Markus Christmann

Mechanisms of human DNA repair: an update.

The human genome, comprising three billion base pairs coding for 30000-40000 genes, is constantly attacked by endogenous reactive metabolites, therapeutic drugs and a plethora of environmental mutagens that impact its integrity. Thus it is obvious that the stability of the genome must be under continuous surveillance. This is accomplished by DNA repair mechanisms, which have evolved to remove or to tolerate pre-cytotoxic, pre-mutagenic and pre-clastogenic DNA lesions in an error-free, or in some cases, error-prone way. Defects in DNA repair give rise to hypersensitivity to DNA-damaging agents, accumulation of mutations in the genome and finally to the development of cancer and various metabolic disorders. The importance of DNA repair is illustrated by DNA repair deficiency and genomic instability syndromes, which are characterised by increased cancer incidence and multiple metabolic alterations. Up to 130 genes have been identified in humans that are associated with DNA repair. This review is aimed at updating our current knowledge of the various repair pathways by providing an overview of DNA-repair genes and the corresponding proteins, participating either directly in DNA repair, or in checkpoint control and signaling of DNA damage.

O6-Methylguanine-DNA methyltransferase (MGMT) in normal tissues and tumors: Enzyme activity, promoter methylation and immunohistochemistry

O(6)-Methylguanine-DNA methyltransferase (MGMT) is a suicide enzyme that repairs the pre-mutagenic, pre-carcinogenic and pre-toxic DNA damage O(6)-methylguanine. It also repairs larger adducts on the O(6)-position of guanine, such as O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine and O(6)-chloroethylguanine. These adducts are formed in response to alkylating environmental pollutants, tobacco-specific carcinogens and methylating (procarbazine, dacarbazine, streptozotocine, and temozolomide) as well as chloroethylating (lomustine, nimustine, carmustine, and fotemustine) anticancer drugs. MGMT is therefore a key node in the defense against commonly found carcinogens, and a marker of resistance of normal and cancer cells exposed to alkylating therapeutics. MGMT also likely protects against therapy-related tumor formation caused by these highly mutagenic drugs. Since the amount of MGMT determines the level of repair of toxic DNA alkylation adducts, the MGMT expression level provides important information as to cancer susceptibility and the success of therapy. In this article, we describe the methods employed for detecting MGMT and review the literature with special focus on MGMT activity in normal and neoplastic tissues. The available data show that the expression of MGMT varies greatly in normal tissues and in some cases this has been related to cancer predisposition. MGMT silencing in tumors is mainly regulated epigenetically and in brain tumors this correlates with a better therapeutic response. Conversely, up-regulation of MGMT during cancer treatment limits the therapeutic response. In malignant melanoma, MGMT is not related to the therapeutic response, which is due to other mechanisms of inherent drug resistance. For most cancers, studies that relate MGMT activity to therapeutic outcome following O(6)-alkylating drugs are still lacking.

Long-term activation of SAPK/JNK, p38 kinase and fas-L expression by cisplatin is attenuated in human carcinoma cells that acquired drug resistance.

Tumor cells chronically exposed to cisplatin (cDDP) acquire cDDP resistance that impacts tumor therapy. To elucidate the mechanism of acquired cDDP resistance (ACR), we compared HeLa cells that gained ACR upon chronic cDDP treatment with the parental strain. We show that ACR is due to a lower level of induced apoptosis. Further, upon cDDP treatment, the levels of Fas, Bax and Bid remained unchanged, whereas Bcl‐2 and p‐Bad were reduced at late times (120 hr) after treatment. At early times, Fas ligand (fas‐L) expression was significantly enhanced in sensitive compared to resistant cells and remained upregulated up to the onset of apoptosis. Thus, activation of the Fas system is critical, which is in line with the finding that in sensitive cells, caspase‐8 along with caspase‐9 and ‐3 were activated by cDDP. cDDP provoked the activation of stress‐activated protein kinase/c‐Jun N‐terminal kinase (SAPK/JNK) and p38 kinase dose‐dependently, with significantly lower levels in ACR cells than in the sensitive parental line. cDDP induces c‐Jun and AP‐1 activity, as measured by a reporter gene assay, which was again attenuated in ACR cells. Time course analysis revealed that SAPK/JNK and p38 kinase activity was sustained upregulated (> 72 hr postexposure), which occurred at much higher level in sensitive than in ACR cells. Inhibition of either JNK or p38 kinase (by JNK inhibitor II and SB 203580, respectively) attenuated cDDP‐induced apoptosis, supporting the role of JNK and p38 kinase in the cDDP response. Since several independently derived cDDP‐resistant cell lines displayed attenuated MAPK signaling, sustained SAPK/JNK and p38 kinase activation may be a general mechanism of cDDP‐induced cell death. ACR cells displayed a reduced level of DNA damage, indicating long‐term stimulation of SAPK/JNK and p38 kinase is triggered by nonrepaired cDDP‐induced DNA lesions.

Transcriptional regulation of human DNA repair genes following genotoxic stress: trigger mechanisms, inducible responses and genotoxic adaptation

DNA repair is the first barrier in the defense against genotoxic stress. In recent years, mechanisms that recognize DNA damage and activate DNA repair functions through transcriptional upregulation and post-translational modification were the focus of intensive research. Most DNA repair pathways are complex, involving many proteins working in discrete consecutive steps. Therefore, their balanced expression is important for avoiding erroneous repair that might result from excessive base removal and DNA cleavage. Amelioration of DNA repair requires both a fine-tuned system of lesion recognition and transcription factors that regulate repair genes in a balanced way. Transcriptional upregulation of DNA repair genes by genotoxic stress is counteracted by DNA damage that blocks transcription. Therefore, induction of DNA repair resulting in an adaptive response is only visible through a narrow window of dose. Here, we review transcriptional regulation of DNA repair genes in normal and cancer cells and describe mechanisms of promoter activation following genotoxic exposures through environmental carcinogens and anticancer drugs. The data available to date indicate that 25 DNA repair genes are subject to regulation following genotoxic stress in rodent and human cells, but for only a few of them, the data are solid as to the mechanism, homeostatic regulation and involvement in an adaptive response to genotoxic stress.

Survival and Death Strategies in Glioma Cells: Autophagy, Senescence and Apoptosis Triggered by a Single Type of Temozolomide-Induced DNA Damage

Apoptosis, autophagy, necrosis and cellular senescence are key responses of cells that were exposed to genotoxicants. The types of DNA damage triggering these responses and their interrelationship are largely unknown. Here we studied these responses in glioma cells treated with the methylating agent temozolomide (TMZ), which is a first-line chemotherapeutic for this malignancy. We show that upon TMZ treatment cells undergo autophagy, senescence and apoptosis in a specific time-dependent manner. Necrosis was only marginally induced. All these effects were completely abrogated in isogenic glioma cells expressing O6-methylguanine-DNA methyltransferase (MGMT), indicating that a single type of DNA lesion, O6-methylguanine (O6MeG), is able to trigger all these responses. Studies with mismatch repair mutants and MSH6, Rad51 and ATM knockdowns revealed that autophagy induced by O6MeG requires mismatch repair and ATM, and is counteracted by homologous recombination. We further show that autophagy, which precedes apoptosis, is a survival mechanism as its inhibition greatly ameliorated the level of apoptosis following TMZ at therapeutically relevant doses (<100 µM). Cellular senescence increases with post-exposure time and, similar to autophagy, precedes apoptosis. If autophagy was abrogated, TMZ-induced senescence was reduced. Therefore, we propose that autophagy triggered by O6MeG adducts is a survival mechanism that stimulates cells to undergo senescence rather than apoptosis. Overall, the data revealed that a specific DNA adduct, O6MeG, has the capability of triggering autophagy, senescence and apoptosis and that the decision between survival and death is determined by the balance of players involved. The data also suggests that inhibition of autophagy may ameliorate the therapeutic outcome of TMZ-based cancer therapy.

Cisplatin-Induced Apoptosis Involves Membrane Fluidification via Inhibition of NHE1 in Human Colon Cancer Cells

We have previously shown that cisplatin triggers an early acid sphingomyelinase (aSMase)-dependent ceramide generation concomitantly with an increase in membrane fluidity and induces apoptosis in HT29 cells. The present study further explores the role and origin of membrane fluidification in cisplatin-induced apoptosis. The rapid increase in membrane fluidity following cisplatin treatment was inhibited by membrane-stabilizing agents such as cholesterol or monosialoganglioside-1. In HT29 cells, these compounds prevented the early aggregation of Fas death receptor and of membrane lipid rafts on cell surface and significantly inhibited cisplatin-induced apoptosis without altering drug intracellular uptake or cisplatin DNA adducts formation. Early after cisplatin treatment, Na+/H+ membrane exchanger-1 (NHE1) was inhibited leading to intracellular acidification, aSMase was activated, and ceramide was detected at the cell membrane. Treatment of HT29 cells with Staphylococcus aureus sphingomyelinase increased membrane fluidity. Moreover, pretreatment with cariporide, a specific inhibitor of NHE1, inhibited cisplatin-induced intracellular acidification, aSMase activation, ceramide membrane generation, membrane fluidification, and apoptosis. Finally, NHE1-expressing PS120 cells were more sensitive to cisplatin than NHE1-deficient PS120 cells. Altogether, these findings suggest that the apoptotic pathway triggered by cisplatin involves a very early NHE1-dependent intracellular acidification leading to aSMase activation and increase in membrane fluidity. These events are independent of cisplatin-induced DNA adducts formation. The membrane exchanger NHE1 may be another potential target of cisplatin, increasing cell sensitivity to this compound.

Nuclear Translocation of Mismatch Repair Proteins MSH2 and MSH6 as a Response of Cells to Alkylating Agents

Mammalian mismatch repair has been implicated in mismatch correction, the prevention of mutagenesis and cancer, and the induction of genotoxicity and apoptosis. Here, we show that treatment of cells specifically with agents inducing O6-methylguanine in DNA, such asN-methyl-N′-nitro-N-nitrosoguanidine and N-methyl-N-nitrosourea, elevates the level of MSH2 and MSH6 and increases GT mismatch binding activity in the nucleus. This inducible response occurs immediately after alkylation, is long-lasting and dose-dependent, and results from translocation of the preformed MutSα complex (composed of MSH2 and MSH6) from the cytoplasm into the nucleus. It is not caused by an increase in MSH2 gene activity. Cells expressing the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT), thus having the ability to repair O6-methylguanine, showed no translocation of MutSα, whereas inhibition of MGMT by O6-benzylguanine provoked the translocation. The results demonstrate that O6-methylguanine lesions are involved in triggering nuclear accumulation of MSH2 and MSH6. The finding that treatment of cells with O6-methylguanine-generating mutagens results in an increase of MutSα and GT binding activity in the nucleus indicates a novel type of genotoxic stress response.

Differential Sensitivity of Malignant Glioma Cells to Methylating and Chloroethylating Anticancer Drugs: p53 Determines the Switch by Regulating xpc, ddb2, and DNA Double-Strand Breaks

Glioblastoma multiforme is the most severe form of brain cancer. First line therapy includes the methylating agent temozolomide and/or the chloroethylating nitrosoureas [1-(2-chloroethyl)-1-nitrosourea; CNU] nimustine [1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea; ACNU], carmustine [1,3-bis(2-chloroethyl)-1-nitrosourea; BCNU], or lomustine [1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea; CCNU]. The mechanism of cell death after CNU treatment is largely unknown. Here we show that ACNU and BCNU induce apoptosis in U87MG [p53 wild-type (p53wt)] and U138MG [p53 mutant (p53mt)] glioma cells. However, contrary to what we observed previously for temozolomide, chloroethylating drugs are more toxic for p53-mutated glioma cells and induce both apoptosis and necrosis. Inactivation of p53 by pifithrin-alpha or siRNA down-regulation sensitized p53wt but not p53mt glioma cells to ACNU and BCNU. ACNU and BCNU provoke the formation of DNA double-strand breaks (DSB) in glioma cells that precede the onset of apoptosis and necrosis. Although these DSBs are repaired in p53wt cells, they accumulate in p53mt cells. Therefore, functional p53 seems to stimulate the repair of CNU-induced cross-links and/or DSBs generated from CNU-induced lesions. Expression analysis revealed an up-regulation of xpc and ddb2 mRNA in response to ACNU in U87MG but not U138MG cells, indicating p53 regulates a pathway that involves these DNA repair proteins. ACNU-induced apoptosis in p53wt glioma cells is executed via both the extrinsic and intrinsic apoptotic pathway, whereas in p53mt glioma cells, the mitochondrial pathway becomes activated. The data suggest that p53 has opposing effects in gliomas treated with methylating or chloroethylating agents and, therefore, the p53 status should be taken into account when deciding which therapeutic drug to use.

BER, MGMT, and MMR in defense against alkylation-induced genotoxicity and apoptosis

Methylating carcinogens and cytostatic drugs induce different methylation products in DNA. In cells not expressing the repair protein MGMT or expressing it at a low level, O6-methylguanine is the major genotoxic, recombinogenic, and apoptotic lesion. Genotoxicity and apoptosis triggered by O6-methylguanine require mismatch repair (MMR). In cells expressing O6-methylguanine-DNA methyl transferase (MGMT) at a high level or for agents producing low amounts of O6-methylguanine, N-alkylations become the major genotoxic lesions. N-Alkylations are repaired by base excision repair (BER). In mammalian cells, naturally occurring mutants of BER have not been detected, which points to the importance of BER for viability. In order to ascertain the role of BER in cellular defense, BER was modulated either by transfection or mutational inactivation. It has been shown that overexpression of N-methylpurine-DNA glycosylase (MPG) does not protect, but rather sensitizes cells to SN2 agents. This has been interpreted in terms of an imbalance in BER. Regarding abasic site endonuclease (APE), transient but not stable overexpression of the enzyme was achieved upon transfection in CHO cells, which indicates that unphysiologic APE levels are not tolerated by the cell. Besides the repair function, APE (alias Ref-1) exerts redox capability by which the activity of various transcription factors is modulated. Therefore, it is possible that stable overexpression of mammalian APE impairs transcriptional regulation of genes, whereas transient overexpression may exert some protective effect. DNA polymerase beta (Pol beta) transfection was ineffective in conferring resistance to methylmethane sulfonate (MMS). On the other hand, Pol beta-deficient cells proved to be highly sensitive to methylation-induced chromosomal aberrations and reproductive cell death. The dramatic hypersensitivity in the killing response is largely due to induction of apoptosis. Obviously, nonrepaired BER intermediates are clastogenic and act as a strong trigger of the apoptotic pathway. The elements of this pathway are currently under investigation.

Human monocytes are severely impaired in base and DNA double-strand break repair that renders them vulnerable to oxidative stress

Monocytes are key players in the immune system. Crossing the blood barrier, they infiltrate tissues and differentiate into (i) macrophages that fight off pathogens and (ii) dendritic cells (DCs) that activate the immune response. A hallmark of monocyte/macrophage activation is the generation of reactive oxygen species (ROS) as a defense against invading microorganisms. How monocytes, macrophages, and DCs in particular respond to ROS is largely unknown. Here we studied the sensitivity of primary human monocytes isolated from peripheral blood and compared them with macrophages and DCs derived from them by cytokine maturation following DNA damage induced by ROS. We show that monocytes are hypersensitive to ROS, undergoing excessive apoptosis. These cells exhibited a high yield of ROS-induced DNA single- and double-strand breaks and activation of the ATR-Chk1-ATM-Chk2-p53 pathway that led to Fas and caspase-8, -3, and -7 activation, whereas macrophages and DCs derived from them were protected. Monocytes are also hypersensitive to ionizing radiation and oxidized low-density lipoprotein. The remarkable sensitivity of monocytes to oxidative stress is caused by a lack of expression of the DNA repair proteins XRCC1, ligase IIIα, poly(ADP-ribose) polymerase-1, and catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), causing a severe DNA repair defect that impacts base excision repair and double-strand break repair by nonhomologous end-joining. During maturation of monocytes into macrophages and DCs triggered by the cytokines GM-CSF and IL-4, these proteins become up-regulated, making macrophages and DCs repair-competent and ROS-resistant. We propose that impaired DNA repair in monocytes plays a role in the regulation of the monocyte/macrophage/DC system following ROS exposure.