Combined treatment with temozolomide and methoxyamine: blocking apurininc/pyrimidinic site repair coupled with targeting topoisomerase IIalpha.

PURPOSE: Methoxyamine has been shown to potentiate the cytotoxic effect of temozolomide both in vitro and in human tumor xenograft models. We postulate that the enhanced cytotoxicity is mediated by methoxyamine-bound apurininc/pyrimidinic (MX-AP) site, a key lesion formed by the combination of temozolomide and methoxyamine. When located within topoisomerase IIalpha (topo II) cleavage sites in DNA, MX-AP sites act as dual lethal targets, not only functionally disrupting the base excision repair (BER) pathway but also potentially poisoning topo II. EXPERIMENTAL DESIGN: Using oligonucleotide substrates, in which a position-specific MX-AP site is located within topo II cleavage sites, we examined the effect of MX-AP site on both AP endonuclease- and topo II-mediated DNA cleavage in vitro. RESULTS: MX-AP sites were refractory to the catalytic activity of AP endonuclease, indicating their ability to block BER. However, they were cleaved by either purified topo II or nuclear extracts from tumor cells expressing high levels of topo II, suggesting that MX-AP sites stimulate topo II-mediated DNA cleavages. In cells, treatment with temozolomide and methoxyamine increased the expression of topo II and enriched the formation of gammaH2AX foci, which were colocalized with up-regulated topo II, confirming that DNA double-strand breaks marked by gammaH2AX foci are associated with topo II in cells. CONCLUSIONS: Our findings identify a molecular mechanism of cell death whereby MX-AP sites that cumulated in cells due to resistance to BER potentially convert topo II into biotoxins, resulting in enzyme-mediated DNA scission and cell death.

Inactivated MGMT by O6-benzylguanine is associated with prolonged G2/M arrest in cancer cells treated with BCNU.

HCT116 and HCT15 cells that highly express O(6)-methylguanine-DNA-methyltransferase (MGMT) displayed a transient cell cycle G2/M arrest in response to exposure to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) alone; however, 70-80% of cells were arrested in G2/M after treatment with O(6)-benzylguanine (BG) and BCNU. Cells accumulated in G2/M showed elevated levels of an inactive form of cyclin B1/p-Cdc2 (Tyr15) complex that was not associated with activation of Chk1/p-Cdc25C and was independent of p53/p21 status. The most prominent feature of cell death was the appearance of enlarged and multinucleated cells that was related to the inhibition of mitotic entry. In contrast, BG-resistant cell lines, HCT116 BBR and HCT15 BBR cells that contain mutations K165E and K165N of MGMT, respectively, displayed a normal cell cycle progression with a slight and transient increase in G2/M arrest at 24 h after treatments with either BCNU alone or BG combined with BCNU. The differences in the ability to progress toward G2/M after treatment with BG and BCNU between cells expressing wild-type MGMT and mutated MGMT were confirmed in CHO cells transfected with human wild type and K165E mutant MGMT cDNA, respectively. Thus, our findings suggest that BG-inactivated MGMT may be linked to cell signaling events, forcing cells into a permanent G2/M arrest in response to the DNA damages induced by BCNU.

In vivo selection of MGMT(P140K) lentivirus-transduced human NOD/SCID repopulating cells without pretransplant irradiation conditioning.

Infusion of transduced hematopoietic stem cells into nonmyeloablated hosts results in ineffective in vivo levels of transduced cells. To increase the proportion of transduced cells in vivo, selection based on P140K O6-methylguanine-DNA-methyltransferase (MGMT[P140K]) gene transduction and O6-benzylguanine/1,3-bis(2-chloroethyl)-1-nitrosourea (BG/BCNU) treatment has been devised. In this study, we transduced human NOD/SCID repopulating cells (SRCs) with MGMT(P140K) using a lentiviral vector and infused them into BG/BCNU-conditioned NOD/SCID mice before rounds of BG/BCNU treatment as a model for in vivo selection. Engraftment was not observed until the second round of BG/BCNU treatment, at which time human cells emerged to compose up to 20% of the bone marrow. Furthermore, 99% of human CFCs derived from NOD/SCID mice were positive for provirus as measured by PCR, compared with 35% before transplant and 11% in untreated irradiation-preconditioned mice, demonstrating selection. Bone marrow showed BG-resistant O6-alkylguanine-DNA-alkyltransferase (AGT) activity, and CFUs were stained intensely for AGT protein, indicating high transgene expression. Real-time PCR estimates of the number of proviral insertions in individual CFUs ranged from 3 to 22. Selection resulted in expansion of one or more SRC clones containing similar numbers of proviral copies per mouse. To our knowledge, these results provide the first evidence of potent in vivo selection of MGMT(P140K) lentivirus-transduced human SRCs following BG/BCNU treatment.

Blockage of abasic site repair enhances antitumor efficacy of 1,3-bis-(2-chloroethyl)-1-nitrosourea in colon tumor xenografts.

Methoxyamine (MX) has been shown to potentiate the antitumor effect of temozolomide (TMZ) in human tumor xenograft models. This potentiation is due to the reactivity of MX with apurinic/pyrimidinic (AP) sites in DNA, which are formed following DNA glycosylase removal of TMZ-induced methyl-purine adducts. MX-bound AP sites cannot be further processed by base excision repair (BER), resulting in cell death. On the basis of this finding, we investigated in vivo whether MX enhanced therapeutic efficacy of other agents, such as 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) that generates AP sites during DNA repair. Nude mice carrying human colon tumor xenografts, HCT116 and HCT116-Ch3, were treated by a single injection (i.p) of BCNU alone (30 mg/kg) or MX (2 mg/kg) combined with BCNU. The effect on tumor growth of BCNU alone was very moderate. Combined administration of MX and BCNU produced significant inhibition of tumor growth. Tumor growth delays were 14 +/- 3 days in HCT116 and 16 +/- 2 days in HCT116-Ch3 tumors, respectively (P < 0.05 versus control or BCNU alone groups). Similar results were also observed in SW480 and DLD1 tumors. Importantly, no systemic toxicity was noted with BCNU and MX. In contrast, BCNU (at dose of 25 mg/kg) combined with O(6)-benzylguanine (BG), an inhibitor of O(6)-alkylguanine-DNA alkyltransferase (AGT) being tested in clinical trials, caused toxic death in all treated mice. However, a lower dose BCNU (10 mg/kg) combined with BG and MX had significant antitumor effect without toxic death. Thus, targeting BER with MX is a promising strategy to improve the antitumor activity of BCNU and perhaps other DNA-damaging agents.