Trends in Molecular Medicine
Volume 12, Issue 9, September 2006, Pages 440-450
Journal home page for Trends in Molecular Medicine

Review
DNA damage-induced cell death by apoptosis

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Following the induction of DNA damage, a prominent route of cell inactivation is apoptosis. During the last ten years, specific DNA lesions that trigger apoptosis have been identified. These include O6-methylguanine, base N-alkylations, bulky DNA adducts, DNA cross-links and DNA double-strand breaks (DSBs). Repair of these lesions are important in preventing apoptosis. An exception is O6-methylguanine–thymine lesions, which require mismatch repair for triggering apoptosis. Apoptosis induced by many chemical genotoxins is the consequence of blockage of DNA replication, which leads to collapse of replication forks and DSB formation. These DSBs are thought to be crucial downstream apoptosis-triggering lesions. DSBs are detected by ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia and Rad3 related) proteins, which signal downstream to CHK1, CHK2 (checkpoint kinases) and p53. p53 induces transcriptional activation of pro-apoptotic factors such as FAS, PUMA and BAX. Many tumors harbor mutations in p53. There are p53 backup systems that involve CHK1 and/or CHK2-driven E2F1 activation and p73 upregulation, which in turn transcribes BAX, PUMA and NOXA. Another trigger of apoptosis upon DNA damage is the inhibition of RNA synthesis, which leads to a decline in the level of critical gene products such as MKP1 (mitogen-activated protein kinase phosphatase). This causes sustained activation of JNK (Jun kinase) and, finally, AP-1, which stimulates death-receptor activation. DNA damage-triggered signaling and execution of apoptosis is cell-type- and genotoxin-specific depending on the p53 (p63 and p73) status, death-receptor responsiveness, MAP-kinase activation and, most importantly, DNA repair capacity. Because most clinical anti-cancer drugs target DNA, increasing knowledge on DNA damage-triggered signaling leading to cell death is expected to provide new strategies for therapeutic interventions.

Introduction

The most vulnerable material in the cell, because it cannot simply be replaced, is DNA. DNA damage can happen due to spontaneous base hydrolysis and during natural stress such as inflammation. In most cases, DNA damage arises from exogenous sources. Thus, DNA is the main target of environmental genotoxins [e.g. alkylating compounds, polycyclic aromatic hydrocarbons, biphenyls, heterocyclic amines, ultraviolet (UV) light and ionizing radiation]. DNA is also the main target of most cytotoxic anticancer drugs that react either directly with DNA through reactive metabolites or indirectly through the incorporation into DNA (nucleotide analogs), or by blockade of DNA-metabolizing functions such as DNA polymerases or topoisomerases.

Two cellular strategies have evolved for coping with DNA damage: (i) DNA damage is repaired or tolerated; or (ii) cells that harbor DNA damage are removed from the population by death. Non-repaired DNA damage often has harmful consequences that manifest as chromosomal changes, gene mutations and malignant transformation. These genotoxic endpoints have been studied intensively and have led to a growing understanding of the critical lesions and molecular mechanisms involved [1]. With regard to cytotoxicity, however, knowledge is still incomplete; this is because cells respond to genotoxins in a complex way by evoking cellular processes that might ultimately lead to DNA repair, damage fixation as mutations or damage elimination by various routes of cell death 2, 3. Cell death that follows DNA damage is not merely a consequence of inactivation of the genome. It is rather based on complex enzymatic reactions that might lead to apoptosis, necrosis, autophagy and other forms of cell death (Table 1). This review focuses on the apoptotic response that follows the induction of DNA damage.

Section snippets

Types of DNA damage that trigger apoptosis

An important aspect of DNA-damaging agents is that they not only target DNA but also cause damage to other cellular components. Therefore, targets other than DNA must be taken into account. For instance, UV light and alkylating agents cause activation of EGF (epidermal growth factor) receptor. This triggers DNA-damage-independent immediate-early responses, involving the mitogen-activated protein (MAP)-kinase pathway [4], which has a role in apoptosis. Therefore, how important is DNA damage in

Apoptosis triggered by O6-methylguanine

Many carcinogens in the environment [8], tobacco smoke [9] and food [10], in addition to endogenous metabolic products [11], methylate DNA. Paradoxically, similar methylating agents (procarbazine, dacarbazine, streptozotocin and temozolomide) are used in cancer therapy. An important killing lesion for all these compounds is O6-methylguanine (O6MeG) (Figure 1). At present, O6MeG provides perhaps the best understood example for a well-defined DNA lesion that triggers apoptosis. O6MeG is repaired

Apoptosis triggered by N-methylation lesions

N-methylated bases that are induced by alkylating agents such as N3-methyladenine and N7-methylguanine are cytotoxic; they trigger apoptosis when present at high levels. These lesions are removed by BER (Figure 1). Contrary to NER, no human disorders due to defective BER are known, indicating the importance of this repair pathway. Deficiency in the late steps of BER [e.g. by lacking DNA polymerase β or the BER proteins XRCC1 (X-ray cross-complementing 1) and ligase III] sensitizes cells to

Apoptosis induced by O6-chloroethylating agents

O6-chloroethylating agents are anticancer drugs that, unlike cyclophosphamide, melphalan, cisplatin and many others, are mono-functional but show similar high cytotoxicity than bi-functional cross-linking agents. Examples of O6-chloroethylating compounds are carmustine, nimustine, lomustine and fotemustine. A minor DNA lesion of these compounds is O6-chloroethylguanine. This lesion is repaired by MGMT through direct chloroethyl-group transfer, which effectively protects against the cytotoxicity

Apoptosis induced by ultraviolet light

UV light is a paradigmatic example of a genotoxin that exerts both DNA damage-dependent and DNA damage-independent responses. Mammalian NER-deficient mutants are hypersensitive to UV light, which is mainly due to the induction of apoptosis [25]. UV light induces a broad spectrum of DNA lesions that include bulky adducts, oxidative lesions, DNA single-strand breaks and intra-strand cross-links. Because NER removes cyclobutane pyrimidine dimers (CPDs) and (6–4) photoproducts, these lesions must

Apoptosis induced by bulky lesion-inducing chemical genotoxins

An archetypal example for an agent that causes bulky DNA adducts is benzo(a)pyrene [B(a)P]. This major carcinogen in cigarette smoke and industrial combustion reacts, upon metabolic conversion by cytochrome P450, with DNA at the exocyclic amino group of deoxyguanosine, forming a N2-BP (N2-benzo(a) pyrene) adduct. The adduct provokes severe steric alteration in DNA that impairs DNA-dependent metabolic processes, which include DNA replication and transcription. This adduct can be removed from DNA

Apoptosis induced by DNA cross-linking agents

From the different cross-linking agents that are used in tumor therapy, only cisplatin is discussed here. Cisplatin [cis-diaminedichloro-platinum (II), cDDP] is a highly cytotoxic agent that mainly reacts with the N7 of guanine, forming inter- and intra-strand DNA cross-links [31]. These lesions are repaired both by NER and by the even more-complex inter-strand cross-link repair pathway, which involves components of NER and homologous recombination (HR). Cells that are defective in NER are

Sustained JNK–p38 kinase activation triggers the death receptor

Similar to UV light, cisplatin causes JNK and p38 kinase activation quickly after treatment of cells, being already detectable 30 minutes after exposure. In contrast to UV, however, the induction is not transient but long-lasting, persisting for several days. This sustained activation of JNK and p38 kinase by cisplatin treatment, which is accompanied by sustained upregulation of AP-1 (activator protein-1) and FAS-L, is important to explain why apoptosis occurs late, starting 2–4 days following

DNA damage signaling: the ATM–ATR–p53–CHK connection

How are specific DNA lesions recognized and how is signaling delivered downstream to the apoptotic machinery? It is unclear whether a general detection mechanism exists to identify all DNA lesions or whether multiple sensor mechanisms, each recognizing a specific lesion, are operating. In recent years, a great deal of information has been obtained on sensor mechanisms detecting DSBs, which are the most lethal DNA lesions. These sensors can signal repair mechanisms or, if this fails, they

DSB repair protects against apoptosis

If non-repaired DSBs are the main trigger of apoptosis (by evoking ATM–ATR signaling), then the repair of DSBs is a highly important (upstream) anti-apoptotic mechanism. DSBs that are induced by ionizing radiation and presumably also those generated by collapsed replication forks in response to chemical genotoxins are repaired by HR and non-homologous end joining (NHEJ). HR, which occurs in late S phase and G2 phase of the cell cycle is error-free, whereas NHEJ, which occurs predominantly in

Role of p53

As mentioned above, DSBs activate ATM, the phosphorylation target of which is p53. Upon phosphorylation, p53 becomes stabilized and blocks proliferation by upregulation of p21, which triggers G1  S arrest. It is believed that at low levels of DSBs only a minor fraction of p53 that is sufficient to drive the transcription of the p21 gene is activated, finally causing cell-cycle arrest. With high levels of DSBs, however, p53 accumulates above a particular threshold and, thus, can activate

p53-independent DNA damage-induced apoptosis

Many tumor cell lines that have been studied to date undergo apoptosis following DNA damage induction to a greater extent if they express wild-type p53. This has recently been shown for glioblastoma cells that have been treated with the methylating anti-cancer drug temozolomide, used in glioma therapy, which showed a clearly better response if p53 was not mutated [17]. However, p53-mutated cells do not lose their ability to undergo apoptosis completely; they are just less efficient in this

Involvement of caspase-2 in DNA damage-signaled apoptosis

Another mechanism of p53-independent apoptosis involves caspase-2. Caspase-2 is the only pro-caspase constitutively present in the nucleus 69, 70. It is required for etoposide-, cisplatin- and UV-light-induced apoptosis [71]. Furthermore, germ cells and oocytes from caspase-2 knockout mice are more resistant to doxorubicin than germ cells and oocytes of caspase-2 wild-type mice [72]. Caspase-2-induced apoptosis also requires caspase-9 activation [73], indicating that caspase-2 acts through the

Role of AKT/PKB protein kinase in DNA damage-triggered apoptosis

The protein AKT [also known as protein kinase B (PKB)] is a serine–threonine kinase that has a central role in cellular signaling. Its activation occurs by phosphatidylinositol 3-kinase (PI3-kinase), which becomes activated by EGF-receptor activation. It provokes suppression of apoptosis [77]. Inhibition of apoptosis by AKT leads to increased survival, which has been shown in many cancer cell types 78, 79, 80. Apoptosis can be suppressed by AKT in at least three ways: (i) AKT can directly cause

Outlook: some open questions

DNA repair-deficient experimental cell systems and hereditary diseases provide compelling evidence that specific DNA lesions are crucial in triggering apoptosis. The majority of primary DNA lesions, however, do not induce apoptosis directly. They either block transcription, thus causing depletion of essential proteins, or are converted by DNA replication into critical secondary lesions that trigger the apoptotic machinery. It is therefore clear that DNA replication is an important element in

Acknowledgements

This work was supported by DFG (Ka 724/13–1 and SFB 432/B7).

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