Poly(ADP-ribose) synthesis in blocked and damaged cells and its relation to carcinogenesis.

There is compelling evidence showing that repair of DNA damage depends on the synthesis of poly(ADP-ribose) molecules at specific sites on histones and other proteins in nuclei of injured cells. In the present study, we have studied the effect of long-term exposure of mouse cells to nicotinamide and various cell cycle blockers on the ability of the cells to increase their levels of poly(ADP-ribose) in response to DNA methylation damage with dimethylsulfate (DMS). Of the cell cycle blockers used, hydroxyurea (HU) and cytosine arabinofuranoside (ara-C) inhibit DNA elongation at the replication fork and cause the appearance of small DNA molecules, whereas butyrate and colcemid block cells without interfering with DNA synthesis. In addition, cells were treated with nicotinamide, an inhibitor of poly(ADP-ribose) polymerase but also a precursor in NAD+ biosynthesis. Long-term exposure of cultured cells to these agents was followed with a short-term damage with DMS. The size-class distribution and concentration of poly(ADP-ribose) molecules were determined using high resolution polyacrylamide gel electrophoresis and were found to contain 1 to more than 30 ADP-ribosyl groups. On the contrary, histones from blocked cell nuclei were found to be mono- and oligo(ADP-ribosyl)ated. Thus, the large molecules of poly(ADP-ribose) were associated with nonhistone proteins and most likely with the enzyme pADP-R polymerase. DMS damage caused an increase in the levels of poly(ADP-ribose) that was synthesized in lysates from cells treated with any one drug alone. On the other hand, a dramatic decrease in total protein poly(ADP-ribosyl)ation resulted from DMS damage of cells treated with nicotinamide + HU or nicotinamide + ara-C. This decrease was not observed in combinations of nicotinamide with cell-cycle blockers that do not directly interfere with DNA synthesis (butyrate and colcemid). We suggest that nicotinamide + ara-C or nicotinamide + HU might be used as effective antineoplastic treatments by virtue of their ability simultaneously to damage DNA and reduce poly(ADP-ribosyl)ation and presumably DNA repair. A model for the facilitation of DNA repair by poly(ADP-ribosyl)ation of histones and of poly(ADP-ribose) polymerase is presented.     

The Histone Methyltransferase SMYD2 Methylates PARP1 and Promotes Poly(ADP-ribosyl)ation Activity in Cancer Cells

Poly(ADP-ribose) polymerase-1 (PARP1) catalyzes the poly(ADP-ribosyl)ation of protein acceptors using NAD⁺ as the substrate is now considered as an important target for development of anticancer therapy. PARP1 is known to be post-translationally modified in various ways including phosphorylation and ubiquitination, but the physiological role of PARP1 methylation is not well understood. Herein we demonstrated that the histone methyltransferase SMYD2, which plays critical roles in human carcinogenesis, mono-methylated PARP1. We confirmed lysine 528 to be a target of SMYD2-dependent PARP1 methylation by LC-MS/MS and Edman Degradation analyses. Importantly, methylated PARP1 revealed enhanced poly(ADP-ribose) formation after oxidative stress, and positively regulated the poly(ADP-ribosyl)ation activity of PARP1. Hence, our study unveils a novel mechanism of PARP1 in human cancer through its methylation by SMYD2.     

Spermine-induced variations in the adenosine 5'-diphosphate ribosylation patterns of nuclear proteins from rat liver and hepatoma.

Rat liver and hepatoma nuclei were incubated in vitro with [3H]nicotinamide adenine dinucleotide to allow synthesis of a polymer of adenosine diphosphoribose subunits joined in an 1',2' ribose-ribose linkage. The addition of 1 mM spermine altered the adenosine 5'-diphosphate (ADP) ribosylation patterns of nuclear proteins in hepatoma, host liver, and regenerating liver. Spermine-treated nuclei showed a greater incorporation of ADP-ribose into H1 histones and nonhistone nuclear proteins with isoelectric points between pH 3.0 and 6.0 when separated on polyacrylamide gels. Conversely, a large reduction in ADP ribosylation was seen in core histones (H2A, H2B, and H3) from the same nuclei. The proportion of ADP-ribose incorporated into histones was reduced in the nuclei from proliferating cells relative to their respective control livers. These results imply that polyamines, which are higher in concentration in rapidly dividing cells, may elicit a regulatory function by causing the preferential ADP ribosylation of H1 histones, as well as the more acidic of the nuclear proteins.     

Expression and functionality of histone H2A variants in cancer

Regulation of gene expression includes the replacement of canonical histones for non-allelic histone variants, as well as their multiple targeting by postranslational modifications. H2A variants are highly conserved between species suggesting they execute important functions that cannot be accomplished by canonical histones. Altered expression of many H2A variants is associated to cancer. MacroH2A variants are enriched in heterocromatic foci and are necessary for chromatin condensation. MacroH2A1.1 and macroH2A1.2 are two mutually exclusive isoforms. MacroH2A1.1 and macroH2A2 inhibit proliferation and are associated with better cancer prognosis; while macroH2A1.2 is associated to cancer progression. H2AX variant functions as a sensor of DNA damage and defines the cellular response towards DNA repair or apoptosis; therefore, screening approaches and therapeutic options targeting H2AX have been proposed. H2A.Z is enriched in euchromatin, acting as a proto-oncogene with established roles in hormone responsive cancers and overexpressed in endocrine-resistant disease. Other H2A family members have also been found altered in cancer, but their function remains unknown. Substantial progress has been made to understand histone H2A variants, their contribution to normal cellular function and to cancer development and progression. Yet, implementation of high resolution mass spectrometry is needed to further our knowledge on highly homologous H2A variants expression and function.     

Acceptors for the poly ADP-ribosylation modification of chromatin structure are altered by carcinogen-induced DNA damage

Poly(ADP-Rib) polymerase is activated by strand breaks in DNAand appears to play an important role in DNA repair. The enzymecatalyses the poly ADP-ribosylation of histones and non-histoneproteins, yet the contribution of these major alterations inchromatin composition have, as yet, not been critically evaluatedwith regard to DNA strand breaks. In the present study, theeffects of N-methyl-N-nitrosourea (MNU) upon the poly ADP-ribosylationof nuclear protein acceptors have been identified and quantifiedat the oligonucleosomal level of chromatin. Treatment of HeLacells with MNU (4.5 mM) for 1 h resulted in a reduction in thecellular NAD pool (30%), a 2–3 fold stimulation of polyADP-ribosylation in isolated nuclei and in isolated oligonucleosomes.Of acceptors modified, the automodification of the polymerasewas stimulated at least 3-fold. Analysis of the acid-solubleacceptors showed a stimulation in the modification of the corehistones and a 2-fold increase in histone H1 poly ADP-ribosylation.This modification causes a novel crosslinking of the latterhistone, and this has been studied as it relates to DNA strandbreaks in the present work. In vivo treatment with MNU resultedin the synthesis of longer chain or more complex polymer speciesat the expense of the shorter chained ADP-ribose moieties.     

Resistance of H1 histone to proteolytic attack in chromatin from rat-ascites hepatoma

From rat-liver and ascites-hepatoma chromatins, NaCl-soluble fractions were prepared. The 0.35 M NaCl-soluble fraction from the hepatoma (AH) chromatin contained much non-histone protein of high-molecular weight, compared with the fraction from the rat-liver (RL) chromatin. The 0.35 M NaCl-insoluble, but 2 M NaCl/5 M urea-soluble fraction was composed mainly of 5 classes of histones. These histones were quantitatively not different between AH and RL chromatins. However, H1 histone was rather protease-resistant in AH chromatin, but not in RL chromatin. The proteolytic capacity was also lower in AH chromatin. In addition, in the micrococcal-nuclease digest of AH nuclei, the oligonucleosomes were considerably retained even by long-time digestion, but not in that of RL nuclei.     

Comparison of benzo [a]pyrene-diol-epoxide binding to histone H2A with different carboxy-terminal regions

We have compared (?)-7r,8t-dihydroxy-9t,10t-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene(BPDE-I) binding to three vertebrate histones H2A with differentC-terminal regions. HPLC analyses of core histones preparedfrom nuclei exposed to [³H]BPDE-I showed that rat liver andchicken erythrocyte histones H2A were heavily labeled by [³H]BPDE-Ibut Xenopus laevis liver histone H2A was not. This result wasconfirmed by HPLC analyses of V8-protease digestsof BPDE-I boundto histone H2A purified from the three different nuclei. Thereare significantamino acid sequence differences only in the C-terminalregions of the different histones H2A, where rat liver and chickenerythrocyte histones H2A contain two and one histidine residues,respectively, while the amino acid sequence of Xenopus histoneH2A contains no histidine. Pre-treatment of the in situ BPDE-I-modifiedH2A.2 from rat liver withcarboxypeptidase B, which should removethe C-terminal lysine from the protein, resulted in increasedretention times on reverse-phase HPLC for the adduct-contaningpeptides upon subsequent V8-protease digestion. This suggestedthat the site(s) of BPDE-I modification are located primarilyin the C-terminal octapeptide of rat H2A.2. To confirm this,C-terminal V8-peptides of thedifferent histones H2A were isolatedand reacted with BPDE-I at physiological pH in vitro. The HPLCanalyses of the reaction mixtures indicated that the C-terminalpeptide of rat liver and chicken erythrocyte histones H2A wasa target site for BPDE-I binding in nuclei. It is suggestedthat the nucleophilic target amino acid for BPDE-I binding inhistone H2A may be a histidine located close to the C terminus.     

Poly ADP-ribosylation and DNA strand breakage in SV40 minichromosomes

Poly ADP-ribosylation and DNA strand breakage in response totreatment with the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine(MNNG) were studied on SV40 minichromosomes in SV40-infected,permeabilized CV-1 monkey cells. After an initial sharp increasein poly ADP-ribosylation, strand breakage and poly ADPR increasedproportionately with increasing dose of MNNG. This suggestsa cause-effect relationship between the two reactions. The majorpoly ADP-ribose acceptor of minichromosomes was core histoneH2B. In contrast, H2B, H2A, H1 and protein A24 were poly ADP-ribosylatedin the nuclear chromatin of the same cells.     

A translationally regulated Tousled kinase phosphorylates histone H3 and confers radioresistance when overexpressed

The gene Tousled of Arabidopsis Thaliana encodes a protein kinase which, when mutated, results in abnormal flower development. From a library of mRNAs that are translationally upregulated by overexpression of the translation initiation factor 4E, we identified a mammalian Tousled Like kinase (TLK1B). The human TLK1B mRNA contains a 5'UTR 1088-nt-long with two upstream AUG codons, and was found to be very inhibitory for translation. The TLK1B protein localizes almost exclusively to the nuclei. TLK1B overexpression in mammalian cells rendered them more resistant to ionizing radiation (IR). Purified TLK1B phosphorylated histone H3 at S(10) with high specificity both in a mix of core histones and in isolated chromatin, suggesting that histone H3 is a physiological substrate for TLK1B. Moreover, overexpression of TLK1B in transfected cells resulted in a higher degree of H3 phosphorylation. Expression of TLK1B in a yeast strain that harbors a temperature-sensitive mutation of the major H3 kinase, Ipl1, complemented the growth defect; restored normal levels of histone H3 phosphorylation; and increased their resistance to IR. Phosphorylation of H3 has been linked to the activation of the immediate-early genes upon mitogenic stimulation, and to chromatin condensation during mitotic/meiotic events. A possible role for TLK1B in radioprotection is discussed.     

Nitrosourea interaction with chromatin and effect on poly(adenosine diphosphate ribose) polymerase activity.

Poly(adenosine diphosphate ribose) polymerase, a chromatin-bound enzyme, was stimulated 150 to 200% after treatment of HeLa cells with methylnitrosourea (MNU). In contrast, a slight inhibitory effect on enzyme activity was observed after treatment of cells with various concentrations of chloroethylnitrosoureas. To define precisely the differential effects of nitrosoureas on the enzyme activity, their interactions with chromatin substructure were studied. A nonrandom, in vivo alkylation of chromatin DNA by equimolar concentrations of MNU and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) was revealed by digestion of nuclei from drug-treated cells with micrococcal nuclease and DNase I. [methyl-14C]MNU interacted preferentially with the more accessible regions of chromatin, the internucleosome linkers, whereas, the [chloroethyl-14C]CCNU alkylated the nucleosomal core DNA to a greater extent. These two drugs also differed in their extent of covalent modification of histone and nonhistone chromosomal protein. The binding of MNU to histones was greater than of CCNU. CCNU mainly affected nonhistone proteins. This difference in the reactivity of methyl and chloroethyl nitrosoureas with chromatin may relate to their differential effect on poly(adenosine diphosphate ribose) polymerase activity, as well as to their carcinogenic and antitumor properties.     

Impairment of prostate cancer cell growth by a selective and reversible lysine-specific demethylase 1 inhibitor

Post-translational modifications of histones by chromatin modifying enzymes regulate chromatin structure and gene expression. As deregulation of histone modifications contributes to cancer progression, inhibition of chromatin modifying enzymes such as histone demethylases is an attractive therapeutic strategy to impair cancer growth. Lysine-specific demethylase 1 (LSD1) removes mono- and dimethyl marks from lysine 4 or 9 of histone H3. LSD1 in association with the androgen receptor (AR) controls androgen-dependent gene expression and prostate tumor cell proliferation, thus highlighting LSD1 as a drug target. By combining protein structure similarity clustering and in vitro screening, we identified Namoline, a γ-pyrone, as a novel, selective and reversible LSD1 inhibitor. Namoline blocks LSD1 demethylase activity in vitro and in vivo. Inhibition of LSD1 by Namoline leads to silencing of AR-regulated gene expression and severely impairs androgen-dependent proliferation in vitro and in vivo. Thus, Namoline is a novel promising starting compound for the development of therapeutics to treat androgen-dependent prostate cancer.     

Synergistic cytotoxicity of N-methyl-N'-nitro-N-nitrosoguanidine and absence of poly(ADP-ribose) glycohydrolase involves chromatin decondensation

DNA-alkylating agents in combination with poly (ADP-ribose) (PAR) synthesis inhibitors are a promising treatment for cancer. In search of other efficacious alternatives, we hypothesized that the absence of poly(ADP-ribose) glycohydrolase (PARG), which leads to the inhibition of PAR hydrolysis, would lead to increased DNA alkylation after treatment with low doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). At a sublethal dose, MNNG shows synergistic cytotoxicity in PARG-null embryonic trophoblast stem (TS) cells. The PAR modifications of histone H1 and histone H2B are much more pronounced in PARG null-TS cells exposed to MNNG, suggesting their relevance in the efficacy of this combination therapy. Because the PAR modification of these chromatin binding proteins leads to chromatin remodeling, a possible mechanism for the observed synergistic effects involves the subsequent decondensation of chromatin, which may cause the genomic DNA to be more accessible to MNNG alkylation. Further analysis demonstrated chromatin decondensation in PARG null-TS cells as visualized by electron microscopy. In addition, treatment with MNNG led to an increase in O6- methylguanine levels in PARG null-TS cells compared to wild-type, which demonstrates increased DNA alkylation in the absence of PARG. Taken together, we provide compelling evidence that the absence of PARG leads to chromatin decondensation, which in turn leads to increased amounts of DNA alkylation and cell death induced by low doses of MNNG. Therefore, combination therapy of PARG inhibition and a DNA- alkylating agent is a potential treatment to induce the death of cancer cells.     

Changes in levels of ADP-ribose polymers in rat liver during 2-acetylaminofluorene-induced hepatocarcinogenesis

The exposure of rats to the carcinogen 2-acetylaminofluorene(2-AAF) results in the accumulation of DNA-damaging adducts.The inability of cells to repair such damage adequately is aputative causal event in chemical carcinogenesis. It has beenshown that one cellular response to DNA damage that leads toDNA repair is poly(ADP-ribosyl)ation of nuclear proteins. Toexamine the possible existence of an altered poly(ADP-ribosyl)ationresponse to 2-AAF-mediated damage of rat liver DNA, tissue ADP-ribosepolymer levels were determined during various stages of 2-AAF-mediatedcarcinogenesis. 2-AAF was administered to rats in a discontinuousfeeding regimen comprised of five consecutive cycles, each cycleconsisting of 3 weeks on 2-AAF diet followed by 1 week of recoveryon a control diet without 2-AAF. During cycle one of 2-AAF administration,rat liver ADP-ribose polymer levels increased 3-fold over thatfound in livers of rats fed only the control diet. In contrast,when rats were administered the non-genotoxic liver mitogen4-AAF for one cycle, no significant elevation occurred in ADP-ribosepolymer levels. Elevated ADP-ribose polymer production was alsoobserved during cycles two and three of 2-AAF administration.However, during cycles four and five of 2-AAF administration,a period when rats administered 2-AAF acquire a high risk forhepatocarcinogenesis, an altered pattern of ADP-ribose polymerproduction occurred in rat livers. ADP-ribose polymer levelsin these rat livers remained low, similar to levels found incontrol rat livers, despite the administration of 2-AAF. Whenthe livers from rats fed either one or five cycles of 2-AAFwere analyzed for possible decreases in the levels of tissueNAD+, the substrate for poly(ADP-ribose) polymerase, no changesin relative abundance were found. In addition, analysis of poly(ADP-ribose)polymerase activity showed no decrease at five cycles of 2-AAFadministration. These results indicated that at late stagesof 2-AAF-induced hepatocarcinogenesis, 2-AAF does not inducean expected increase in ADP-ribose polymer levels, and suggestedthat significant changes in DNA repair may occur at a time justpreceding an increased risk for developing liver cancer.