Jörg Fahrer

Quantitative analysis of the binding affinity of poly(ADP-ribose) to specific binding proteins as a function of chain length

Poly(ADP-ribose) (PAR) is synthesized by poly(ADP-ribose) polymerases in response to genotoxic stress and interacts non-covalently with DNA damage checkpoint and repair proteins. Here, we present a variety of techniques to analyze this interaction in terms of selectivity and affinity. In vitro synthesized PAR was end-labeled using a carbonyl-reactive biotin analog. Binding of HPLC-fractionated PAR chains to the tumor suppressor protein p53 and to the nucleotide excision repair protein XPA was assessed using a novel electrophoretic mobility shift assay (EMSA). Long ADP-ribose chains (55-mer) promoted the formation of three specific complexes with p53. Short PAR chains (16-mer) were also able to bind p53, yet forming only one defined complex. In contrast, XPA did not interact with short polymer, but produced a single complex with long PAR chains (55-mer). In addition, we performed surface plasmon resonance with immobilized PAR chains, which allowed establishing binding constants and confirmed the results obtained by EMSA. Taken together, we developed several new protocols permitting the quantitative characterization of PAR–protein binding. Furthermore, we demonstrated that the affinity of the non-covalent PAR interactions with specific binding proteins (XPA, p53) can be very high (nanomolar range) and depends both on the PAR chain length and on the binding protein.

DEK Is a Poly(ADP-Ribose) Acceptor in Apoptosis and Mediates Resistance to Genotoxic Stress

ABSTRACT DEK is a nuclear phosphoprotein implicated in oncogenesis and autoimmunity and a major component of metazoan chromatin. The intracellular cues that control the binding of DEK to DNA and its pleiotropic functions in DNA- and RNA-dependent processes have remained mainly elusive so far. Our recent finding that the phosphorylation status of DEK is altered during death receptor-mediated apoptosis suggested a potential involvement of DEK in stress signaling. In this study, we show that in cells committed to die, a portion of the cellular DEK pool is extensively posttranslationally modified by phosphorylation and poly(ADP-ribosyl)ation. Through interference with DEK expression, we further show that DEK promotes the repair of DNA lesions and protects cells from genotoxic agents that typically trigger poly(ADP-ribose) polymerase activation. The posttranslational modification of DEK during apoptosis is accompanied by the removal of the protein from chromatin and its release into the extracellular space. Released modified DEK is recognized by autoantibodies present in the synovial fluids of patients affected by juvenile rheumatoid arthritis/juvenile idiopathic arthritis. These findings point to a crucial role of poly(ADP-ribosyl)ation in shaping DEKs autoantigenic properties and in its function as a promoter of cell survival.

O6 -methylguanine-DNA methyltransferase in the defense against N -nitroso compounds and colorectal cancer

Colorectal cancer (CRC) is among the leading causes of cancer death worldwide, involving multiple dietary and non-dietary risk factors. A growing body of evidence suggests that N-nitroso compounds (NOC) play a pivotal role in the etiology of CRC. NOC are present in food and are also formed endogenously in the large intestine. Upon metabolic activation and also spontaneously, they form electrophilic species that methylate the DNA, producing N-methylated purines and O(6)-methylguanine, the latter of which bears high mutagenic and carcinogenic potential. Methylated DNA bases are removed by base excision repair initiated by the alkyladenine-DNA glycosylase, the family of AlkB homologs proteins, and the suicide enzyme O(6)-methylguanine-DNA methyltransferase (MGMT), which is the main focus of this review. We present animal models with a deficiency of MGMT that display a tremendously enhanced sensitivity toward alkylation-induced colorectal carcinogenesis, highlighting its role in the protection against the cytotoxic and mutagenic effects of alkylating agents. In line with these studies, MGMT was linked to the formation of human sporadic CRC. Colorectal tumors and precursor lesions frequently display epigenetic inactivation of MGMT resulting from promoter hypermethylation, which is tightly associated with the occurrence of G:C to A:T transition mutations in the KRAS oncogene. We also discuss clinical data, which identified the MGMT status of CRC patients as promising parameter for the treatment of metastasized CRC using alkylating anticancer drugs such as temozolomide.

Cytolethal distending toxin (CDT) is a radiomimetic agent and induces persistent levels of DNA double-strand breaks in human fibroblasts.

Cytolethal distending toxin (CDT) is a unique genotoxin produced by several pathogenic bacteria. The tripartite protein toxin is internalized into mammalian cells via endocytosis followed by retrograde transport to the ER. Upon translocation into the nucleus, CDT catalyzes the formation of DNA double-strand breaks (DSBs) due to its intrinsic endonuclease activity. In the present study, we compared the DNA damage response (DDR) in human fibroblasts triggered by recombinant CDT to that of ionizing radiation (IR), a well-known DSB inducer. Furthermore, we dissected the pathways involved in the detection and repair of CDT-induced DNA lesions. qRT-PCR array-based mRNA and western blot analyses showed a partial overlap in the DDR pattern elicited by CDT and IR, with strong activation of both the ATM-Chk2 and the ATR-Chk1 axis. In line with its in vitro DNase I-like activity on plasmid DNA, neutral and alkaline Comet assay revealed predominant induction of DSBs in CDT-treated fibroblasts, whereas irradiation of cells generated higher amounts of SSBs and alkali-labile sites. Using confocal microscopy, the dynamics of the DSB surrogate marker γ-H2AX was monitored after pulse treatment with CDT or IR. In contrast to the fast induction and disappearance of γ-H2AX-foci observed in irradiated cells, the number of γ-H2AX-foci induced by CDT were formed with a delay and persisted. 53BP1 foci were also generated following CDT treatment and co-localized with γ-H2AX foci. We further demonstrated that ATM-deficient cells are very sensitive to CDT-induced DNA damage as reflected by increased cell death rates with concomitant cleavage of caspase-3 and PARP-1. Finally, we provided novel evidence that both homologous recombination (HR) and non-homologous end joining (NHEJ) protect against CDT-elicited DSBs. In conclusion, the findings suggest that CDT functions as a radiomimetic agent and, therefore, is an attractive tool for selectively inducing persistent levels of DSBs and unveiling the associated cellular responses.

Site-specific noncovalent interaction of the biopolymer poly(ADP-ribose) with the Werner syndrome protein regulates protein functions.

Werner syndrome is a premature aging disorder that is caused by defects in the Werner protein (WRN). WRN is a member of the RecQ helicase family and possesses helicase and exonuclease activities. It is involved in various aspects of DNA metabolism such as DNA repair, telomere maintenance, and replication. Poly(ADP-ribose) polymerase 1 (PARP1) is also involved in these processes by catalyzing the formation of the nucleic-acid-like biopolymer poly(ADP-ribose) (PAR). It was previously shown that WRN interacts with PARP1 and that WRN activity is inhibited by PARP1. Using several bioanalytical approaches, here we demonstrate that the enzymatic product of PARP1, i.e., PAR, directly interacts with WRN physically and functionally. First, WRN binds HPLC-size-fractionated short and long PAR in a noncovalent manner. Second, we identified and characterized a PAR-binding motif (PBM) within the WRN sequence and showed that several basic and hydrophobic amino acids are of critical importance for mediating the PAR binding. Third, PAR-binding inhibits the DNA-binding, the helicase and the exonuclease activities of WRN in a concentration-dependent manner. On the basis of our results we propose that the transient nature of PAR produced by living cells would provide a versatile and swiftly reacting control system for WRNs function. More generally, our work underscores the important role of noncovalent PAR-protein interactions as a regulatory mechanism of protein function.

The disulfide compound α-lipoic acid and its derivatives: A novel class of anticancer agents targeting mitochondria

The endogenous disulfide α-lipoic acid (LA) is an essential mitochondrial co-factor. In addition, LA and its reduced counterpart dihydro lipoic acid form a potent redox couple with antioxidative functions, for which it is used as dietary supplement and therapeutic. Recently, it has gained attention due to its cytotoxic effects in cancer cells, which is the key aspect of this review. We initially recapitulate the dietary occurrence, gastrointestinal absorption and pharmacokinetics of LA, illustrating its diverse antioxidative mechanisms. We then focus on its mode of action in cancer cells, in which it triggers primarily the mitochondrial pathway of apoptosis, whereas non-transformed primary cells are hardly affected. Furthermore, LA impairs oncogenic signaling and displays anti-metastatic potential. Novel LA derivatives such as CPI-613, which target mitochondrial energy metabolism, are described and recent pre-clinical studies are presented, which demonstrate that LA and its derivatives exert antitumor activity in vivo. Finally, we highlight clinical studies currently performed with the LA analog CPI-613. In summary, LA and its derivatives are promising candidates to complement the arsenal of established anticancer drugs due to their mitochondria-targeted mode of action and non-genotoxic properties.

DNA Double Strand Breaks as Predictor of Efficacy of the Alpha-Particle Emitter Ac-225 and the Electron Emitter Lu-177 for Somatostatin Receptor Targeted Radiotherapy

Rationale Key biologic effects of the alpha-particle emitter Actinium-225 in comparison to the beta-particle emitter Lutetium-177 labeled somatostatin-analogue DOTATOC in vitro and in vivo were studied to evaluate the significance of γH2AX-foci formation. Methods To determine the relative biological effectiveness (RBE) between the two isotopes (as - biological consequence of different ionisation-densities along a particle-track), somatostatin expressing AR42J cells were incubated with Ac-225-DOTATOC and Lu-177-DOTATOC up to 48 h and viability was analyzed using the MTT assay. DNA double strand breaks (DSB) were quantified by immunofluorescence staining of γH2AX-foci. Cell cycle was analyzed by flow cytometry. In vivo uptake of both radiolabeled somatostatin-analogues into subcutaneously growing AR42J tumors and the number of cells displaying γH2AX-foci were measured. Therapeutic efficacy was assayed by monitoring tumor growth after treatment with activities estimated from in vitro cytotoxicity. Results Ac-225-DOTATOC resulted in ED50 values of 14 kBq/ml after 48 h, whereas Lu-177-DOTATOC displayed ED50 values of 10 MBq/ml. The number of DSB grew with increasing concentration of Ac-225-DOTATOC and similarly with Lu-177-DOTATOC when applying a factor of 700-fold higher activity compared to Ac-225. Already 24 h after incubation with 2.5–10 kBq/ml, Ac-225-DOTATOC cell-cycle studies showed up to a 60% increase in the percentage of tumor cells in G2/M phase. After 72 h an apoptotic subG1 peak was also detectable. Tumor uptake for both radio peptides at 48 h was identical (7.5%ID/g), though the overall number of cells with γH2AX-foci was higher in tumors treated with 48 kBq Ac-225-DOTATOC compared to tumors treated with 30 MBq Lu-177-DOTATOC (35% vs. 21%). Tumors with a volume of 0.34 ml reached delayed exponential tumor growth after 25 days (44 kBq Ac-225-DOTATOC) and after 21 days (34 MBq Lu-177-DOTATOC). Conclusion γH2AX-foci formation, triggered by beta- and alpha-irradiation, is an early key parameter in predicting response to internal radiotherapy.

Enhanced Histone Deacetylase Activity in Malignant Melanoma Provokes RAD51 and FANCD2-Triggered Drug Resistance

DNA-damaging anticancer drugs remain a part of metastatic melanoma therapy. Epigenetic reprogramming caused by increased histone deacetylase (HDAC) activity arising during tumor formation may contribute to resistance of melanomas to the alkylating drugs temozolomide, dacarbazine, and fotemustine. Here, we report on the impact of class I HDACs on the response of malignant melanoma cells treated with alkylating agents. The data show that malignant melanomas in situ contain a high level of HDAC1/2 and malignant melanoma cells overexpress HDAC1/2/3 compared with noncancer cells. Furthermore, pharmacologic inhibition of class I HDACs sensitizes malignant melanoma cells to apoptosis following exposure to alkylating agents, while not affecting primary melanocytes. Inhibition of HDAC1/2/3 caused sensitization of melanoma cells to temozolomide in vitro and in melanoma xenografts in vivo HDAC1/2/3 inhibition resulted in suppression of DNA double-strand break (DSB) repair by homologous recombination because of downregulation of RAD51 and FANCD2. This sensitized cells to the cytotoxic DNA lesion O(6)-methylguanine and caused a synthetic lethal interaction with the PARP-1 inhibitor olaparib. Furthermore, knockdown experiments identified HDAC2 as being responsible for the regulation of RAD51. The influence of class I HDACs on DSB repair by homologous recombination and the possible clinical implication on malignant melanoma therapy with temozolomide and other alkylating drugs suggests a combination approach where class I HDAC inhibitors such as valproic acid or MS-275 (entinostat) appear to counteract HDAC- and RAD51/FANCD2-mediated melanoma cell resistance. Cancer Res; 76(10); 3067-77. ©2016 AACR.

The eucalyptus oil ingredient 1,8-cineol induces oxidative DNA damage

The natural compound 1,8-cineol, also known as eucalyptol, is a major constituent of eucalyptus oil. This epoxy-monoterpene is used as flavor and fragrance in consumer goods as well as medical therapies. Due to its anti-inflammatory properties, 1,8-cineol is also applied to treat upper and lower airway diseases. Despite its widespread use, only little is known about the genotoxicity of 1,8-cineol in mammalian cells. This study investigates the genotoxicity and cytotoxicity of 1,8-cineol in human and hamster cells. First, we observed a significant and concentration-dependent increase in oxidative DNA damage in human colon cancer cells, as detected by the Formamidopyrimidine-DNA glycosylase (Fpg)-modified alkaline comet assay. Pre-treatment of cells with the antioxidant N-acetylcysteine prevented the formation of Fpg-sensitive sites after 1,8-cineol treatment, supporting the notion that 1,8-cineol induces oxidative DNA damage. In the dose range of DNA damage induction, 1,8-cineol did neither reduce the viability of colon cancer cells nor affected their cell cycle distribution, suggesting that cells tolerate 1,8-cineol-induced oxidative DNA damage by engaging DNA repair. To test this hypothesis, hamster cell lines with defects in BRCA2 and Rad51, which are essentials players of homologous recombination (HR)-mediated repair, were treated with 1,8-cineol. The monoterpene induced oxidative DNA damage and subsequent DNA double-strand breaks in the hamster cell lines tested. Intriguingly, we detected a significant concentration-dependent decrease in viability of the HR-defective cells, whereas the corresponding wild-type cell lines with functional HR were not affected. Based on these findings, we conclude that 1,8-cineol is weakly genotoxic, inducing primarily oxidative DNA damage, which is most likely tolerated in DNA repair proficient cells without resulting in cell cycle arrest and cell death. However, cells with deficiency in HR were compromised after 1,8-cineol treatment, suggesting a protective role of HR in response to high doses of 1,8-cineol.

Lipoic acid inhibits the DNA repair protein O 6-methylguanine-DNA methyltransferase (MGMT) and triggers its depletion in colorectal cancer cells with concomitant autophagy induction.

Alkylating agents are present in food and tobacco smoke, but are also used in cancer chemotherapy, inducing the DNA lesion O (6)-methylguanine. This critical adduct is repaired by O (6)-methylguanine-DNA methyltransferase (MGMT), resulting in MGMT inactivation and degradation. In the present study, we analyzed the effects of the natural disulfide compound lipoic acid (LA) on MGMT in vitro and in colorectal cancer cells. We show that LA, but not its reduced form dihydrolipoic acid, potently inhibits the activity of recombinant MGMT by interfering with its catalytic Cys-145 residue, which was partially reversible by N-acetyl cysteine. Incubation of HCT116 colorectal cancer cells with LA altered their glutathione pool and caused a decline in MGMT activity. This was mirrored by LA-induced depletion of MGMT protein, which was not attributable to changes in MGMT messenger RNA levels. Loss of MGMT protein coincided with LA-induced autophagy, a process resulting in lysosomal degradation of proteins, including presumably MGMT. LA-stimulated autophagy in a p53-independent manner as revealed by the response of isogenic HCT116 cell lines. Knockdown of the crucial autophagy component beclin-1 and chemical inhibitors blocked LA-induced autophagy, but did not abrogate LA-triggered MGMT degradation. Concomitant with MGMT depletion, LA pretreatment resulted in enhanced O (6)-methylguanine levels in DNA. It also increased the cytotoxicity of the alkylating anticancer drug temozolomide in temozolomide-resistant colorectal cancer cells. Taken together, our study showed that the natural compound LA inhibits MGMT and induces autophagy. Furthermore, LA enhanced the cytotoxic effects of temozolomide, which makes it a candidate for a supplement in cancer therapy.