Impaired DNA repair in mouse monocytes compared to macrophages and precursors.

Previously we showed that human monocytes isolated from peripheral blood display downregulation of several DNA repair proteins, including XRCC1, ligase III, PARP-1 and DNA-PKCS, resulting in a deficiency of DNA repair, while in macrophages derived from monocytes the repair protein expression and DNA repair is restored. To see whether this is a specific phenomenon of human monocytes and macrophages, we assessed the expression of these repair genes in mice. We also addressed the question at which differentiation step in bone marrow cells downregulation of DNA repair gene expression occurs. The study revealed that mouse monocytes, similar to human, lack the expression of XRCC1, ligase III, PARP-1 and DNA-PKCS. If mice were treated with total body irradiation, they showed significant apoptosis in bone marrow monocytes, but not in peritoneal macrophages. This was also observed after treatment with the methylating anticancer drug temozolomide, resulting in high death rate of monocytes, but not macrophages. Monocytes arise from hematopoietic stem cells. Even the early stem cell fraction (LT-HSC) expressed detectable amounts of XRCC1, which was transiently upregulated, achieving the highest expression level in CMP (common myeloid progenitor) and, during the subsequent differentiation process, downregulated up to a non-detectable level in monocytes. The immediate monocyte precursor GMP also expressed ligase III, PARP-1 and DNA-PKCS. All these repair genes lacking in monocytes were upregulated again in macrophages. The sensitivity of monocytes, macrophages and precursor cells roughly correlated with their XRCC1 expression level. Monocytes, but not macrophages, also displayed strong γH2AX focal staining, indicating the presence of non-repaired DNA double-strand breaks following total body irradiation. Overall, the data revealed that murine monocytes exhibit the same DNA repair-impaired phenotype and high sensitivity compared to macrophages as observed in human. Therefore, the repair deficiency previously described for human monocytes appears to be a general property of this cell type.

Nijmegen breakage syndrome protein (NBN) causes resistance to methylating anticancer drugs such as temozolomide.

Methylating agents are first-line therapeutics for gliomas and malignant melanomas. They attack DNA at various sites, and both O(6)-methylguanine and N-methylated base adducts contribute to the killing response. The mechanism of cellular defense against these agents primarily involves O(6)-methylguanine-DNA methyltransferase (MGMT) and base excision repair (BER). Here, we determined whether a key protein involved in DNA double-strand break (DSB) recognition and signaling, nibrin (NBN alias NBS-1), plays a role in the cellular defense against methylating agents. Comparing NBN mutated fibroblasts and lymphoblastoid cells from patients suffering from Nijmegen breakage syndrome, we show that NBN mutants are clearly more sensitive to N-methyl-N'-nitro-N-nitrosoguanidine and temozolomide than the corresponding wild-type cells. Hypersensitivity was due to the induction of both apoptosis and necrosis. The mismatch repair proteins MSH2, MSH6, MLH1, and PMS2 were expressed at a similar level in the cell lines and BER was not affected by NBN mutation. Because MGMT expression abrogated the hypersensitivity of NBN mutated cells, we conclude that O(6)-methylguanine-derived lesions are responsible for triggering the response. Down-regulation of NBN in melanoma cells by small interfering RNA rendered them more sensitive to temozolomide, suggesting that NBN is a novel modulator of temozolomide sensitivity. Because NBN is part of the MRN complex, which recognizes DSBs, the data strongly indicate that MRN is critically involved in DSB processing after O(6)-methylguanine induction. The data provide first evidence that NBN is involved in the cellular defense against O(6)-methylguanine-inducing agents such as temozolomide and identify NBN as a critical target of methylating anticancer drug resistance.

Artesunate enhances the therapeutic response of glioma cells to temozolomide by inhibition of homologous recombination and senescence.

Glioblastoma multiforme (GBM), a malignant brain tumor with a dismal prognosis, shows a high level of chemo- and radioresistance and, therefore, attempts to sensitize glioma cells are highly desired. Here, we addressed the question of whether artesunate (ART), a drug currently used in the treatment of malaria, enhances the killing response of glioblastoma cells to temozolomide (TMZ), which is the first-line therapeutic for GBM. We measured apoptosis, necrosis, autophagy and senescence, and the extent of DNA damage in glioblastoma cells. Further, we determined the tumor growth in nude mice. We show that ART enhances the killing effect of TMZ in glioblastoma cell lines and in glioblastoma stem-like cells. The DNA double-strand break level induced by TMZ was not clearly enhanced in the combined treatment regime. Also, we did not observe an attenuation of TMZ-induced autophagy, which is considered a survival mechanism. However, we observed a significant effect of ART on homologous recombination (HR) with downregulation of RAD51 protein expression and HR activity. Further, we found that ART is able to inhibit senescence induced by TMZ. Since HR and senescence are pro-survival mechanisms, its inhibition by ART appears to be a key node in enhancing the TMZ-induced killing response. Enhancement of the antitumor effect of TMZ by co-administration of ART was also observed in a mouse tumor model. In conclusion, the amelioration of TMZ-induced cell death upon ART co-treatment provides a rational basis for a combination regime of TMZ and ART in glioblastoma therapy.

CD133 Expression Is Not Synonymous to Immunoreactivity for AC133 and Fluctuates throughout the Cell Cycle in Glioma Stem-Like Cells.

A transmembrane protein CD133 has been implicated as a marker of stem-like glioma cells and predictor for therapeutic response in malignant brain tumours. CD133 expression is commonly evaluated by using antibodies specific for the AC133 epitope located in one of the extracellular domains of membrane-bound CD133. There is conflicting evidence regarding the significance of the AC133 epitope as a marker for identifying stem-like glioma cells and predicting the degree of malignancy in glioma cells. The reasons for discrepant results between different studies addressing the role of CD133/AC133 in gliomas are unclear. A possible source for controversies about CD133/AC133 is the widespread assumption that expression patterns of the AC133 epitope reflect linearly those of the CD133 protein. Consequently, the readouts from AC133 assessments are often interpreted in terms of the CD133 protein. The purpose of this study is to determine whether and to what extent do the readouts obtained with anti-AC133 antibody correspond to the level of CD133 protein expressed in stem-like glioma cells. Our study reveals for the first time that CD133 expressed on the surface of glioma cells is poorly immunoreactive for AC133. Furthermore, we provide evidence that the level of CD133 occupancy on the surface of glioma cells fluctuates during the cell cycle. Our results offer a new explanation for numerous inconsistencies regarding the biological and clinical significance of CD133/AC133 in human gliomas and call for caution in interpreting the lack or presence of AC133 epitope in glioma cells.

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.

Contribution of ATM and ATR to the resistance of glioblastoma and malignant melanoma cells to the methylating anticancer drug temozolomide.

The major cytotoxic DNA adduct induced by temozolomide and other methylating agents used in malignant glioma and metastasized melanoma therapy is O(6)-methylguanine (O(6)-MeG). This primary DNA damage is converted by mismatch repair into secondary lesions, which block replication and in turn induce DNA double-strand breaks that trigger the DNA damage response (DDR). Key upstream players in the DDR are the phosphoinositide 3-kinases ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3 related (ATR). Here, we addressed the question of the importance of ATM and ATR in the cell death response following temozolomide. We show that (i) ATM- and ATR-mutated cells are hypersensitive to temozolomide, (ii) O(6)-MeG triggers ATM and ATR activation, (iii) knockdown of ATM and ATR enhances cell kill in gliobalstoma and malignant melanoma cells with a stronger and significant effect in ATR knockdown cells, (iv) ATR, but not ATM, knockdown abolished phosphorylation of H2AX, CHK1, and CHK2 in glioma cells, and (v) temozolomide-induced cell death was more prominently enhanced by pharmacologic inhibition of CHK1 compared with CHK2. The data suggest that ATM and, even better, ATR inhibition is a useful strategy in sensitizing cancer cells to temozolomide and presumably also other anticancer drugs.