Normal human cells undergo a finite number of cell divisions and ultimately enter a nondividing state called replicative senescence. It has been proposed that telomere shortening is the molecular clock that triggers senescence. To test this hypothesis, two telomerase-negative normal human cell types, retinal pigment epithelial cells and foreskin fibroblasts, were transfected with vectors encoding the human telomerase catalytic subunit. In contrast to telomerase-negative control clones, which exhibited telomere shortening and senescence, telomerase-expressing clones had elongated telomeres, divided vigorously, and showed reduced staining for β-galactosidase, a biomarker for senescence. Notably, the telomerase-expressing clones have a normal karyotype and have already exceeded their normal life-span by at least 20 doublings, thus establishing a causal relationship between telomere shortening and in vitro cellular senescence. The ability to maintain normal human cells in a phenotypically youthful state could have important applications in research and medicine.

/pdf/extension-of-life-span-by-introduction-of-telomerase-into-4ucjiz7vl1.pdf

Extension of life-span by introduction of telomerase into normal human cells

Poly(ADP-ribose) polymerase (PARP1) facilitates DNA repair by binding to DNA breaks and attracting DNA repair proteins to the site of damage. Nevertheless, PARP1-/- mice are viable, fertile and do not develop early onset tumours. Here, we show that PARP inhibitors trigger gamma-H2AX and RAD51 foci formation. We propose that, in the absence of PARP1, spontaneous single-strand breaks collapse replication forks and trigger homologous recombination for repair. Furthermore, we show that BRCA2-deficient cells, as a result of their deficiency in homologous recombination, are acutely sensitive to PARP inhibitors, presumably because resultant collapsed replication forks are no longer repaired. Thus, PARP1 activity is essential in homologous recombination-deficient BRCA2 mutant cells. We exploit this requirement in order to kill BRCA2-deficient tumours by PARP inhibition alone. Treatment with PARP inhibitors is likely to be highly tumour specific, because only the tumours (which are BRCA2-/-) in BRCA2+/- patients are defective in homologous recombination. The use of an inhibitor of a DNA repair enzyme alone to selectively kill a tumour, in the absence of an exogenous DNA-damaging agent, represents a new concept in cancer treatment.

Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase

There is convincing evidence that cellular prooxidant states--that is, increased concentrations of active oxygen and organic peroxides and radicals--can promote initiated cells to neoplastic growth. Prooxidant states can be caused by different classes of agents, including hyperbaric oxygen, radiation, xenobiotic metabolites and Fenton-type reagents, modulators of the cytochrome P-450 electron-transport chain, peroxisome proliferators, inhibitors of the antioxidant defense, and membrane-active agents. Many of these agents are promoters or complete carcinogens. They cause chromosomal damage by indirect action, but the role of this damage in carcinogenesis remains unclear. Prooxidant states can be prevented or suppressed by the enzymes of the cellular antioxidant defense and low molecular weight scavenger molecules, and many antioxidants are antipromoters and anticarcinogens. Finally, prooxidant states may modulate the expression of a family of prooxidant genes, which are related to cell growth and differentiation, by inducing alterations in DNA structure or by epigenetic mechanisms, for example, by polyadenosine diphosphate-ribosylation of chromosomal proteins.

https://science.sciencemag.org/content/sci/227/4685/375.full.pdf

Prooxidant states and tumor promotion.

Apoptosis is a morphologically and biochemically distinct form of cell death that occurs under a variety of physiological and pathological conditions. In the present study, the proteolytic cleavage of poly(ADP-ribose) polymerase (pADPRp) during the course of chemotherapy-induced apoptosis was examined. Treatment of HL-60 human leukemia cells with the topoisomerase II-directed anticancer agent etoposide resulted in morphological changes characteristic of apoptosis. Endonucleolytic degradation of DNA to generate nucleosomal fragments occurred simultaneously. Western blotting with epitope-specific monoclonal and polyclonal antibodies revealed that these characteristic apoptotic changes were accompanied by early, quantitative cleavage of the M(r) 116,000 pADPRp polypeptide to an M(r) approximately 25,000 fragment containing the amino-terminal DNA-binding domain of pADPRp and an M(r) approximately 85,000 fragment containing the automodification and catalytic domains. Activity blotting revealed that the M(r) approximately 85,000 fragment retained basal pADPRp activity but was not activated by exogenous nicked DNA. Similar cleavage of pADPRp was observed after exposure of HL-60 cells to a variety of chemotherapeutic agents including cis-diaminedichloroplatinum(II), colcemid, 1-beta-D-arabinofuranosylcytosine, and methotrexate; to gamma-irradiation; or to the protein synthesis inhibitors puromycin or cycloheximide. Similar changes were observed in MDA-MB-468 human breast cancer cells treated with trifluorothymidine or 5-fluoro-2'-deoxyuridine and in gamma-irradiated or glucocorticoid-treated rat thymocytes undergoing apoptosis. Treatment with several compounds (tosyl-L-lysine chloromethyl ketone, tosyl-L-phenylalanine chloromethyl ketone, N-ethylmaleimide, iodoacetamide) prevented both the proteolytic cleavage of pADPRp and the internucleosomal fragmentation of DNA. The results suggest that proteolytic cleavage of pADPRp, in addition to being an early marker of chemotherapy-induced apoptosis, might reflect more widespread proteolysis that is a critical biochemical event early during the process of physiological cell death.

https://cancerres.aacrjournals.org/content/canres/53/17/3976.full.pdf

Specific Proteolytic Cleavage of Poly(ADP-ribose) Polymerase: An Early Marker of Chemotherapy-induced Apoptosis

Poly(ADP-ribosyl)ation is a post-translational modification of proteins. During this process, molecules of ADP-ribose are added successively on to acceptor proteins to form branched polymers. This modification is transient but very extensive in vivo, as polymer chains can reach more than 200 units on protein acceptors. The existence of the poly(ADP-ribose) polymer was first reported nearly 40 years ago. Since then, the importance of poly(ADP-ribose) synthesis has been established in many cellular processes. However, a clear and unified picture of the physiological role of poly(ADP-ribosyl)ation still remains to be established. The total dependence of poly(ADP-ribose) synthesis on DNA strand breaks strongly suggests that this post-translational modification is involved in the metabolism of nucleic acids. This view is also supported by the identification of direct protein-protein interactions involving poly(ADP-ribose) polymerase (113 kDa PARP), an enzyme catalysing the formation of poly(ADP-ribose), and key effectors of DNA repair, replication and transcription reactions. The presence of PARP in these multiprotein complexes, in addition to the actual poly(ADP-ribosyl)ation of some components of these complexes, clearly supports an important role for poly(ADP-ribosyl)ation reactions in DNA transactions. Accordingly, inhibition of poly(ADP-ribose) synthesis by any of several approaches and the analysis of PARP-deficient cells has revealed that the absence of poly(ADP-ribosyl)ation strongly affects DNA metabolism, most notably DNA repair. The recent identification of new poly(ADP-ribosyl)ating enzymes with distinct (non-standard) structures in eukaryotes and archaea has revealed a novel level of complexity in the regulation of poly(ADP-ribose) metabolism.

/pdf/poly-adp-ribosyl-ation-reactions-in-the-regulation-of-bcg8wwt2pc.pdf

Poly(adp-ribosyl)ation reactions in the regulation of nuclear functions

Chromatin proteins are covalently modified by at least five different processes; in no case has the precise physiological function been established. One of these post-synthetic, covalent modifications is effected by the enzyme poly(ADP-ribose) polymerase, which uses the coenzyme NAD+ to ADP-ribosylate chromatin proteins. The modification consists largely of mono(ADP-ribose), but long, homopolymer chains of (ADP-ribose) are also present. Various physiological functions have been suggested for (ADP-ribose)n. Here we demonstrate that one function of (ADP-ribose)n is to participate in the cellular recovery from DNA damage. Specific inhibitors of poly(ADP-ribose) polymerase prevent rejoining of DNA strand breaks caused by dimethyl sulphate and cytotoxicity is enhanced thereby. The rejoining of strand breaks is prevented also by nutritionally depleting the cells of NAD.

(ADP-ribose)n participates in DNA excision repair.

The DNA repair proteins XRCC1 and DNA ligase III are physically associated in human cells and directly interact in vitro and in vivo. Here, we demonstrate that XRCC1 is additionally associated with DNA polymerase-beta in human cells and that these polypeptides also directly interact. We also present data suggesting that poly (ADP-ribose) polymerase can interact with XRCC1. Finally, we demonstrate that DNA ligase III shares with poly (ADP-ribose) polymerase the novel function of a molecular DNA nick-sensor, and that the DNA ligase can inhibit activity of the latter polypeptide in vitro. Taken together, these data suggest that the activity of the four polypeptides described above may be co-ordinated in human cells within a single multiprotein complex.

XRCC1 Polypeptide Interacts with DNA Polymerase β and Possibly Poly (ADP-Ribose) Polymerase, and DNA Ligase III Is a Novel Molecular ‘Nick-Sensor’ In Vitro

Nuclear ADP-ribosyl transferase (ADPRT)1,2 catalyses the transfer of ADP-ribose from NAD+ to chromatin proteins to form mono-, oligo- and poly (ADP-ribose)-modified chromatin proteins3–7. The enzyme is entirely dependent on DNA for activity8 and is activated by nicks in the DNA9–11. Both radiation and alkylating agents deplete cells of NAD which is used to make (ADP-ribose)n (refs 1, 2, 12, 15–17), and this depletion is prevented by inhibitors of ADPRT13,15,18–25. ADPRT inhibitors also retard DNA strand-rejoining after damage1,2,26,27, and potentiate the lethality of DNA-damaging agents1,2,28. N-methyl-N′-nitro-N-nitrosoguanidine, an alkylating agent, transiently increases intracellular (ADP-ribose)n over 100-fold17. These data suggest that (ADP-ribose)n biosynthesis is necessary for effective repair of some types of DNA damage1. The question remains of which step in DNA repair requires ADPRT activity. The enzyme inhibitors do not prevent incision events1,2,27 nor do they inhibit repair replication29–31. Here we describe experiments which support the hypothesis that ADP-ribosylation activates DNA ligase activity. We suggest that ADPRT has a general function in the control of the breaking and rejoining of DNA.

Regulation of DNA ligase activity by poly(ADP-ribose).

The nuclear enzyme ADP-ribosyl transferase (ADPRT) catalyses the formation of poly(ADP-ribose)-modified chromatin proteins from NAD+ (refs 1–5) and is entirely dependent on DNA6 containing nicks7–11. Nuclear ADPRT activity is required for efficient DNA excision repair12,13, probably because it regulates DNA ligase activity14. Indirect evidence has suggested that ADPRT activity may also be involved in control of gene expression and cell differentiation15–21. We report here an obligatory involvement of ADPRT activity in the differentiation of muscle cells. Inhibitors of ADPRT activity reversibly inhibit both fusion of myoblasts to form multi-nucleate muscle fibres and the differentiation-specific increase in creatine phos-phokinase (CPK) activity. These two markers of differentiation can also be reversibly inhibited by depriving the cells of nicotinamide and thus lowering their cellular NAD content. Specific gene expression sometimes requires gene rearrangements or DNA transposition; this implies that DNA strand-breaking and rejoining might be involved in gene expression. We also describe the appearance during cytodifferentiation of single-strand DNA breaks which are not due to a general deficiency in DNA repair.

DNA strand breaks and ADP-ribosyl transferase activation during cell differentiation.

Both N-methylN-nitrosourea and γ-radiation lower cellular NAD in mouse leukaemia cells (L-1210) in a dose-dependent way. The minimum NAD level is reached 2 h after a brief exposure to N-methyl-N-nitrosourea, but within 15 min of y-irradiation. The cells remain metabolically active; they are able to recover their control NAD levels and are impermeable to trypan blue.



Several inhibitors of poly(ADP-ribose) polymerase inhibit the drop in cellular NAD caused by these two agents: 2 mM 5-methylnicotinamide, 1 mM theophylline or 1 mM theobromine inhibit the effect of N-methyl-N-nitrosourea on cellular NAD level; 200 uM thymidine, 500 uM 5-methyl-nicotinamide, 500 αM theophylline and 500 αM theobromine prevent the lowering of cellular NAD by y-irradiation. The extent to which the drop in cellular NAD is inhibited is dependent on both the concentration of cytotoxic agent and of polymerase inhibitor. Caffeine will inhibit the drop in NAD but only at 10 mM, while nicotonic acid is ineffective even at this dose.



The activity of poly(ADP-ribose) polymerase in permeabilized cells immediately after y-radiation increases with dose up to 12 krad, giving a maximal 3.4-fold stimulation of the enzyme activity, whereas the degradation of NAD under conditions optimal for NAD glycohydrolase does not change. The activity of the polymerase shows a close temporal correlation with the NAD drop following both γ-radiation and N-methyl-N-nitrosourea. The enzyme activity is maximal when the NAD content is decreasing at the highest rate and has returned to normal levels when it ceases falling.



In permeabilized cells we can distinguish poly(ADP-ribose) polymerase and NAD glycohydrolase activity by their differential response to inhibitors. The polymerase is sensitive to 5-methylnicotin-amide, theophylline, theobromine and thymidine; the NAD glycohydrolase is sensitive to 5-methyl-nicotinamide and theophylline, but not to theobromine and thymidine.



We propose that the decrease in cellular NAD level produced by y-radiation and by N-methyl-N-nitrosourea is caused by an increased flux through poly(ADP-ribose) mediated by an increased activity of poly(ADP-ribose) polymerase. This consequently lowers the cellular NAD level. This hypothesis implies an involvement of (ADP-ribose)n in the cellular response to cytotoxic drugs.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1979.tb04225.x

Sydney Shall

Papers

(ADP-ribose)n participates in DNA excision repair.

XRCC1 Polypeptide Interacts with DNA Polymerase β and Possibly Poly (ADP-Ribose) Polymerase, and DNA Ligase III Is a Novel Molecular ‘Nick-Sensor’ In Vitro

Regulation of DNA ligase activity by poly(ADP-ribose).

DNA strand breaks and ADP-ribosyl transferase activation during cell differentiation.

The involvement of poly(ADP-ribose) polymerase in the degradation of NAD caused by gamma-radiation and N-methyl-N-nitrosourea.