-dose radiation exposure is not increased above that observed in sham-irradiated mice. Associated with the reduction in constitutive DNA-strand break accumulation was a decrease in resting levels of the DNA-repair-associated enzyme poly(ADP-ribose) transferase (ADPRT). These results are consistent with the interpretation that cumulative DNA damage and associated DNA-repair activity in unstimulated cells are both reduced after chronic LDR exposure.     

Growth-phase-dependent response to DNA damage in poly(ADP-ribose) polymerase deficient cell lines: basis for a new hypothesis describing the role of poly(ADP-ribose) polymerase in DNA replication and repair

We have studied the role of poly(ADP-ribose) polymerase in the repair of DNA damage induced by x-ray and N-methyl N-nitro-N-nitrosoguanidine (MNNG) by using V79 chinese hamster cells, and two derivative mutant cell lines, ADPRT54 and ADPRT351, that are deficient in poly(ADP-ribose) polymerase activity. Under exponentially growing conditions these mutant cell lines are hypersensitive to x-irradiation and MNNG compared to their parental V79 cells which could be interpreted to suggest that poly(ADP-ribose) polymerase is involved in the repair of DNA damage. However, the level of DNA strand breaks induced by x-irradiation and MNNG and their rates of repair are similar in all the cell lines, thus suggesting that it may not be the difference in strand break formation or in its rate of repair that is contributing to the enhanced cell killing in exponentially growing poly(ADP-ribose) polymerase deficient cell lines. In contrast, under growth-arrested conditions, all three cell lines become similarly sensitive to both x-irradiation and MNNG, thus suggesting that poly(ADP-ribose) polymerase may not be involved in the repair of DNA damage in growth-arrested cells. These paradoxical results could be interpreted to suggest that poly(ADP-ribose) polymerase is involved in DNA repair in a cell-cycle-dependent fashion, however, it is functionally active throughout the cell cycle. To resolve this dilemma and explain these results and those obtained by many others, we propose that the normal function of poly(ADP-ribose) polymerase is to prevent DNA recombination processes and facilitate DNA ligation.     

ADP-ribosylation is involved in the integration of foreign DNA into the mammalian cell genome.

The most commonly used DNA transfection method, which employs the calcium phosphate co-precipitation of the donor DNA, involves several discrete steps (1,2). These include the uptake of the donor DNA by the recipient cells, the transport of the DNA to the nucleus, transient expression prior to integration into the host cell genome, concatenation and integration of the transfected DNA into the host cell genome and finally the stable expression of the integrated genes (2,3). Both the concatenation and the integration of the donor DNA into the host genome involve the formation and ligation of DNA strand-breaks. In the present study we demonstrate that the nuclear enzyme, adenosine diphosphoribosyl transferase (ADPRT, E.C. 2.4.2.30), which is dependent on the presence of DNA strand breaks for its activity (4,5) and necessary for the efficient ligation of DNA strand-breaks in eukaryotic cells (4,6), is required for the integration of donor DNA into the host genome. However, ADPRT activity does not influence the uptake of DNA into the cell, its episomal maintenance or replication, nor its expression either before or after integration into the host genome. These observations strongly suggest the involvement of ADPRT activity in eukaryotic DNA recombination events.     

The dynamic nature of DNA-strand breaks present in differentiating muscle cells and quiescent lymphocytes

Cellular differentiation in a number of eukaryotic systems is associated with changes in the number of DNA-strand breaks and involves the activity of adenosine diphosphoribosyl transferase (ADPRT). DNA-strand breaks are essential for activation of nuclear ADPRT, the activity of which is required for efficient religation of DNA-strand breaks. In this study we demonstrate the dynamic nature of DNA-strand breaks formed in the genome of differentiating avian skeletal muscle cells and quiescent human lymphocytes. Inhibition of ADPRT activity blocks DNA-strand ligation in both cell types and leads to the accumulation of a higher number of strand breaks.     

ADP-核糖基转移酶的抑制对γ线所致肿瘤细胞DNA损伤的加强作用

本文探讨了ADP—核糖基转移酶(ADPRT)的活性、DNA单链断裂(SSB)重接和细胞潜在致死质损伤修复(PLDR)三者的关系。证明了ADPRT的特异性抑制剂3—氨基苯甲酰胺(3AB)能阻抑γ线所致的小鼠腹水瘤细胞DNA SSB的重接和PLDR。为增强放射治疗的效果提供了可能的新途径。     

DNA strand breaks: the DNA template alterations that trigger p53-dependent DNA damage response pathways.

The tumor suppressor protein p53 serves as a critical regulator of a G1 cell cycle checkpoint and of apoptosis following exposure of cells to DNA-damaging agents. The mechanism by which DNA-damaging agents elevate p53 protein levels to trigger G1/S arrest or cell death remains to be elucidated. In fact, whether damage to the DNA template itself participates in transducing the signal leading to p53 induction has not yet been demonstrated. We exposed human cell lines containing wild-type p53 alleles to several different DNA-damaging agents and found that agents which rapidly induce DNA strand breaks, such as ionizing radiation, bleomycin, and DNA topoisomerase-targeted drugs, rapidly triggered p53 protein elevations. In addition, we determined that camptothecin-stimulated trapping of topoisomerase I-DNA complexes was not sufficient to elevate p53 protein levels; rather, replication-associated DNA strand breaks were required. Furthermore, treatment of cells with the antimetabolite N(phosphonoacetyl)-L-aspartate (PALA) did not cause rapid p53 protein increases but resulted in delayed increases in p53 protein levels temporally correlated with the appearance of DNA strand breaks. Finally, we concluded that DNA strand breaks were sufficient for initiating p53-dependent signal transduction after finding that introduction of nucleases into cells by electroporation stimulated rapid p53 protein elevations. While DNA strand breaks appeared to be capable of triggering p53 induction, DNA lesions other than strand breaks did not. Exposure of normal cells and excision repair-deficient xeroderma pigmentosum cells to low doses of UV light, under conditions in which thymine dimers appear but DNA replication-associated strand breaks were prevented, resulted in p53 induction attributable to DNA strand breaks associated with excision repair. Our data indicate that DNA strand breaks are sufficient and probably necessary for p53 induction in cells with wild-type p53 alleles exposed to DNA-damaging agents.     

3-氨基苯酰胺对氧化应激诱导的HeLa细胞端粒DNA链断裂重连接作用的影响

端粒是位于真核细胞染色体末端的DNA-蛋白质复合体,在维持染色体稳定上起着重要的作用,并且与细胞的衰老和凋亡有着密切的关系.在各种DNA损伤中,单链断裂(single-strand breaks, SSBs)是最常见的类型之一,既可直接通过内源活性氧或离子化辐射产生,也可间接地在DNA代谢或碱基切除修复期间产生.已知多聚(ADP-核糖)聚合酶［poly(ADPribose) polymerase, PARP］在SSBs修复中起着极为重要的作用.本实验观察了PARP抑制剂3-氨基苯酰胺(3-aminobenzamide, 3-AB)对氧化应激诱导的HeLa细胞端粒DNA链断裂重连接的效应以及对过氧化氢(H2O2)抑制HeLa细胞增殖的影响.结果表明3-AB能够显著地抑制氧化应激诱导的HeLa细胞端粒DNA链断裂后的重连接作用,并能增强H2O2对HeLa细胞增殖的抑制作用,提示PARP参与了端粒DNA链断裂损伤的修复过程.     

Radiation-induced DNA single-strand breaks in freshly isolated human leukocytes.

Single-strand breaks are a major form of DNA damage caused by ionizing radiation, and measurement of strand breaks has long been used as an index of overall cellular DNA damage. Most assays for DNA single-strand breaks in cells rely on measuring fractionated DNA samples following alkali denaturation. Quantification is usually achieved by prelabeling cells with radioactive DNA precursors; however, this is not possible in the situation of nondividing cells or freshly isolated tissue. It has previously been demonstrated that the alkali unwinding assay of DNA strand breaks can be quantified by blotting the recovered DNA on nylon membranes and hybridizing with radiolabeled sequence-specific probes. We report here improvements to the technique, which include hot alkali denaturation of DNA samples prior to blotting and the use of carrier DNA that is non-complementary to the radiolabeled probe. Our method allows both single- and double-stranded DNA to be quantified with the same efficiency, thereby improving the sensitivity and reproducibility of the assay, and allows calibration for determination of absolute levels of DNA strand breaks in cells. We also used this method to assay radiation-induced DNA strand breaks in freshly isolated human leukocytes and found them to have a strand break induction rate of 1815 strand breaks/cell/Gy.     

Effect of inhibitors of poly(ADP-ribose) polymerase on the heat response of HeLa S3 cells

The purpose of this study was to investigate a possible involvement of poly(ADP-ribosyl)ation reactions in hyperthermic cell killing and hyperthermic DNA strand-break induction and repair in HeLa S3 cells. The inhibitors of poly(ADP-ribose) polymerase, 3-aminobenzamide (3AB) and 4-aminobenzamide (4AB), were used as tools in this study. Both inhibitors could sensitize the cells for hyperthermic cell killing equally well, although 3AB is known to be a more effective enzyme inhibitor. The heat sensitization at the level of cell killing could be reversed when the compounds were still present during a 4-h postincubation at 37 degrees C. More heat-induced DNA strand breaks were formed in the presence of 3AB and 4AB. Repair of strand breaks was inhibited during the postincubation at 37 degrees C. Thus the effect of 3AB and 4AB on DNA strand-break repair was different from the cited effect on cell survival. It is concluded that the sensitizing effect of 3AB and 4AB on hyperthermic cell killing is not caused by inhibition of poly(ADP-ribose) polymerase and is also not related to repair of DNA strand breaks.     

Involvement of polyadp-ribose polymerase in the initiation of phytohemagglutinin induced human lymphocyte proliferation

Nicotinamide (10 mM) or 3-aminobenzamide (5 mM) added at the onset of phytohemagglutinin (PHA) treated human lymphocyte cultures provoke a marked inhibition of the PHA induced DNA synthesis and cell proliferation as well as of poly(ADPR) polymerase activity. When the inhibitors of poly(ADPR) polymerase are added at a later stage of culture (48 h) no inhibition of the stimulation of DNA synthesis and cell proliferation by PHA in human lymphocyte cultures is observed. The intervention of ADP ribosylation at the initiation of DNA synthesis is suggested.