methylnitronitrosoguanidine and Glioblastoma

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.

Topics: Antineoplastic Agents, Alkylating; Ataxia Telangiectasia Mutated Proteins; Cell Line, Tumor; Checkpoint Kinase 1; Dacarbazine; DNA Breaks, Double-Stranded; DNA Modification Methylases; DNA Repair Enzymes; Drug Resistance, Neoplasm; Gene Knockdown Techniques; Glioblastoma; Guanine; Humans; Melanoma; Methylnitronitrosoguanidine; Phosphorylation; Protein Kinases; Signal Transduction; Temozolomide; Tumor Suppressor Proteins

Glioblastomas (GBM) are lethal brain tumors that are highly resistant to therapy. The only meaningful improvement in therapeutic response came from use of the S(N)1-type alkylating agent temozolomide in combination with ionizing radiation. However, no genetic markers that might predict a better response to DNA alkylating agents have been identified in GBMs, except for loss of O(6-)methylguanine-DNA methyltransferase via promoter methylation. In this study, using genetically defined primary murine astrocytes as well as human glioma lines, we show that loss of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) confers sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a functional analogue of temozolomide. We find that MNNG induces replication-associated DNA double-strand breaks (DSB), which are inefficiently repaired in PTEN-deficient astrocytes and trigger apoptosis. Mechanistically, this is because PTEN-null astrocytes are compromised in homologous recombination (HR), which is important for the repair of replication-associated DSBs. Our results suggest that reduced levels of Rad51 paralogs in PTEN-null astrocytes might underlie the HR deficiency of these cells. Importantly, the HR deficiency of PTEN-null cells renders them sensitive to the poly(ADP-ribose) polymerase (PARP) inhibitor ABT-888 due to synthetic lethality. In sum, our results tentatively suggest that patients with PTEN-null GBMs (about 36%) may especially benefit from treatment with DNA alkylating agents such as temozolomide. Significantly, our results also provide a rational basis for treating the subgroup of patients who are PTEN deficient with PARP inhibitors in addition to the current treatment regimen of radiation and temozolomide.

Topics: Animals; Antineoplastic Agents, Alkylating; Astrocytes; Benzimidazoles; Brain Neoplasms; Cyclin-Dependent Kinase Inhibitor p16; Dacarbazine; DNA Damage; DNA Repair; Glioblastoma; Methylnitronitrosoguanidine; Mice; Mice, Transgenic; Poly(ADP-ribose) Polymerase Inhibitors; PTEN Phosphohydrolase; Recombination, Genetic; Temozolomide

Methylating drugs such as temozolomide (TMZ) are widely used in the treatment of brain tumors including malignant glioblastoma. The mechanism of TMZ-induced glioblastoma cell death and apoptosis, however, is not fully understood. Here, we tested the potential involvement of AMP-activated protein kinase (AMPK) in this process. We found that methylating agents TMZ and N-methyl-N'-nitro-N-nitrosoguanidine induce AMPK activation in primary cultured human glioblastoma and glioblastoma cell lines. TMZ-induced O(6)-methylguanine production is involved in AMPK activation. O(6)-benzylguanine, an O(6)-methylguanine-DNA methyltransferase inhibitor, enhances TMZ-induced O(6)-methylguanine production, leading to enhanced reactive oxygen species production, which serves as an upstream signal for AMPK activation. Activation of AMPK is involved in TMZ-induced glioblastoma cell death and apoptosis. AMPK inhibitor (Compound C) or AMPKα siRNA knockdown inhibits TMZ-induced glioblastoma cell death and apoptosis, whereas AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside enhances it. In further studies, we found that activation of AMPK is involved in TMZ-induced p53 activation and subsequent p21, Noxa, and Bax up-regulation. Activation of AMPK by TMZ also inhibits mTOR complex 1 (mTORC1) signaling and promotes anti-apoptosis protein Bcl-2 down-regulation, which together mediate TMZ-induced pro-cell apoptosis effects. Our study suggests that activation of AMPK by TMZ contributes to glioblastoma cell apoptosis, probably by promoting p53 activation and inhibiting mTORC1 signaling.

Topics: AMP-Activated Protein Kinases; Antineoplastic Agents, Alkylating; Apoptosis; bcl-2-Associated X Protein; Cell Line, Tumor; Dacarbazine; DNA Modification Methylases; Enzyme Activation; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Glioblastoma; Guanine; Humans; Mechanistic Target of Rapamycin Complex 1; Methylnitronitrosoguanidine; Multiprotein Complexes; Proteins; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Signal Transduction; Temozolomide; TOR Serine-Threonine Kinases; Tumor Suppressor Protein p53

The alkylation repair deficient (Mer- phenotype) cells produce high levels of proteolytic enzyme plasminogen activator (PA) after treatment with alkylation agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Both, in Escherichia coli and in mammalian cells O6-methylguanine (O6-MeG) is repaired by analogues O6-methylguanine-DNA methyltransferase (MGMT). In E. coli MGMT is product of ada gene. To investigate the effect of bacterial MGMT expression on the induction of PA activity in human cells, we have transfected ada-alkB operon into Mer- human A1235 cells that are known to produce high levels of PA after MNNG treatment. We have shown here that A4 and A8 transformants that harbour ada gene become resistant to killing by MNNG. In addition, MNNG produced induction of extracellular PA activity was much less pronounced in A4 and A8 transformants (induction ratio 3.42 and 3.74, respectively) than in control A1235 and Aneo-1 cells (induction ratio 11.04 and 9.11, respectively). However, changes of intracellular PA activity were not significant. It appears, therefore, that induction of extracellular PA activity is inversely related to the cell capacity to repair the DNA lesions induced by alkylation agents.

Topics: Bacterial Proteins; Cell Survival; DNA Damage; DNA Repair; Enzyme Induction; Escherichia coli; Glioblastoma; Humans; Methylnitronitrosoguanidine; Mutagens; O(6)-Methylguanine-DNA Methyltransferase; Plasminogen Activators; Recombinant Proteins; Transfection; Tumor Cells, Cultured