*Correspondence address: Dr Alexander Bürkle, Department of Gerontology, University of Newcastle upon Tyne, IHE, Wolfson Research Centre, Newcastle General Hospital, Westgate Road, Newcastle upon Tyne, NE4 6BE, UK
Tel: +44-191-256 3324; Fax: +44-191-219 5074 E-mail: Alexander.Buerkle@ncl.ac.uk

Key words: DNA strand breaks / poly(ADP-ribose) / DNA repair / genomic instability / cancer therapy / tumour cell resistance

Summary
Activation of poly(ADP-ribose) polymerase-1 (PARP-1) is an immediate cellular reaction to DNA strand breakage as induced by alkylating agents, ionising radiation or oxidants. The resulting formation of protein-coupled poly(ADP-ribose) facilitates survival of proliferating cells under conditions of DNA damage, probably via its contribution to DNA base-excision repair. Furthermore, based on recent results there is a role emerging for PARP-1 as a negative regulator of genomic instability in cells under genotoxic stress. Regarding possible applications for clinical cancer therapy with DNA-damaging agents, it appears that both inhibition and up-regulation of the poly(ADP-ribosyl)ation response in the malignant cells to be eradicated are promising strategies to improve the outcome of such therapy, albeit for different reasons.

1. Poly(ADP-ribosyl)ation and poly(ADP-ribose) polymerases
Catalytic activation of the 113-kDa nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1; EC 2.4.2.30) is one of the immediate cellular responses to DNA damage as inflicted by alkylating agents, ionising radiation or oxidants [for recent reviews see: 1-3]. This highly conserved, abundant enzyme uses NAD+ as substrate to carry out posttranslational modification of a number of nuclear proteins, including itself, with poly(ADP-ribose). Catalytic activity depends on binding of the enzyme to nicks or double-strand breaks in DNA, which are recognised by two zinc fingers within the DNA-binding domain of the enzyme. Poly(ADP-ribose) has a short half-life under conditions of DNA breakage, due to its rapid degradation by poly(ADP-ribose) glycohydrolase (PARG) (Fig. 1A). Studies both on cell cultures and on PARP-1 knock-out mice have revealed a significant biological role of poly(ADP-ribosyl)ation in the recovery of proliferating cells from DNA damage as induced, e.g., by alkylation or ionising radiation [4-8] and this has been linked with an involvement of PARP-1 in DNA base-excision repair [9-12]. In striking contrast to these cytoprotective functions of PARP-1 in cells exposed to low doses of genotoxic agents, there is a risk of acute cell death by PARP-1 overactivation, due to the depletion of NAD+ pools. This cell death mechanism has been observed mostly in non-proliferative cell types, such as b-cells in pancreatic islets [13,14] and neurones [15,16]. Furthermore, both poly(ADP-ribose) formation and PARP-1 cleavage by caspases are prominent features of apoptotic cell death [17,18].

In line with the above-mentioned cytoprotective function of PARP-1, a positive correlation of poly(ADP-ribosyl)ation capacity of mononuclear leukocytes with life span of mammalian species [19,20] and also an association of high poly(ADP-ribosyl)ation capacity with longevity in humans [21] have been established. Very recently, further links with ageing and longevity were made with the observations that telomeres in PARP-1 knockout mice are shorter than in wild-type mice [22] and that PARP-1 mRNA levels in fibroblasts from old human donors are lower compared to those from young ones [23].

Over the last two years, the existence of several additional polypeptides synthesising poly(ADP-ribose) has been demonstrated [24-29]. These new members of the "PARP family" seem to account for about 25% total poly(ADP-ribose) production in mouse 3T3 fibroblasts [30]. Knowledge on the biological functions of these novel poly(ADP-ribose) polymerases is still rather limited.

2. Cellular sensitisation by PARP(-1) inhibition and its possible exploitation for tumour therapy
During the last two decades, a large number of interventional studies in cell cultures and animals have been performed aiming at abrogation of poly(ADP-ribose) formation. A wide range of strategies has been employed, such as low-molecular weight inhibitors [e.g. 4], selection of PARP-deficient cell clones [31], PARP-1 antisense RNA [9], expression of dominant negative PARP-1 [5,6] and PARP-1 gene disruption [32,7,33]. Unanimously, the result of such interventions was a sensitisation of the cells to the cytotoxic effects of monofunctional alkylating agents or ionising radiation, while little if any effect on survival was recorded in cells not exposed to DNA damage. This very robust finding has provided a basis for the concept of incorporating low-molecular weight PARP inhibitors into existing protocols of cytotoxic cancer therapy with DNA-damaging agents (e.g., ionising radiation, alkylating agents, agents inducing formation of oxygen free radicals), anticipating that addition of PARP inhibitors would render conventional treatment more efficacious and might help solving the frequent and serious problem of tumour cell resistance. Since the first generation of PARP inhibitors (nicotinamide, benzamide and their derivatives) lacked potency and specificity and had a number of additional side effects, it was necessary to develop better compounds. In the meantime this goal has been achieved, and highly potent and water-soluble inhibitors have very recently become available [34]. It will now be very interesting to see in preclinical studies if PARP inhibition can indeed add to the in-vivo efficacy of cytotoxic cancer treatment regimens.

3. Induction of above-normal poly(ADP-ribose) levels and its possible exploitation for tumour therapy
Recently cell culture transfection experiments have been performed to create the reverse biochemical situation, i.e. to raise damage-induced cellular poly(ADP-ribose) formation above the normal level. This was achieved by overexpressing wild-type PARP-1 in stably transfected cultures using either constitutive [35,36] or conditional promoters [37]. Surprisingly, even at low levels of DNA damage PARP-1 overexpression did not increase survival under conditions of genotoxic stress, but led rather to a sensitisation of the cells to the cytotoxic effects of alkylating agents or g-radiation, which was apparently not due to NAD+ depletion [35-37]. These results suggested that the normal level of damage-induced cellular poly(ADP-ribose) represents an optimum with regard to cell survival under conditions of genotoxic stress [35-36]. In contrast to the effect on cytotoxicity, PARP-1 overexpression had a strong suppressive effect on the induction of sister-chromatid exchange (SCE) by the alkylating agent MNNG [37]. Viewed together with substantial evidence from the literature showing that abrogation of PARP(-1) activity leads to upregulation of DNA alkylation-induced SCE, PARP-1 thus emerges as a regulator of alkylation-induced SCE formation, imposing a control that is strictly negative and commensurate with the enzyme activity level [37]. The dissociation of the effects of DNA damage on cytotoxicity and SCE formation by above-normal poly(ADP-ribose) levels may, perhaps paradoxically at first glance, provide a platform for developing yet another poly(ADP-ribose)-centred strategy to optimise the outcome of conventional cytotoxic tumour therapy. This is based on the following reasoning: One of the hallmarks of tumour cells is the instability of their genome (as represented by formation of chromosomal breaks and aberrations, SCE, gene rearrangements, deletions or amplifications). In fact genomic instability can be regarded as a driving force for the process of carcinogenesis, including the tumour progression to higher levels of malignancy, since it is is a source of phenotypic variation of the tumour cells [38-40]. The latter increases the chance for tumour cell survival in an unfavourable microenvironment created by the host's immune system, by limited nutrient supply resulting from inadequate tumour perfusion, or by metabolic blockade caused by cytostatic agents used in chemotherapy. DNA damage, on the other hand, is an important trigger of genomic instability, and thus conventional cytotoxic tumour therapy employing DNA-damaging agents may actually add to genomic instability and boost further malignant progression in those tumour cells that survive a given cycle of cytotoxic treatment. In this context, interventions to prevent genomic instability can be expected to enhance the overall therapeutic success. Raising the cellular poly(ADP-ribose) levels to above-normal levels therefore appears to be an interesting target, as it can preserve the (desirable) cell-killing effect of a DNA-damaging agent while the SCE-inducing effect (viewed as a prototypic manifestation of genomic instability) will be suppressed [37]. In practical terms, several ways to achieve this goal can be envisaged: Pharmaceuticals might be developed that should increase in living cells the activity of PARP-1 at a given number of DNA strand breaks. Another option could be to target poly(ADP-ribose) catabolism, which is mainly mediated by PARG [41]. Specific inhibitors of this enzyme that would be able to efficiently penetrate living cells (yet to be developed) can be expected to raise cellular poly(ADP-ribose) levels as well and might show the same desirable effect of "freezing" the process of genomic instability. As is the case with PARP inhibitors, such "poly(ADP-ribose) potentiators" could then be combined with classical cytotoxic therapy (Fig. 1B).

In conclusion, while research on poly(ADP-ribosyl)ation has been conducted for more than three decades, the interest in this topic, concerning both basic science and possible applications, is currently booming rather than declining. At this time, both PARP-1 and the novel PARPs as well as PARG promise to be exciting research topics for yet some more years to come.

Acknowledgements
Work of my group cited in this chapter was supported by grants from the Deutsche Forschungsgemeinschaft [Bu 698/2-1 through -4] and from the EU Commission [Concerted Action Programme on „Molecular gerontology: the identification of links between ageing and the onset of age-related diseases [MOLGERON]"; BMH1 CT94 1710].

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Legend to Figure

Fig. 1. Proposed model for the role of poly(ADP-ribosyl)ation in the regulation of genomic instability. PARP-1, poly(ADP-ribose) polymerase-1; PARG, poly(ADP-ribose) glycohydrolase; arrows, stimulation or biochemical reaction; T-shaped lines, inhibition. A, Normal situation. B, Cellular poly(ADP-ribose) raised above normal levels by either over-activity of PARP-1 or (hypothetically) by inhibition of PARG. For details see text.

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