The chloroethylating anticancer drug ACNU induces FRA1 that is involved in drug resistance of glioma cells

AbstractFRA1 belongs, together with c-Fos and FosB, to the family of Fos proteins that form with members of the ATF and Jun family the transcription factor AP-1 (activator protein 1). Previously we showed that c-Fos protects mouse embryonic fibroblasts against the cytotoxic effects of ultraviolet (UV) light by induction of the endonuclease XPF, leading to enhanced nucleotide excision repair (NER) activity. Here, we analyzed the regulation of FRA1 in glioma cells treated with the anticancer drug nimustine (ACNU) and its role in ACNU-induced toxicity. We show that FRA1 is upregulated in glioblastoma cells following ACNU on mRNA and protein levels. Knockdown of FRA1 by either siRNA or shRNA clearly sensitized glioma cells towards ACNU-induced cell death. Despite decreased AP-1 binding activity upon FRA1 knockdown, this effect is independent on regulation of the AP-1 target genes fasL, ercc1 and xpf. In addition, FRA1 knockdown does not affect DNA repair capacity. However, lack of FRA1 attenuated the ACNU-induced phosphorylation of CHK1 and led to a reduced arrest of cells in G2/M and, thereby, presumably leads to enhanced cell death in the subsequent cell cycle

Rad51 and BRCA2 - New Molecular Targets for Sensitizing Glioma Cells to Alkylating Anticancer Drugs

First line chemotherapeutics for brain tumors (malignant gliomas) are alkylating agents such as temozolomide and nimustine. Despite growing knowledge of how these agents work, patients suffering from this malignancy still face a dismal prognosis. Alkylating agents target DNA, forming the killing lesion O6-alkylguanine, which is converted into DNA double-strand breaks (DSBs) that trigger apoptosis. Here we assessed whether inhibiting repair of DSBs by homologous recombination (HR) or non-homologous end joining (NHEJ) is a reasonable strategy for sensitizing glioma cells to alkylating agents. For down-regulation of HR in glioma cells, we used an interference RNA (iRNA) approach targeting Rad51 and BRCA2, and for NHEJ we employed the DNA-PK inhibitor NU7026. We also assessed whether inhibition of poly(ADP)ribosyltransferase (PARP) by olaparib would enhance the killing effect. The data show that knockdown of Rad51 or BRCA2 greatly sensitizes cells to DSBs and the induction of cell death following temozolomide and nimustine (ACNU). It did not sensitize to ionizing radiation (IR). The expression of O6-methylguanine-DNA methyltransferase (MGMT) abolished all these effects, indicating that O6-alkylguanine induced by these drugs is the primary lesion responsible for the formation of DSBs and increased sensitivity of glioma cells following knockdown of Rad51 and BRCA2. Inhibition of DNA-PK only slightly sensitized to temozolomide whereas a significant effect was observed with IR. A triple strategy including siRNA and the PARP inhibitor olaparib further improved the killing effect of temozolomide. The data provides evidence that down-regulation of Rad51 or BRCA2 is a reasonable strategy for sensitizing glioma cells to killing by O6-alkylating anti-cancer drugs. The data also provide proof of principle that a triple strategy involving down-regulation of HR, PARP inhibition and MGMT depletion may greatly enhance the therapeutic effect of temozolomide

Delayed c-Fos activation in human cells triggers XPF induction and an adaptive response to UVC-induced DNA damage and cytotoxicity

The oncoprotein c-Fos has been commonly found differently expressed in cancer cells. Our previous work showed that mouse cells lacking the immediate-early gene c-fos are hypersensitive to ultraviolet (UVC) light. Here, we demonstrate that in human diploid fibroblasts UV-triggered induction of c-Fos protein is a delayed and long-lasting event. Sustained upregulation of c-Fos goes along with transcriptional stimulation of the NER gene xpf, which harbors an AP-1 binding site in the promoter. Data gained on c-Fos knockdown and c-Fos overexpressing human cells provide evidence that c-Fos/AP-1 stimulates upregulation of XPF, thereby increasing the cellular repair capacity protecting from UVC-induced DNA damage. When these cells are pre-exposed to a low non-toxic UVC dose and challenged with a subsequent high dose of UVC irradiation, they show accelerated repair of UVC-induced DNA adducts and reduced cell kill. The data indicate a protective role of c-Fos induction by triggering an adaptive response pathway

Rac1-Regulated Endothelial Radiation Response Stimulates Extravasation and Metastasis That Can Be Blocked by HMG-CoA Reductase Inhibitors

Radiotherapy (RT) plays a key role in cancer treatment. Although the benefit of ionizing radiation (IR) is well established, some findings raise the possibility that irradiation of the primary tumor not only triggers a killing response but also increases the metastatic potential of surviving tumor cells. Here we addressed the question of whether irradiation of normal cells outside of the primary tumor augments metastasis by stimulating the extravasation of circulating tumor cells. We show that IR exposure of human endothelial cells (EC), tumor cells (TC) or both increases TC-EC adhesion in vitro. IR-stimulated TC-EC adhesion was blocked by the HMG-CoA reductase inhibitor lovastatin. Glycyrrhizic acid from liquorice root, which acts as a Sialyl-Lewis X mimetic drug, and the Rac1 inhibitor NSC23766 also reduced TC-EC adhesion. To examine the in vivo relevance of these findings, tumorigenic cells were injected into the tail vein of immunodeficient mice followed by total body irradiation (TBI). The data obtained show that TBI dramatically enhances tumor cell extravasation and lung metastasis. This pro-metastatic radiation effect was blocked by pre-treating mice with lovastatin, glycyrrhizic acid or NSC23766. TBI of mice prior to tumor cell transplantation also stimulated metastasis, which was again blocked by lovastatin. The data point to a pro-metastatic trans-effect of RT, which likely rests on the endothelial radiation response promoting the extravasation of circulating tumor cells. Administration of the widely used lipid-lowering drug lovastatin prior to irradiation counteracts this process, likely by suppressing Rac1-regulated E-selectin expression following irradiation. The data support the concern that radiation exposure might increase the extravasation of circulating tumor cells and recommend co-administration of lipid-lowering drugs to avoid this adverse effect of ionizing radiation

Cisplatin sensitivity of testis tumour cells is due to deficiency in interstrand-crosslink repair and low ERCC1-XPF expression

<p>Abstract</p> <p>Background</p> <p>Cisplatin based chemotherapy cures over 80% of metastatic testicular germ cell tumours (TGCT). In contrast, almost all other solid cancers in adults are incurable once they have spread beyond the primary site. Cell lines derived from TGCTs are hypersensitive to cisplatin reflecting the clinical response. Earlier findings suggested that a reduced repair capacity might contribute to the cisplatin hypersensitivity of testis tumour cells (TTC), but the critical DNA damage has not been defined. This study was aimed at investigating the formation and repair of intrastrand and interstrand crosslinks (ICLs) induced by cisplatin in TTC and their contribution to TTC hypersensitivity.</p> <p>Results</p> <p>We observed that repair of intrastrand crosslinks is similar in cisplatin sensitive TTC and resistant bladder cancer cells, whereas repair of ICLs was significantly reduced in TTC. γH2AX formation, which serves as a marker of DNA breaks formed in response to ICLs, persisted in cisplatin-treated TTC and correlated with sustained phosphorylation of Chk2 and enhanced PARP-1 cleavage. Expression of the nucleotide excision repair factor ERCC1-XPF, which is implicated in the processing of ICLs, is reduced in TTC. To analyse the causal role of ERCC1-XPF for ICL repair and cisplatin sensitivity, we over-expressed ERCC1-XPF in TTC by transient transfection. Over-expression increased ICL repair and rendered TTC more resistant to cisplatin, which suggests that ERCC1-XPF is rate-limiting for repair of ICLs resulting in the observed cisplatin hypersensitivity of TTC.</p> <p>Conclusion</p> <p>Our data indicate for the first time that the exceptional sensitivity of TTC and, therefore, very likely the curability of TGCT rests on their limited ICL repair due to low level of expression of ERCC1-XPF.</p

Both base excision repair and O-6-methylguanine-DNA methyltransferase protect against methylation-induced colon carcinogenesis

Methylating agents are widely distributed environmental carcinogens. Moreover, they are being used in cancer chemotherapy. The primary target of methylating agents is DNA, and therefore, DNA repair is the first-line barrier in defense against their toxic and carcinogenic effects. Methylating agents induce in the DNA O[superscript 6]-methylguanine (O[superscript 6]MeG) and methylations of the ring nitrogens of purines. The lesions are repaired by O[superscript 6]-methylguanine-DNA methyltransferase (Mgmt) and by enzymes of the base excision repair (BER) pathway, respectively. Whereas O[superscript 6]MeG is well established as a pre-carcinogenic lesion, little is known about the carcinogenic potency of base N-alkylation products such as N3-methyladenine and N3-methylguanine. To determine their role in cancer formation and the role of BER in cancer protection, we checked the response of mice with a targeted gene disruption of Mgmt or N-alkylpurine-DNA glycosylase (Aag) or both Mgmt and Aag, to azoxymethane (AOM)-induced colon carcinogenesis, using non-invasive mini-colonoscopy. We demonstrate that both Mgmt- and Aag-null mice show a higher colon cancer frequency than the wild-type. With a single low dose of AOM (3 mg/kg) Aag-null mice showed an even stronger tumor response than Mgmt-null mice. The data provide evidence that both BER initiated by Aag and O[superscript 6]MeG reversal by Mgmt are required for protection against alkylation-induced colon carcinogenesis. Further, the data indicate that non-repaired N-methylpurines are not only pre-toxic but also pre-carcinogenic DNA lesions.Deutsche Forschungsgemeinschaft (DFG) (FOR 527)Deutsche Forschungsgemeinschaft (DFG) (DFG KA 724/13-3)Deutsche Forschungsgemeinschaft (DFG) (WI 3304/1-1

DNA breaks and chromosomal aberrations arise when replication meets base excision repair

Exposures that methylate DNA potently induce DNA double-strand breaks (DSBs) and chromosomal aberrations, which are thought to arise when damaged bases block DNA replication. Here, we demonstrate that DNA methylation damage causes DSB formation when replication interferes with base excision repair (BER), the predominant pathway for repairing methylated bases. We show that cells defective in the N-methylpurine DNA glycosylase, which fail to remove N-methylpurines from DNA and do not initiate BER, display strongly reduced levels of methylation-induced DSBs and chromosomal aberrations compared with wild-type cells. Also, cells unable to generate single-strand breaks (SSBs) at apurinic/apyrimidinic sites do not form DSBs immediately after methylation damage. In contrast, cells deficient in x-ray cross-complementing protein 1, DNA polymerase β, or poly (ADP-ribose) polymerase 1 activity, all of which fail to seal SSBs induced at apurinic/apyrimidinic sites, exhibit strongly elevated levels of methylation-induced DSBs and chromosomal aberrations. We propose that DSBs and chromosomal aberrations after treatment with N-alkylators arise when replication forks collide with SSBs generated during BER

Immunological and mass spectrometry-based approaches to determine thresholds of the mutagenic DNA adduct O 6 -methylguanine in vivo

© 2018, Springer-Verlag GmbH Germany, part of Springer Nature. N-nitroso compounds are alkylating agents, which are widespread in our diet and the environment. They induce DNA alkylation adducts such as O 6 -methylguanine (O 6 -MeG), which is repaired by O 6 -methylguanine-DNA methyltransferase (MGMT). Persistent O 6 -MeG lesions have detrimental biological consequences like mutagenicity and cytotoxicity. Due to its pivotal role in the etiology of cancer and in cytotoxic cancer therapy, it is important to detect and quantify O 6 -MeG in biological specimens in a sensitive and accurate manner. Here, we used immunological approaches and established an ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) to monitor O 6 -MeG adducts. First, colorectal cancer (CRC) cells were treated with the methylating anticancer drug temozolomide (TMZ). Immunofluorescence microscopy and an immuno-slot blot assay, both based on an adduct-specific antibody, allowed for the semi-quantitative, dose-dependent assessment of O 6 -MeG in CRC cells. Using the highly sensitive and specific UPLC–MS/MS, TMZ-induced O 6 -MeG adducts were quantified in CRC cells and even in peripheral blood mononuclear cells exposed to clinically relevant TMZ doses. Furthermore, all methodologies were used to detect O 6 -MeG in wildtype (WT) and MGMT-deficient mice challenged with the carcinogen azoxymethane. UPLC–MS/MS measurements and dose–response modeling revealed a non-linear formation of hepatic and colonic O 6 -MeG adducts in WT, whereas linear O 6 -MeG formation without a threshold was observed in MGMT-deficient mice. Collectively, the UPLC–MS/MS analysis is highly sensitive and specific for O 6 -MeG, thereby allowing for the first time for the determination of a genotoxic threshold upon exposure to O 6 -methylating agents. We envision that this method will be instrumental to monitor the efficacy of methylating chemotherapy and to assess dietary exposures