A Matter of Life and Cell Death

STUFF OF LIFE

PARP-1 activity was first detected in 1963, when Pierre Chambon and colleagues added nicotinamide mononucleotide (NMN) to rat liver nuclear extracts and observed the production of a new DNA-dependent polyadenylic acid,³ which was identified later as poly(ADP-ribose) (PAR). But two decades passed before poly(ADP-ribosyl)ation was implicated in DNA repair, after PARP inhibitors were found to sensitize cells to DNA-damaging agents.

Yet while the role proposed for PARP-1 in DNA repair is an important one, PARP-1 deficiency is not lethal to mice, says Keith Caldecott of the University of Sussex Genome Damage and Stability Center. "That had always been a bit of a puzzle," he says, especially as knocking out PARP-1's partner molecule, X-ray cross-complementing factor1 (XRCC1), does produce a lethal phenotype.

It turned out that more than one PARP exists. The detection of residual DNA-dependent PARP activity in embryonic PARP-1-knockout mouse fibroblasts led to the discovery of PARP-2 (18 PARP family members are now recognized⁴), and mice lacking both PARP-1 and PARP-2 are lethally affected, says Caldecott. de Murcia's paper delves into the role of PARP-2. The French group found that PARPs 1 and 2 form heterodimers, and that PARP-2 interacts with known partners of PARP-1 (XRCC1, DNA polymerase β, and DNA ligase III), although de Murcia says that the two molecules preferentially modify different histones (H1 and H2B, respectively). Treatment of PARP-2-deficient cells displayed a delay in the resealing of DNA strand breaks similar to that found in PARP-1 knockouts.¹

AGENT OF DEATH

While de Murcia's study is, according to Caldecott, "a very nitty-gritty type of paper" dealing with the mechanism of DNA repair, Dawson's paper "raises new possibilities for how PARP might be impacting on DNA-damage detection, and the interface with apoptosis."

Szabó says that during the first twenty years of PARP research, the emphasis was on the molecule's function as an early target for cleavage by caspases during apoptosis. It is a line of investigation that he says turned out to be "kind of a blind alley," because though PARP cleavage remains an important marker for apoptotic activity, PARP is now not implicated in its execution. But Dawson's work implicates PARP in the execution of a cell-death pathway that she says shares similarities with apoptosis.

© 2004 Elsevier Ltd.

PARP-1 and AIF may act in glutamate-induced excitotoxicity. Glutamate increases Ca²⁺ influx, activating neuronal nitric oxide syntase (nNOS). Mitochondrial uptake of CA²⁺ and preoxynitrite (ONOO^-) block mitochondrial respiration increasing reactive oxygen species and decreasing energy production. In the nucleus ONOO^- damages DNA activating PARP-1 which results in energy depletion and translocation of AIF into the nucleus. (adapted from S.J. Hong etal., Trends Pharmocol Sci, 25:259–64, 2004.)

Her paper came about in the context of her research on the pathologic death of brain neurons, particularly in relation to ischemia-related injury such as stroke, and degenerative diseases such as Parkinson. She had been working on an excitotoxicity pathway studied since the 1970s, in which the neurotransmitter glutamate kick-starts a chain of events leading, via the NMDA receptor, calcium, nitric oxide, and peroxynitrite, to cell necrosis.

In the early 1990s, Dawson had been part of a team that had found PARP inhibitors protecting cells against nitric oxide-induced death. "That paper was published in Science, but the PARP community was not impressed." And for good reason, Dawson says, as the drugs had not been PARP-specific. "PARP-knockout mice were really the clincher," she says. "They were protected against experimental stroke, experimental heart attack, arthritis, Parkinson's disease ... much more than any other manipulation at that time."

Dawson says that electron micrographs of cells following an exci-totoxic challenge revealed morphological features of both classic apoptosis and necrosis. There was no evidence of apoptotic bodies, for example, but neither did the cells simply burst open in a necrotic fashion. "We didn't know what to do with this," she says. The break came when the team spotted similar electron micrographs in a paper describing apoptosis-inducing factor (AIF), a protein released from mitochondria during apoptosis that condenses chromatin and fragments DNA.⁵ So Dawson recloned AIF, made antibodies, and found that AIF did indeed move from the mitochondria to the nucleus following PARP activation with NMDA exposure of mouse fibroblasts, a process that was prevented by PARP inhibition.

A MATTER OF DEGREE

PARP-1's involvement in both cell repair and cell death raises the question of how to reconcile these apparently contradictory functions. "It's possible that this paradox is resolved essentially by the way the cell determines how much DNA damage it will tolerate before it triggers cell death, says Caldecott. "So PARP would act as a sort of rheostat." Szabó says it is a line of thought that goes back twenty years, when Nathan Berger proposed his "suicide hypothesis"⁶ whereby over-activation of PARP can lead to depletion of its NAD+ substrate, and hence an energy crisis that induces cell necrosis.

Dawson maintains that her PARP-1-dependent cell-death pathway is not necrotic. Nor is it apoptotic, she says. Like the morphology, the biochemistry has a mixed necrotic-apoptotic flavor. On the one hand, NAD depletion suggests necrosis; on the other, apoptotic caspases are activated (although inhibiting them has no protective effect.) Dawson speaks of it in terms of a pathologic pathway that uses components of the programmed machinery of apoptosis.

Dawson accepts that NAD+ deletion is likely to contribute to the death of cells in her PARP-1-dependent system, but points out that the importance of AIF in the process is demonstrated by the protective effect of its neutralization with AIF-specific antibodies.

Regardless of what the pathway is called, Dawson says that PARP-1-mediated translocation of AIF has now been demonstrated in other cell types such as heart myocytes and neurones. "In any ischemia-mediated cell death in any organ system, PARP-1 is one of the major players."

Given PARP-1's involvement in both protective and pathogenic processes, it is not surprising that PARP-1 inhibitors are attracting considerable industrial interest with a view to redirecting cells away from undesirable fates.⁷ Szabó says that Fujisawa, Glaxo-SmithKline, Guilford, Novartis, Ono, Pfizer, as well as his own Inotek, all have programs, with some molecules now at the clinical phase of development.

In the case of degenerative diseases and ischemia-related death of postmitotic cells, such inhibitors are expected to rescue cells from necrosis or other caspase-independent cell-death pathways. For cancerous cells, PARP-1 inhibitors might promote the efficacy of DNA-damaging antitumor drugs by preventing DNA repair. Recent trials with such an antitumor agent (temozolomide) and a PARP inhibitor produced complete regression of human xenograft tumors in mice.⁸

And then there is PARP-2 to consider, not to mention the other 16 PARP-family members. "We just got some really funny results on that," is all Dawson will say regarding a possible regulatory role for PARP-2 in AIF-mediated cell death. "We're still trying to figure it out."