Advertisement

Survival and Proliferation of Cells Expressing Caspase-uncleavable Poly(ADP-ribose) Polymerase in Response to Death-inducing DNA Damage by an Alkylating Agent*

Open AccessPublished:December 24, 1999DOI:https://doi.org/10.1074/jbc.274.52.37097
      To determine whether caspase-3-induced cleavage of poly(ADP-ribose) polymerase (PARP), a DNA damage-sensitive enzyme, alters the balance between survival and death of the cells following DNA damage, we created stable cell lines that express either caspase-uncleavable mutant or wild type PARP in the background of PARP (−/−) fibroblasts. The survival and apoptotic responses of these cells were compared after exposure toN-methyl-N′-nitro-N-nitrosoguanidine (MNNG), a DNA-damaging agent that activates PARP, or to tumor necrosis factor-α, which causes apoptosis without initial DNA damage. In response to MNNG, the cells with caspase-uncleavable PARP were very resistant to loss of viability or induction of apoptosis. Most significantly, ∼25% of these cells survived and retained clonogenicity at a level of DNA damage that eliminated the cells with wild type PARP or PARP (−/−) cells. Expression of caspase-uncleavable PARP could not protect the cells from death induced by tumor necrosis factor, although there was a slower progression of apoptotic events in these cells. Therefore, one of the functions for cleavage of PARP during apoptosis induced by alkylating agents is to prevent survival of the extensively damaged cells.
      PARP
      poly(ADP-ribose) polymerase
      PARP-D/A
      caspase-uncleavable D214A mutant PARP
      PARP-wt
      wild type PARP
      DHQ
      1,5-dihydroxyisoquinoline
      MNNG
      N-methyl-N′-nitro-N-nitrosoguanidine
      pADPr
      polymer of ADP-ribose
      TNF-α
      tumor necrosis factor-α
      FITC
      fluorescein isothiocyanate
      TUNEL
      terminal dUTP nick-end labeling
      PBS
      phosphate-buffered saline
      Poly(ADP-ribose) polymerase (PARP),1 EC 2.4.2.30) a DNA repair-associated enzyme is involved in two diametrically opposite responses to DNA damage, i.e. DNA repair along with other survival responses at lower levels of DNA damage and cell death responses after saturating levels of DNA damage. One of the immediate responses to DNA damage in the higher eukaryotic cells is catalytic activation of PARP, which results in consumption of NAD and formation of the polymers of ADP-ribose (pADPr) on PARP itself and a few selected nuclear proteins that are involved in chromatin architecture and DNA-related metabolisms. This rapid reaction by PARP has been implicated in DNA base excision repair and in the maintenance of genomic integrity after DNA damage (reviewed in Refs.
      • Lindahl T.
      • Satoh M.S.
      • Poirier G.G.
      • Klungland A.
      ,
      • Lautier D.
      • Lagueux J.
      • Thibodeau J.
      • Menard L.
      • Poirier G.G.
      ,
      • de Murcia G.
      • Menissier-de Murcia J.
      ,
      • Oei S.L.
      • Griesenbeck J.
      • Schweiger M.
      ,
      • Oliver F.J.
      • Menissier-de Murcia J.
      • de Murcia G.
      ). Recently identified homologs of PARP that can synthesize pADPr raise the possibility that some of the survival functions of full-length PARP may be partially attributable to the PARP homologs (
      • Babiychuk E.
      • Cottrill P.B.
      • Storozhenko S.
      • Fuangthong M.
      • Chen Y.
      • O'Farrell M.K.
      • Van Montagu M.
      • Inze D.
      • Kushnir S.
      ,
      • Amé J.C.
      • Rolli V.
      • Schreiber V.
      • Niedergang C.
      • Apiou F.
      • Decker P.
      • Muller S.
      • Hoger T.
      • Menissier-de Murcia J.
      • de Murcia G.
      ,
      • Shieh W.M.
      • Amé J.C.
      • Wilson M.V.
      • Wang Z.Q.
      • Koh D.W.
      • Jacobson M.K.
      • Jacobson E.L.
      ,
      • Berghammer H.
      • Ebner M.
      • Marksteiner R.
      • Auer B.
      ,
      • Johansson M.
      ,
      • Smith S.
      • Giriat I.
      • Schmitt A.
      • de Lange T.
      ). However, PARP (−/−) mice or cells that possess at least one active PARP homolog (
      • Amé J.C.
      • Rolli V.
      • Schreiber V.
      • Niedergang C.
      • Apiou F.
      • Decker P.
      • Muller S.
      • Hoger T.
      • Menissier-de Murcia J.
      • de Murcia G.
      ,
      • Shieh W.M.
      • Amé J.C.
      • Wilson M.V.
      • Wang Z.Q.
      • Koh D.W.
      • Jacobson M.K.
      • Jacobson E.L.
      ,
      • Berghammer H.
      • Ebner M.
      • Marksteiner R.
      • Auer B.
      ) display high susceptibility to DNA-damaging agents, consistent with a dominant if not exclusive role for full-length PARP in the cellular responses to DNA damage. Two independently created PARP (−/−) mice with interruptions in different exons were shown to be highly susceptible to DNA-damaging agents such as γ-rays and alkylating agents (
      • Menissier-de Murcia J.
      • Niedergang C.
      • Trucco C.
      • Ricoul M.
      • Dutrillaux B.
      • Mark M.
      • Javier-Olivier M.F.
      • Masson M.
      • Dierich A.
      • LeMeur M.
      • Walztinger C.
      • Chambon P.
      • de Murcia G.
      ,
      • Wang Z.-Q.
      • Stingl L.
      • Morrison C.
      • Jantsch M.
      • Los M.
      • Schultz-Osthoff K.
      • Wagner E.F.
      ). Despite the initial lack of consensus among different studies with PARP (−/−) models, it is now clearly emerging that PARP (−/−) cells have significant deficiencies in response to DNA damage. These cells inherently possess shorter telomere and unstable chromosomes (
      • Fagagna F.
      • Hande M.P.
      • Tong W.M.
      • Lansdorp P.M.
      • Wang Z.Q.
      • Jackson S.P.
      ), and in response to genotoxic stress, they exhibit impaired proliferation, increased chromosomal abnormalities, G2/M block in cell cycle, and reduced capacity to repair the DNA damaged by alkylating agents (
      • Menissier-de Murcia J.
      • Niedergang C.
      • Trucco C.
      • Ricoul M.
      • Dutrillaux B.
      • Mark M.
      • Javier-Olivier M.F.
      • Masson M.
      • Dierich A.
      • LeMeur M.
      • Walztinger C.
      • Chambon P.
      • de Murcia G.
      ,
      • Wang Z.-Q.
      • Stingl L.
      • Morrison C.
      • Jantsch M.
      • Los M.
      • Schultz-Osthoff K.
      • Wagner E.F.
      ,
      • Wang Z.Q.
      • Auer B.
      • Stingl L.
      • Berghammer H.
      • Haidacher D.
      • Schweiger M.
      • Wagner E.F.
      ,
      • Trucco C.
      • Oliver F.J.
      • de Murcia G.
      • Menissier-de Murcia J.
      ) (reviewed in Refs.
      • Dantzer F.
      • Schreiber V.
      • Niedergang C.
      • Trucco C.
      • Flatter E.
      • De La Rubia G.
      • Oliver J.
      • Rolli V.
      • Menissier-de Murcia J.
      • de Murcia G.
      ,
      • Le Rhun Y.
      • Kirkland J.B.
      • Shah G.M.
      ,
      • Jeggo P.A.
      ). Recently, PARP (−/−) cells were shown to be specifically defective in long patch repair pathways of DNA base excision repair (
      • Dantzer F.
      • Schreiber V.
      • Niedergang C.
      • Trucco C.
      • Flatter E.
      • De La Rubia G.
      • Oliver J.
      • Rolli V.
      • Menissier-de Murcia J.
      • de Murcia G.
      ). Thus, in response to low or moderate levels of DNA damage, the activity of PARP could help in DNA repair and cell survival. However, PARP may have a completely different function(s) when death responses are initiated.
      During the initial phase of apoptotic death, when the majority of the cellular proteins remain intact, PARP is one of the earliest proteins to be specifically cleaved to form two fragments of ∼89 and 24 kDa (
      • Kaufmann S.H.
      • Desnoyers S.
      • Ottaviano Y.
      • Davidson N.E.
      • Poirier G.G.
      ). The 89-kDa fragment, although capable of the basal enzymatic activity due to presence of the catalytic domain (
      • Simonin F.
      • Menissier-de Murcia J.
      • Poch O.
      • Muller S.
      • Gradwohl G.
      • Molinete M.
      • Penning C.
      • Keith G.
      • de Murcia G.
      ), cannot be stimulated by the DNA strand breaks (
      • Kaufmann S.H.
      • Desnoyers S.
      • Ottaviano Y.
      • Davidson N.E.
      • Poirier G.G.
      ,
      • Shah G.M.
      • Kaufmann S.H.
      • Poirier G.G.
      ). The apoptosis-specific cleavage of PARP at the DEVD↓G site was shown to be due to a protease resembling interleukin-1β-converting enzyme (
      • Lazebnik Y.A.
      • Kaufmann S.H.
      • Desnoyers S.
      • Poirier G.G.
      • Earnshaw W.C.
      ) that was identified as CPP32, Yama, or Apopain and later classified as caspase 3 (
      • Fernandes-Alnemri T.
      • Litwack G.
      • Alnemri E.S.
      ,
      • Tewari M.
      • Quan L.T.
      • O'Rourke K.
      • Desnoyers S.
      • Zeng Z.
      • Beidler D.R.
      • Poirier G.G.
      • Salvesen G.S.
      • Dixit V.M.
      ,
      • Nicholson D.W.
      • Ali A.
      • Thornberry N.A.
      • Vaillancourt J.P.
      • Ding C.K.
      • Gallant M.
      • Gareau Y.
      • Griffin P.R.
      • Labelle M.
      • Lazebnik Y.A.
      • Munday N.A.
      • Raju S.M.
      • Smulson M.E.
      • Yamin T.-T.
      • Yu V.L.
      • Miller D.K.
      ,
      • Alnemri E.S.
      • Livingston D.J.
      • Nicholson D.W.
      • Salvesen G.
      • Thornberry N.A.
      • Wong W.W.
      • Yuan J.
      ). Although PARP cleavage has served as a sensitive analytical reporter for the activation of caspases or for the onset of apoptosis in various models of cell death, the physiological role for this cleavage is not known (
      • Duriez P.J.
      • Shah G.M.
      ,
      • Scovassi A.I.
      • Dengri M.
      • Donzelli M.
      • Rossi L.
      • Bernardi R.
      • Mandarino A.
      • Frouin I.
      • Negri C.
      ). For many of the ∼50 other caspase targets, their cleavage serves different functions, such as inactivation of anti-apoptotic factors, activation of proapoptotic factors, or disassembly of cellular structures (
      • Thornberry N.A.
      • Lazebnik Y.
      ). However, for some substrates such as PARP, DNA-dependent protein kinase, and DNA replication factor C, it has been assumed that disabling their normal survival functions in DNA repair and replication would facilitate cell death (
      • Thornberry N.A.
      • Lazebnik Y.
      ,
      • Rosen A.
      • Casciola-Rosen L.
      ). Additionally, the cleaved fragments of PARP might acquire novel functions that could actively contribute to apoptosis. The caspase-induced cleavage of PARP might result in production of an enzyme with altered substrate specificity or subcellular localization. This altered enzyme might actively potentiate the apoptotic response. The 24-kDa fragment of PARP has been shown to bind to apoptotic fragments of DNA (
      • Smulson M.E.
      • Pang D.
      • Jung M.
      • Dimtchev A.
      • Chasovskikh S.
      • Spoonde A.
      • Simbulan-Rosenthal C.
      • Rosenthal D.
      • Yakovlev A.
      • Dritschilo A.
      ), and this could facilitate death decisions by competing with any remaining intact PARP or other proteins for binding to strand breaks.
      The catalytic function of PARP is also implicated in cell death responses following massive amounts of DNA damage that are encountered during certain pathological conditions. A prolonged and high level of catalytic activation of PARP under these circumstances has been suggested to lead to cell death by energy deprivation due to depletion of NAD and ATP (
      • Berger N.A.
      ). This role of PARP is strongly supported by recent studies demonstrating resistance of PARP (−/−) neuronal, cardiac and pancreatic cells to death caused by extensive DNA damage with ischemia-reperfusion or streptozotocin (
      • Burkart V.
      • Wang Z.Q.
      • Radons J.
      • Heller B.
      • Herceg Z.
      • Stingl L.
      • Wagner E.F.
      • Kolb H.
      ,
      • Eliasson M.J.L.
      • Sampei K.
      • Mandir A.S.
      • Hurn P.D.
      • Traystman R.J.
      • Bao J.
      • Pieper A.
      • Wang Z.Q.
      • Dawson T.M.
      • Snyder S.H.
      • Dawson V.L.
      ,
      • Pieper A.A.
      • Brat D.J.
      • Krug D.K.
      • Watkins C.C.
      • Gupta A.
      • Blackshaw S.
      • Verma A.
      • Wang Z.Q.
      • Snyder S.H.
      ,
      • Masutani M.
      • Suzuki H.
      • Kamada N.
      • Watanabe M.
      • Ueda O.
      • Nozaki T.
      • Jishage K.
      • Watanabe T.
      • Sugimoto T.
      • Nakagama H.
      • Ochiya T.
      • Sugimura T.
      ). This function of PARP in cell death would be eliminated when it is cleaved by caspases, because the 89-kDa fragment of PARP cannot be stimulated in the presence of DNA breaks (
      • Kaufmann S.H.
      • Desnoyers S.
      • Ottaviano Y.
      • Davidson N.E.
      • Poirier G.G.
      ,
      • Shah G.M.
      • Kaufmann S.H.
      • Poirier G.G.
      ).
      Thus, PARP has been implicated in both the survival and death responses following DNA damage. PARP cleavage by caspases could play a crucial role in shifting the equilibrium between its two roles, and this can be explored in a model where PARP is rendered resistant to cleavage by caspase 3. The caspase-resistant PARP could strengthen the cellular responses to DNA damage, resulting in delayed cell death and possible survival of the damaged cells. On the other hand, uncleavable PARP could accelerate death by continued depletion of NAD and ATP. To distinguish between these alternatives, we have created stable cell lines that express either caspase-uncleavable mutant or wild type PARP in a PARP (−/−) fibroblast background. Our study for the first time compared the death and survival responses of these stable cell lines after exposure to alkylating DNA damage that activates PARP as part of the survival response or to tumor necrosis factor-α (TNF-α), which can cause apoptosis without initial DNA damage. We hypothesized that response of the cells expressing caspase-uncleavable PARP would be different in these two situations. In response to alkylating DNA damage, uncleaved PARP might have both survival and death functions, whereas in response to TNF-α it might serve only the death functions. Three recently reported studies used a similar approach and focused mainly on the death responses of cells expressing caspase-uncleavable PARP (
      • Oliver F.J.
      • de la Rubia G.
      • Rolli V.
      • Ruiz-Ruiz M.C.
      • de Murcia G.
      • Murcia J.M.
      ,
      • Herceg Z.
      • Wang Z.Q.
      ,
      • Boulares A.H.
      • Yakovlev A.G.
      • Ivanova V.
      • Stoica B.A.
      • Wang G.
      • Iyer S.
      • Smulson M.
      ). As described later, one of these studies used a transient transfection approach (
      • Oliver F.J.
      • de la Rubia G.
      • Rolli V.
      • Ruiz-Ruiz M.C.
      • de Murcia G.
      • Murcia J.M.
      ), and the other two studies using stable cell lines focused mainly on the death responses after exposure to TNF-α or staurosporine (
      • Herceg Z.
      • Wang Z.Q.
      ,
      • Boulares A.H.
      • Yakovlev A.G.
      • Ivanova V.
      • Stoica B.A.
      • Wang G.
      • Iyer S.
      • Smulson M.
      ), treatments that are not likely to involve survival functions of PARP induced by DNA damage. We report here that in response to moderate levels of DNA damage by the alkylating agent MNNG, cells expressing caspase-uncleavable PARP exhibit reduced cell death and increased cell survival under conditions where cells with wild type PARP are eliminated. When death is induced in the absence of initial DNA damage by TNF-α, the presence of the caspase-uncleavable PARP could not prevent the ultimate apoptotic demise of the cell, and in fact, the progression of apoptosis is slower than in the cells expressing wild type PARP.

      DISCUSSION

      At present, nearly 50 proteins are known to be cleaved by caspases during apoptotic death. Cleavage of some of these proteins actively promotes death, whereas cleavage of others may be permissive for the death pathway. If cleavage of these substrates were essential for apoptotic progression, then introduction of uncleavable forms of the protein might be expected to exert a significant protective effect. However, no long term protective effects have been reported after introduction of several caspase-resistant substrates, e.g.Bcl-2 (
      • Cheng E.H.
      • Kirsch D.G.
      • Clem R.J.
      • Ravi R.
      • Kastan M.B.
      • Bedi A.
      • Ueno K.
      • Hardwick J.M.
      ), RB1 (
      • Tan X.
      • Wang J.Y.
      ), p21 cip1/waf1 (
      • Levkau B.
      • Koyama H.
      • Raines E.W.
      • Clurman B.E.
      • Herren B.
      • Orth K.
      • Roberts J.M.
      • Ross R.
      ), MEKK-1 (
      • Cardone M.H.
      • Salvesen G.S.
      • Widmann C.
      • Johnson G.
      • Frisch S.M.
      ), PAK-2 (
      • Rudel T.
      • Bokoch G.M.
      ), DFF45 (
      • Wohrl W.
      • Hacker G.
      ), and the lamins (
      • Rao L.
      • Perez D.
      • White E.
      ). Our study demonstrates for the first time that expression of a caspase-uncleavable PARP not only temporarily slows down apoptotic events following DNA damage but also allows some of the cells to recover and proliferate. However, this protection has two limitations. (a) It is most evident at potentially lethal low levels of DNA damage by alkylating agents and not against death induced by saturating amounts of DNA damage (e.g. 100 μmMNNG), and (b) it is not a general protection against other forms of death, e.g. TNF-α-mediated apoptosis.
      There are important differences in the role of PARP in cellular responses to MNNG and to TNF-α. In response to MNNG-induced DNA damage, PARP is activated immediately and participates in DNA repair processes until it is cleaved by the caspases. We have considered two reasons why cells expressing uncleavable PARP survive better when faced with DNA damage induced by MNNG. First, uncleavable PARP might remain fully functional and participate in DNA repair processes after wild type PARP has been inactivated. Second, uncleavable PARP, activated due to the presence of DNA damage, might ADP-ribosylate and inactivate proapoptotic factors, thereby directly inhibiting apoptotic progression. Therefore, inhibition of the catalytic function of uncleavable PARP abrogates its protective effects against MNNG (Table I). In contrast, during TNF-α-mediated death, the absence of initial DNA damage would preclude early and persistent activation of PARP that could help in DNA repair. Furthermore, a brief early burst of PARP activation that does occur during Fas treatment appears to trigger apoptosis rather then help in cell survival (
      • Simbulan-Rosenthal C.M.
      • Rosenthal D.S.
      • Iyer S.
      • Boulares A.H.
      • Smulson M.E.
      ). Thus, the actions of the uncleavable PARP could protect cells against MNNG-induced DNA damage but not during TNF-α-induced apoptosis.
      One question that needs to be addressed is whether the protective effects exerted by the uncleavable PARP in response to DNA damage reflect action of the enzyme before or after activation of the first caspases. Boise and Thompson (
      • Boise L.H.
      • Thompson C.B.
      ) have demonstrated that removal of the apoptotic stimulus even after activation of caspase permits the cells expressing Bcl-xL to recover and resume proliferation. Thus, the first activation of caspases occurs at a stage when the cell survival hangs in the balance, and actions of the uncleavable PARP before and after caspase activation could collectively shift the balance in favor of recovery.
      Protection offered by uncleavable PARP to low levels of DNA damage must be subject to influence by other factors, because only 25% of the cells retain clonogenicity (Fig. 5). This partial penetrance could arise if the susceptibility of cells to apoptosis-inducing signals varies as a function of cell cycle position. In many models of cell death, it has been observed that after receipt of a death-inducing signal, the actual execution of individual cells occurs asynchronously. It could be that execution of the cell after receipt of a lethal signal is a random process, or more likely, it may be influenced by other factors, such as cell cycle position of each cell (
      • Terui Y.
      • Furukawa Y.
      • Kikuchi J.
      • Saito M.
      ). Earlier studies have demonstrated the cell cycle-specific ADP-ribosylation of proteins in response to γ-irradiation (
      • Ramsamooj P.
      • Prasad S.
      • Dritschilo A.
      • Notario V.
      ). Together, our results suggest that rescue of cells by caspase-uncleavable PARP may be efficient only for cells in a specific phase of the cell cycle that receive a low dose of DNA damage of the type that normally activates PARP.
      The higher susceptibility of PARP (−/−) cells to MNNG is in agreement with an earlier study that used another alkylating agent, methyl methane sulfonate to induce the DNA damage (
      • Oliver F.J.
      • de la Rubia G.
      • Rolli V.
      • Ruiz-Ruiz M.C.
      • de Murcia G.
      • Murcia J.M.
      ). However, a selective survival advantage for cells expressing uncleavable PARP, shown here, was not observed in that study (
      • Oliver F.J.
      • de la Rubia G.
      • Rolli V.
      • Ruiz-Ruiz M.C.
      • de Murcia G.
      • Murcia J.M.
      ). Apart from limitations in the experimental analyses of cell death in transient transfections, the earlier study also used a very high dose of 500 μm that was lethal within 1 h. In contrast, we used lower doses of MNNG that caused no initial lethality, whereas the use of stable clones permitted us a more reliable survival estimate by analyzing an entire population of cells with an identical PARP phenotype.
      Importantly, we found that in cells treated with high levels of MNNG (100 μm), the presence of uncleavable PARP exerted neither a protective nor harmful effect. We did not observe a high stimulation of catalytic function of the uncleavable PARP from 24 to 96 h after exposure to high doses of MNNG (data not shown). It is therefore unlikely that consumption of NAD by catalytic activity of the uncleavable PARP was critical in death caused by higher levels of MNNG-induced DNA damage. It is interesting to compare these results with recent demonstrations of the role of NAD consumption by PARP in cell death caused by ischemia-reperfusion or streptozotocin (
      • Burkart V.
      • Wang Z.Q.
      • Radons J.
      • Heller B.
      • Herceg Z.
      • Stingl L.
      • Wagner E.F.
      • Kolb H.
      ,
      • Eliasson M.J.L.
      • Sampei K.
      • Mandir A.S.
      • Hurn P.D.
      • Traystman R.J.
      • Bao J.
      • Pieper A.
      • Wang Z.Q.
      • Dawson T.M.
      • Snyder S.H.
      • Dawson V.L.
      ,
      • Pieper A.A.
      • Brat D.J.
      • Krug D.K.
      • Watkins C.C.
      • Gupta A.
      • Blackshaw S.
      • Verma A.
      • Wang Z.Q.
      • Snyder S.H.
      ,
      • Masutani M.
      • Suzuki H.
      • Kamada N.
      • Watanabe M.
      • Ueda O.
      • Nozaki T.
      • Jishage K.
      • Watanabe T.
      • Sugimoto T.
      • Nakagama H.
      • Ochiya T.
      • Sugimura T.
      ). These pathological conditions induce massive amounts of DNA damage and cause death of specific target cells by both apoptosis and necrosis (
      • Le Rhun Y.
      • Kirkland J.B.
      • Shah G.M.
      ,
      • Burkart V.
      • Wang Z.Q.
      • Radons J.
      • Heller B.
      • Herceg Z.
      • Stingl L.
      • Wagner E.F.
      • Kolb H.
      ,
      • Charron M.J.
      • Bonner-Weir S.
      ). Under these circumstances, all or most of the wild type PARP must remain uncleaved by the caspases, because death of the target cells is linked to NAD and ATP depletion by the highly activated PARP. Together, our results suggest that the extent of DNA damage, type of death-inducing agent, and susceptibility of the target cell might determine whether catalytic or cleavage functions of PARP would be implicated in survival or apoptotic/necrotic death of the cell.
      Our results suggest that stimulation of the TNF-α-induced death pathways is more strongly correlated with caspase cleavage of the wild type PARP than with the catalytic function of the uncleavable PARP. This is observed from the highest (50-fold) susceptibility of PARP-wt cells and intermediate (5-fold) susceptibility of PARP-D/A cells, as compared with PARP (−/−) cells (Fig. 6 A). In contrast to our results, two recent studies reported that cells expressing uncleavable PARP are more prone to TNF-α-induced apoptosis (
      • Herceg Z.
      • Wang Z.Q.
      ,
      • Boulares A.H.
      • Yakovlev A.G.
      • Ivanova V.
      • Stoica B.A.
      • Wang G.
      • Iyer S.
      • Smulson M.
      ) as well as necrosis (
      • Herceg Z.
      • Wang Z.Q.
      ), as compared with PARP-wt or PARP (−/−) cells. However, 6 h after TNF-α treatment, we did not observe necrotic death in the cells expressing uncleavable PARP. Unlike the earlier study (
      • Herceg Z.
      • Wang Z.Q.
      ), we used much lower concentrations of TNF-α (10–160× less) and actinomycin D (4× less), because in our experience treatment with higher doses of actinomycin D (1 μg/ml) was sufficient to elicit death response even in the absence of TNF-α. Thus, a mixed mode of death induced by both actinomycin D and TNF-α in the other studies could explain the differences observed in our studies. It is also possible that together, our studies reflect different snapshots of the TNF-α-treated cells, i.e.reduced apoptosis at 6 h, increased form of necrotic death at 12 h, and complete loss of viability by 24 h.
      In conclusion, the most significant advantage associated with expression of uncleavable PARP was observed when DNA damage was low enough for the cells to remain viable for a significant period of time before commitment to either survival or death. At this stage, cells with wild type PARP succumbed, whereas some of the cells with uncleavable PARP survived. Therefore, PARP activation and its cleavage might influence crucial life and death decisions relatively early in the apoptotic pathway.

      ACKNOWLEDGEMENTS

      We are thankful to following researchers for providing different reagents: Dr. Z. Q. Wang for PARP (−/−) and (+/+) cell lines; Drs. M. Miwa and A. Burkle for monoclonal anti-polymer 10H; Dr. J. H. Kupper for cDNA of human PARP; Dr. G. Poirier for monoclonal anti-PARP C-2–10; and Dr. D. Nicholson for rabbit polyclonal anti-caspase 3-R#MF393. We also thank Dr. Z. Q. Wang for critical reading of the manuscript. M. Dufour at the Flow Cytometry Service of the CHUL Research Center carried out fluorescence-activated cell sorter analysis.

      REFERENCES

        • Lindahl T.
        • Satoh M.S.
        • Poirier G.G.
        • Klungland A.
        Trends Biochem. Sci. 1995; 20: 405-411
        • Lautier D.
        • Lagueux J.
        • Thibodeau J.
        • Menard L.
        • Poirier G.G.
        Mol. Cell. Biochem. 1993; 122: 171-193
        • de Murcia G.
        • Menissier-de Murcia J.
        Trends Biochem. Sci. 1994; 19: 172-176
        • Oei S.L.
        • Griesenbeck J.
        • Schweiger M.
        Rev. Physiol. Biochem. Pharmacol. 1997; 131: 127-173
        • Oliver F.J.
        • Menissier-de Murcia J.
        • de Murcia G.
        Am. J. Hum. Genet. 1999; 64: 1282-1288
        • Babiychuk E.
        • Cottrill P.B.
        • Storozhenko S.
        • Fuangthong M.
        • Chen Y.
        • O'Farrell M.K.
        • Van Montagu M.
        • Inze D.
        • Kushnir S.
        Plant J. 1998; 15: 635-645
        • Amé J.C.
        • Rolli V.
        • Schreiber V.
        • Niedergang C.
        • Apiou F.
        • Decker P.
        • Muller S.
        • Hoger T.
        • Menissier-de Murcia J.
        • de Murcia G.
        J. Biol. Chem. 1999; 274: 17860-17868
        • Shieh W.M.
        • Amé J.C.
        • Wilson M.V.
        • Wang Z.Q.
        • Koh D.W.
        • Jacobson M.K.
        • Jacobson E.L.
        J. Biol. Chem. 1998; 273: 30069-30072
        • Berghammer H.
        • Ebner M.
        • Marksteiner R.
        • Auer B.
        FEBS Lett. 1999; 449: 259-263
        • Johansson M.
        Genomics. 1999; 57: 442-445
        • Smith S.
        • Giriat I.
        • Schmitt A.
        • de Lange T.
        Science. 1998; 282: 1484-1487
        • Menissier-de Murcia J.
        • Niedergang C.
        • Trucco C.
        • Ricoul M.
        • Dutrillaux B.
        • Mark M.
        • Javier-Olivier M.F.
        • Masson M.
        • Dierich A.
        • LeMeur M.
        • Walztinger C.
        • Chambon P.
        • de Murcia G.
        Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7303-7307
        • Wang Z.-Q.
        • Stingl L.
        • Morrison C.
        • Jantsch M.
        • Los M.
        • Schultz-Osthoff K.
        • Wagner E.F.
        Genes Dev. 1997; 11: 2347-2358
        • Fagagna F.
        • Hande M.P.
        • Tong W.M.
        • Lansdorp P.M.
        • Wang Z.Q.
        • Jackson S.P.
        Nat. Genet. 1999; 23: 76-80
        • Wang Z.Q.
        • Auer B.
        • Stingl L.
        • Berghammer H.
        • Haidacher D.
        • Schweiger M.
        • Wagner E.F.
        Genes Dev. 1995; 9: 509-520
        • Trucco C.
        • Oliver F.J.
        • de Murcia G.
        • Menissier-de Murcia J.
        Nucleic Acids Res. 1998; 26: 2644-2649
        • Dantzer F.
        • Schreiber V.
        • Niedergang C.
        • Trucco C.
        • Flatter E.
        • De La Rubia G.
        • Oliver J.
        • Rolli V.
        • Menissier-de Murcia J.
        • de Murcia G.
        Biochimie ( Paris ). 1999; 81: 69-75
        • Le Rhun Y.
        • Kirkland J.B.
        • Shah G.M.
        Biochem. Biophys. Res. Commun. 1998; 245: 1-10
        • Jeggo P.A.
        Curr. Biol. 1998; 8: 49-51
        • Kaufmann S.H.
        • Desnoyers S.
        • Ottaviano Y.
        • Davidson N.E.
        • Poirier G.G.
        Cancer Res. 1993; 53: 3976-3985
        • Simonin F.
        • Menissier-de Murcia J.
        • Poch O.
        • Muller S.
        • Gradwohl G.
        • Molinete M.
        • Penning C.
        • Keith G.
        • de Murcia G.
        J. Biol. Chem. 1990; 265: 19249-19256
        • Shah G.M.
        • Kaufmann S.H.
        • Poirier G.G.
        Anal. Biochem. 1995; 232: 251-254
        • Lazebnik Y.A.
        • Kaufmann S.H.
        • Desnoyers S.
        • Poirier G.G.
        • Earnshaw W.C.
        Nature. 1994; 371: 346-347
        • Fernandes-Alnemri T.
        • Litwack G.
        • Alnemri E.S.
        J. Biol. Chem. 1994; 269: 30761-30764
        • Tewari M.
        • Quan L.T.
        • O'Rourke K.
        • Desnoyers S.
        • Zeng Z.
        • Beidler D.R.
        • Poirier G.G.
        • Salvesen G.S.
        • Dixit V.M.
        Cell. 1995; 81: 801-809
        • Nicholson D.W.
        • Ali A.
        • Thornberry N.A.
        • Vaillancourt J.P.
        • Ding C.K.
        • Gallant M.
        • Gareau Y.
        • Griffin P.R.
        • Labelle M.
        • Lazebnik Y.A.
        • Munday N.A.
        • Raju S.M.
        • Smulson M.E.
        • Yamin T.-T.
        • Yu V.L.
        • Miller D.K.
        Nature. 1995; 376: 37-43
        • Alnemri E.S.
        • Livingston D.J.
        • Nicholson D.W.
        • Salvesen G.
        • Thornberry N.A.
        • Wong W.W.
        • Yuan J.
        Cell. 1996; 87: 171
        • Duriez P.J.
        • Shah G.M.
        Biochem. Cell Biol. 1997; 75: 337-349
        • Scovassi A.I.
        • Dengri M.
        • Donzelli M.
        • Rossi L.
        • Bernardi R.
        • Mandarino A.
        • Frouin I.
        • Negri C.
        Eur. J. Histochem. 1998; 42: 251-258
        • Thornberry N.A.
        • Lazebnik Y.
        Science. 1998; 281: 1312-1316
        • Rosen A.
        • Casciola-Rosen L.
        J. Cell. Biochem. 1997; 64: 50-54
        • Smulson M.E.
        • Pang D.
        • Jung M.
        • Dimtchev A.
        • Chasovskikh S.
        • Spoonde A.
        • Simbulan-Rosenthal C.
        • Rosenthal D.
        • Yakovlev A.
        • Dritschilo A.
        Cancer Res. 1998; 58: 3495-3498
        • Berger N.A.
        Radiat. Res. 1985; 101: 4-15
        • Burkart V.
        • Wang Z.Q.
        • Radons J.
        • Heller B.
        • Herceg Z.
        • Stingl L.
        • Wagner E.F.
        • Kolb H.
        Nat. Med. 1999; 5: 314-319
        • Eliasson M.J.L.
        • Sampei K.
        • Mandir A.S.
        • Hurn P.D.
        • Traystman R.J.
        • Bao J.
        • Pieper A.
        • Wang Z.Q.
        • Dawson T.M.
        • Snyder S.H.
        • Dawson V.L.
        Nat. Med. 1997; 3: 1089-1095
        • Pieper A.A.
        • Brat D.J.
        • Krug D.K.
        • Watkins C.C.
        • Gupta A.
        • Blackshaw S.
        • Verma A.
        • Wang Z.Q.
        • Snyder S.H.
        Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3059-3064
        • Masutani M.
        • Suzuki H.
        • Kamada N.
        • Watanabe M.
        • Ueda O.
        • Nozaki T.
        • Jishage K.
        • Watanabe T.
        • Sugimoto T.
        • Nakagama H.
        • Ochiya T.
        • Sugimura T.
        Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2301-2304
        • Oliver F.J.
        • de la Rubia G.
        • Rolli V.
        • Ruiz-Ruiz M.C.
        • de Murcia G.
        • Murcia J.M.
        J. Biol. Chem. 1998; 273: 33533-33539
        • Herceg Z.
        • Wang Z.Q.
        Mol. Cell. Biol. 1999; 19: 5124-5133
        • Boulares A.H.
        • Yakovlev A.G.
        • Ivanova V.
        • Stoica B.A.
        • Wang G.
        • Iyer S.
        • Smulson M.
        J. Biol. Chem. 1999; 274: 22932-22940
        • Shah G.M.
        • Poirier D.
        • Desnoyers S.
        • Saint-Martin S.
        • Hoflack J.C.
        • Rong P.
        • ApSimon M.
        • Kirkland J.B.
        • Poirier G.G.
        Biochim. Biophys. Acta. 1996; 1312: 1-7
        • Shah G.M.
        • Shah R.G.
        • Poirier G.G.
        Biochem. Biophys. Res. Commun. 1996; 229: 838-844
        • Kawamitsu H.
        • Hoshino H.
        • Okada H.
        • Miwa M.
        • Momoi H.
        • Sugimura T.
        Biochemistry. 1984; 23: 3771-3777
        • Shah G.M.
        • Poirier D.
        • Duchaine C.
        • Brochu G.
        • Desnoyers S.
        • Lagueux J.
        • Verreault A.
        • Hoflack J.C.
        • Kirkland J.B.
        • Poirier G.G.
        Anal. Biochem. 1995; 227: 1-13
        • Froelich C.J.
        • Orth K.
        • Turbov J.
        • Seth P.
        • Gottlieb R.
        • Babior B.
        • Shah G.M.
        • Bleackley R.C.
        • Dixit V.M.
        • Hanna W.
        J. Biol. Chem. 1996; 271: 29073-29079
        • Huot J.
        • Houle F.
        • Rousseau S.
        • Deschesnes R.G.
        • Shah G.M.
        • Landry J.
        J. Cell Biol. 1998; 143: 1361-1373
        • Ruscetti T.
        • Lehnert B.E.
        • Halbrook J.
        • Le Trong H.
        • Hoekstra M.F.
        • Chen D.J.
        • Peterson S.R.
        J. Biol. Chem. 1998; 273: 14461-14467
        • Simbulan-Rosenthal C.M.
        • Rosenthal D.S.
        • Iyer S.
        • Boulares A.H.
        • Smulson M.E.
        J. Biol. Chem. 1998; 273: 13703-13712
        • Jacobson E.L.
        • Smith J.
        • Wielckens K.
        • Hilz H.
        • Jacobson M.K.
        Carcinogenesis. 1985; 6: 715-718
        • Banasik M.
        • Komura H.
        • Shimoyama M.
        • Ueda K.
        J. Biol. Chem. 1992; 267: 1569-1575
        • Enari M.
        • Talanian R.V.
        • Wong W.W.
        • Nagata S.
        Nature. 1996; 380: 723-726
        • Casiano C.A.
        • Ochs R.L.
        • Tan E.M.
        Cell Death Differ. 1998; 5: 183-190
        • Cheng E.H.
        • Kirsch D.G.
        • Clem R.J.
        • Ravi R.
        • Kastan M.B.
        • Bedi A.
        • Ueno K.
        • Hardwick J.M.
        Science. 1997; 278: 1966-1968
        • Tan X.
        • Wang J.Y.
        Trends Cell Biol. 1998; 8: 116-120
        • Levkau B.
        • Koyama H.
        • Raines E.W.
        • Clurman B.E.
        • Herren B.
        • Orth K.
        • Roberts J.M.
        • Ross R.
        Mol. Cell. 1998; 1: 553-563
        • Cardone M.H.
        • Salvesen G.S.
        • Widmann C.
        • Johnson G.
        • Frisch S.M.
        Cell. 1997; 90: 315-323
        • Rudel T.
        • Bokoch G.M.
        Science. 1997; 276: 1571-1574
        • Wohrl W.
        • Hacker G.
        Biochem. Biophys. Res. Commun. 1999; 254: 552-558
        • Rao L.
        • Perez D.
        • White E.
        J. Cell Biol. 1996; 135: 1441-1455
        • Boise L.H.
        • Thompson C.B.
        Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3759-3764
        • Terui Y.
        • Furukawa Y.
        • Kikuchi J.
        • Saito M.
        J. Cell. Physiol. 1995; 164: 74-84
        • Ramsamooj P.
        • Prasad S.
        • Dritschilo A.
        • Notario V.
        Int. J. Oncol. 1996; 8: 803-808
        • Charron M.J.
        • Bonner-Weir S.
        Nat. Med. 1999; 5: 269-270