Synergistic efficacy of O6benzylguanine and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in a human colon cancer xenograft completely resistant to BCNU alone

doi:10.1016/0006-2952(93)90086-C

Biochemical Pharmacology

Volume 45, Issue 2, 26 January 1993, Pages 483-491

https://doi.org/10.1016/0006-2952(93)90086-C Get rights and content

Abstract

The DNA repair protein O⁶alkylguanine-DNA alkyltransferase (alkyltransferase) repairs cytotoxic DNA damage formed by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). High levels of this repair protein cause tumor drug resistance to nitrosoureas. To investigate the ability of a direct alkyltransferase inhibitor, O⁶-benzylguanine, to reverse the nitrosourea resistance of human colon cancer cells, we studied the VACO 6 cell line which has high alkyltransferase and is completely resistant to BCNU at maximal tolerated doses in the xenograft model. O⁶-Benzylguanine at 0.5 $μg mL$ for 1 hr inactivated VACO 6 alkyltransferase by > 98% and reduced the ic₅₀ of BCNU by 3- to 4-fold. Further analysis indicated that these two agents act in a highly synergistic fashion. In xenograft bearing athymic mice, dose-dependent depletion of hepatic and tumor alkyltransferase was noted. To maintain alkyltransferase depletion in the xenograft for at least 24 hr, two doses of 60 $mg kg$ O⁶-benzylguanine were given 1 hr prior and 7 hr after BCNU. Under these conditions, VACO 6 xenografts became responsive to BCNU with significant reductions (P < 0.001) in the tumor growth rate. The combination increased toxicity to the host, reducing the maximum tolerated dose of BCNU by approximately 50%. This study provides definitive evidence that high alkyltransferase activity is responsible for BCNU resistance in human colon cancer xenografts and that with careful drug scheduling, O⁶-benzylguanine can sensitize a tumor which is completely unresponsive to BCNU alone. Further studies which optimize the therapeutic index of BCNU and O⁶-benzylguanine in vivo will define the schedule to be used in broader preclinical studies.

References (30)

SL Gerson et al.
Modulation of nitrosourea resistance in myeloid leukemias
Blood
(1988)
SL Gerson et al.
Modulation of nitrosourea resistance in human colon cancer by O⁶-methylguanine
Biochem Pharmacol
(1992)
KW Kohn
Interstrand cross-linking of DNA by 1,3-bis(2-chloroethyl)-1-nitrosourea and other 1-(2-haloethyl)-1-nitrosoureas
Cancer Res
(1977)
WP Tong et al.
Formation of the cross-link 1-[N³-deoxycyctidyl], 2-[N¹-deoxy-guanosinyl]-ethane in DNA treated with N,N-bis(2-chloroethyl)-N-nitrosourea
Cancer Res
(1982)
JM Bogden et al.
A system in mouse liver for the repair of O⁶-methylguanine lesions in methylated DNA
Nucleic Acids Res
(1981)
AE Pegg
Mammalian O⁶-alkylguanine-DNA alkyl-transferase: Regulation and importance in response to alkylating carcinogenic and therapeutic agents
Cancer Res
(1990)
TP Brent et al.
Formation of DNA interstrand cross-links by the novel chloroethylating agent 2-chloroethyl(methylsulfonyl)-methanesulfonate: Suppression by O⁶-alkylguanine-DNA alkyltransferase purified from human leukemic lymphoblasts
Cancer Res
(1987)
PE Gonzaga et al.
Affinity purification and characterization of human O⁶-alkylguanine-DNA alkyltransferase complexed with BCNU-treated, synthetic oligonucleotide
Nucleic Acids Res
(1989)
DA Scudiero et al.
Sensitivity of human cell strains having different abilities to repair O⁶-methylguanine in DNA to inactivation by alkylating agents including chloroethylnitrosoureas
Cancer Res
(1984)
C Zlotogorski et al.
Pretreatment of human colon tumor cells with DNA methylating agents inhibits their ability to repair chloroethyl monoadducts
Carcinogenesis
(1984)

WJ Bodell et al.

Increased repair of O⁶-alkylguanine DNA adducts in glioma-derived human cells resistant to the cytotoxic and cytogenetic effects of 1,3-bis(2-chloroethyl)-1-nitrosourea

Carcinogenesis

(1986)

DB Yarosh et al.

Repair of O⁶-methylguanine in DNA by demethylation is lacking in Mer⁻ human tumor cell strains

Carcinogenesis

(1983)

TP Brent et al.

O⁶-Alkylguanine-DNA alkyltransferase activity correlates with the therapeutic response of human rhab-domyosareoma xenografts to 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea

C Fujio et al.

Hypersensitivity of human tumor xenografts lacking O⁶-alkylguanine-DNA alkyl-transferase to the anti-tumor agent 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea

Carcinogenesis

(1989)

RO Pieper et al.

Effects of streptozotocin/bis-chloroethylnitrosourea combination therapy on O⁶-methylguanine DNA methyltransferase activity and mRNA levels in HT-29 cells in vitro

Cancer Res

(1991)

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Chemoselection of allogeneic HSC after murine neonatal transplantation without myeloablation or post-transplant immunosuppression
2012, Molecular Therapy
Citation Excerpt :
The observation that transfer of the O6-alkylguanine-DNA alkyltransferase gene into mammalian cells decreased sensitivity to 1,3-bis(2-chloroethyl)nitrosourea (BCNU), a well-established HSC toxin, suggested that alkyltransferases such as MGMT could be used for in vivo chemoselection.8,9 Identification of O6-Benzylguanine (BG)10 as an inhibitor of endogenous MGMT, and the derivation of BG-resistant forms of MGMT (P140K and G156A)11,12 provided dramatic improvements in the durability of this in vivo chemoselection strategy. The MGMTP140K mutant repair enzyme exhibits 1,000-fold resistance to alkylators and nitrosoureas compared with the wild-type MGMT enzyme following treatment with BG.13,14,15
The feasibility of allogeneic transplantation, without myeloablation or post-transplant immunosuppression, was tested using in vivo chemoselection of allogeneic hematopoietic stem cells (HSCs) after transduction with a novel tricistronic lentiviral vector (MGMT^P140K-2A-GFP-IRES-TK (MAGIT)). This vector contains P140K-O⁶-methylguanine-methyltransferase (MGMT^P140K), HSV-thymidine kinase (TK^HSV), and enhanced green fluorescent protein (eGFP) enabling (i) in vivo chemoselection of HSC by conferring resistance to benzylguanine (BG), an inhibitor of endogenous MGMT, and to chloroethylating agents such as 1,3-bis(2-chloroethyl)nitrosourea (BCNU) and, (ii) depletion of proliferating cells such as malignant clones or transduced donor T cells mediating graft versus host disease (GVHD), by expression of the suicide gene TK^HSV and Ganciclovir (GCV) administration. Non-myeloablative transplantation of transduced, syngeneic, lineage-depleted (Lin⁻) BM in neonates resulted in 0.67% GFP⁺ mononuclear cells in peripheral blood. BG/BCNU chemoselection, 4 and 8 weeks post-transplant, produced 50-fold donor cell enrichment. Transplantation and chemoselection of major histocompatibility complex (MHC)-mismatched MAGIT-transduced Lin⁻ BM also produced similar expansion for >40 weeks. The efficacy of this allotransplant approach was validated in Hbb^th3 heterozygous mice by correction of β-thalassemia intermedia, without toxicity or GVHD. Negative selection, by administration of GCV resulted in donor cell depletion without graft ablation, as re-expansion of donor cells was achieved with BG/BCNU treatment. These studies show promise for developing non-ablative allotransplant approaches using in vivo positive/negative selection.
Long-term polyclonal and multilineage engraftment of methylguanine methyltransferase P140K gene-modified dog hematopoietic cells in primary and secondary recipients
2009, Blood
Citation Excerpt :
This was the first indication that MGMT might be a useful strategy for in vivo selection and chemoprotection. Subsequently, specific active site inhibitors of wild-type MGMT were developed, namely O6BG, which sensitized cells in a synergistic fashion when used in conjunction with BCNU.42,43 The development of specific mutants of MGMT that were resistant to O6BG44-49 further improved the positive selection of gene-modified cells relative to cells expressing the wild-type form of MGMT, and also reduced the mutagenesis rate.49
Overexpression of methylguanine methyltransferase P140K (MGMTP140K) has been successfully used for in vivo selection and chemoprotection in mouse and large animal studies, and has promise for autologous and allogeneic gene therapy. We examined the long-term safety of MGMTP140K selection in a clinically relevant dog model. Based on the association of provirus integration and proto-oncogene activation leading to leukemia in the X-linked immunodeficiency trial, we focused our analysis on the distribution of retrovirus integration sites (RIS) relative to proto-oncogene transcription start sites (TSS). We analyzed RIS near proto-oncogene TSS before (n = 157) and after (n = 129) chemotherapy in dogs that received MGMTP140K gene-modified cells and identified no overall increase of RIS near proto-oncogene TSS after chemotherapy. We also wanted to determine whether in vivo selected cells retained fundamental characteristics of hematopoietic stem cells. To that end, we performed secondary transplantation of MGMTP140K gene-modified cells after in vivo selection in dog leukocyte antigen (DLA)–matched dogs. Gene-modified cells achieved multilineage repopulation, and we identified the same gene-modified clone in both dogs more than 800 and 900 days after transplantation. These data suggest that MGMTP140K selection is well tolerated and should allow clinically for selection of gene-corrected cells in genetic or infectious diseases or chemoprotection for treatment of malignancy.
Should concomitant and adjuvant treatment with temozolomide be used as standard therapy in patients with anaplastic glioma?
2006, Critical Reviews in Oncology/Hematology
Malignant gliomas are devastating tumors associated with poor prognosis. Standard treatment has been surgery followed by radiotherapy while the role of chemotherapy has remained controversial. Concomitant and adjuvant treatment with temozolomide has recently been shown to improve survival in patients with glioblastoma. While it seems intuitive to apply this regimen to patients with anaplastic gliomas which have traditionally been considered more chemosensitive, chemotherapy has not been shown to prolong life in patients with anaplastic gliomas. Despite promising preclinical and early clinical results, there is currently not enough level 1 evidence to justify concomitant and adjuvant temozolomide as standard therapy for patients with newly diagnosed anaplastic gliomas. Further investigation is needed to better define the role of chemotherapy in patients with anaplastic gliomas. Trials evaluating chemoradiotherapy as well as targeted therapeutic agents are the subject of further research.
Exploiting the role of O6-methylguanine-DNA-methyltransferase (MGMT) in cancer therapy
2006, Current Opinion in Pharmacology
Improving the efficacy of standard chemotherapy by targeting DNA repair mechanisms remains an important area of research. O⁶-methylguanine-DNA-methyltransferase (MGMT), which repairs alkylating agent damage, is one such target. Downregulation of the gene through epigenetic silencing has been shown to predict response to alkylating agent therapy in selected malignancies. Platinums have also been found to downregulate MGMT expression and this approach is currently under exploration. Another way to deplete O⁶-alkylguanine DNA alkyltransferase (AGT) levels is to modify methylating agent scheduling. Extended dosing has met with early favourable results. However, pseudosubstrates used to inhibit AGT activity have had limited success because of dose-limiting myelotoxicity. Topoisomerase I is ‘trapped’ on DNA by alteration of ligation kinetics following alkylating agent damage, leading to interest in combining AGT inhibitors or O⁶-alkylating agents with topoisomerase I inhibitors. DNA repair by AGT is an interesting target for cancer therapy that remains to be fully evaluated. The best results are likely to be achieved where its inhibition is part of treatment targeting multiple DNA damage processing pathways.
Inactivation of O6-alkylguanine DNA alkyltransferase as a means to enhance chemotherapy
2006, Cancer Treatment Reviews
DNA adducts at the O⁶-position of guanine are a result of the carcinogenic, mutagenic and cytotoxic actions of methylating and chloroethylating agents. The presence of the DNA repair protein O⁶-alkylguanine-DNA alkyltransferase (AGT) renders cells resistant to the biological effects induced by agents that attack at this position. O⁶-Benzylguanine (O⁶-BG) is a low molecular weight substrate of AGT and therefore, results in sensitizing cells and tumors to alkylating agent-induced cytotoxicity and antitumor activity. Presently, chemotherapy regimens of O⁶-BG in combination with BCNU, temozolomide and Gliadel are in clinical development. Other ongoing clinical trials include expression of mutant AGT proteins that confer resistance to O⁶-BG in bone marrow stem cells, in an effort to reduce the potential enhanced toxicity and mutagenicity of alkylating agents in the bone marrow. O⁶-BG has also been found to enhance the cytotoxicity of agents that do not form adducts at the O⁶-position of DNA, including platinating agents. O⁶-BG’s mechanism of action with these agents is not fully understood; however, it is independent of AGT activity or AGT inactivation. A better understanding of the effects of this agent will contribute to its clinical usefulness and the design of better analogs to further improve cancer chemotherapy.
O6-3-[125I]iodobenzyl-2′-deoxyguanosine ([ 125I]IBdG): Synthesis and evaluation of its usefulness as an agent for quantification of alkylguanine-DNA alkyltransferase (AGT)
2005, Bioorganic and Medicinal Chemistry
The development of O⁶-(3-[¹²⁵I]iodobenzyl)-2′-deoxyguanosine ([¹²⁵I]IBdG), the glycosylated analogue of the O⁶-3-iodobenzylguanine (IBG), as an agent for the in vivo mapping of the DNA repair protein alkylguanine-DNA alkyltransferase (AGT) is described. Synthesis of its tin precursor, O⁶-3-trimethylstannylbenzyl-2′-deoxyguanosine (TBdG) was achieved in four steps from deoxyguanosine. Radioiodination of TBdG in a single step gave [¹²⁵I]IBdG in 70–85% isolated radiochemical yield. [¹²⁵I]IBdG bound specifically to pure AGT with an IC₅₀ of 7.1 μM. From paired-label assays, [¹²⁵I]IBdG showed a 2- to 3-fold higher cellular uptake than [¹³¹I]IBG in DAOY medulloblastoma, TE-671 rhabdomyosarcoma, SK-Mel-28 melanoma, and HT-29 colon carcinoma human cell lines. Uptake of both labeled compounds in these cell lines decreased with increasing concentrations of unlabeled O⁶-benzylguanine (BG) when BG was present in the medium during incubation with the labeled compounds. Compared to BG, unlabeled IBdG diminished the uptake of [¹²⁵I]IBdG and [¹³¹I]IBG in DAOY cells more efficiently (IC₅₀ < 1 μM vs >10 μM for BG). There was no significant change in cell-bound activity of [¹²⁵I]IBdG and [¹³¹I]IBG when BG was removed from the incubation medium before incubating cells with the tracers, suggesting that only a very small portion of radioactivity taken up by the cells is AGT bound. This was corroborated by gel-electrophoresis performed on extracts from cells treated with varying amounts of BG and then incubated with [¹²⁵I]IBdG in the presence of BG. No radiolabeled AGT band was discernable by phosphor-imaging, signifying low cellular AGT binding of the radiotracer. In contrast, when cell extracts were prepared from BG pre-treated cells and aliquots were incubated with [¹²⁵I]IBdG subsequently, the intensity of radiolabeled AGT band decreased linearly as a function of BG concentration. This suggests that the low level of [¹²⁵I]IBdG that binds to AGT does so in a concentration dependent manner. These data suggest that IBdG is transported across the cell membrane to a higher degree than IBG. However, to be a practical tracer for quantifying cellular AGT, considerable localization of such derivatives need to occur within the cell nucleus where AGT is present predominantly.

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^∗: Recipient of an Edward Mallinckrodt Jr. Foundation Scholar Award.

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Biochemical Pharmacology

Abstract

References (30)

Modulation of nitrosourea resistance in myeloid leukemias

Blood

Modulation of nitrosourea resistance in human colon cancer by O⁶-methylguanine

Biochem Pharmacol

Interstrand cross-linking of DNA by 1,3-bis(2-chloroethyl)-1-nitrosourea and other 1-(2-haloethyl)-1-nitrosoureas

Cancer Res

Formation of the cross-link 1-[N³-deoxycyctidyl], 2-[N¹-deoxy-guanosinyl]-ethane in DNA treated with N,N-bis(2-chloroethyl)-N-nitrosourea

Cancer Res

A system in mouse liver for the repair of O⁶-methylguanine lesions in methylated DNA

Nucleic Acids Res

Mammalian O⁶-alkylguanine-DNA alkyl-transferase: Regulation and importance in response to alkylating carcinogenic and therapeutic agents

Cancer Res

Formation of DNA interstrand cross-links by the novel chloroethylating agent 2-chloroethyl(methylsulfonyl)-methanesulfonate: Suppression by O⁶-alkylguanine-DNA alkyltransferase purified from human leukemic lymphoblasts

Cancer Res

Affinity purification and characterization of human O⁶-alkylguanine-DNA alkyltransferase complexed with BCNU-treated, synthetic oligonucleotide

Nucleic Acids Res

Sensitivity of human cell strains having different abilities to repair O⁶-methylguanine in DNA to inactivation by alkylating agents including chloroethylnitrosoureas

Cancer Res

Pretreatment of human colon tumor cells with DNA methylating agents inhibits their ability to repair chloroethyl monoadducts

Carcinogenesis

Increased repair of O⁶-alkylguanine DNA adducts in glioma-derived human cells resistant to the cytotoxic and cytogenetic effects of 1,3-bis(2-chloroethyl)-1-nitrosourea

Carcinogenesis

Repair of O⁶-methylguanine in DNA by demethylation is lacking in Mer⁻ human tumor cell strains

Carcinogenesis

O⁶-Alkylguanine-DNA alkyltransferase activity correlates with the therapeutic response of human rhab-domyosareoma xenografts to 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea

Hypersensitivity of human tumor xenografts lacking O⁶-alkylguanine-DNA alkyl-transferase to the anti-tumor agent 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea

Carcinogenesis

Effects of streptozotocin/bis-chloroethylnitrosourea combination therapy on O⁶-methylguanine DNA methyltransferase activity and mRNA levels in HT-29 cells in vitro

Cancer Res

Cited by (103)

Chemoselection of allogeneic HSC after murine neonatal transplantation without myeloablation or post-transplant immunosuppression

Long-term polyclonal and multilineage engraftment of methylguanine methyltransferase P140K gene-modified dog hematopoietic cells in primary and secondary recipients

Should concomitant and adjuvant treatment with temozolomide be used as standard therapy in patients with anaplastic glioma?

Exploiting the role of O<sup>6</sup>-methylguanine-DNA-methyltransferase (MGMT) in cancer therapy

Inactivation of O<sup>6</sup>-alkylguanine DNA alkyltransferase as a means to enhance chemotherapy

O<sup>6</sup>-3-[<sup>125</sup>I]iodobenzyl-2′-deoxyguanosine ([ <sup>125</sup>I]IBdG): Synthesis and evaluation of its usefulness as an agent for quantification of alkylguanine-DNA alkyltransferase (AGT)

Synergistic efficacy of O6benzylguanine and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in a human colon cancer xenograft completely resistant to BCNU alone

Abstract

Blood

Biochem Pharmacol

Interstrand cross-linking of DNA by 1,3-bis(2-chloroethyl)-1-nitrosourea and other 1-(2-haloethyl)-1-nitrosoureas

Cancer Res

Formation of the cross-link 1-[N3-deoxycyctidyl], 2-[N1-deoxy-guanosinyl]-ethane in DNA treated with N,N-bis(2-chloroethyl)-N-nitrosourea

Cancer Res

A system in mouse liver for the repair of O6-methylguanine lesions in methylated DNA

Nucleic Acids Res

Mammalian O6-alkylguanine-DNA alkyl-transferase: Regulation and importance in response to alkylating carcinogenic and therapeutic agents

Cancer Res

Formation of DNA interstrand cross-links by the novel chloroethylating agent 2-chloroethyl(methylsulfonyl)-methanesulfonate: Suppression by O6-alkylguanine-DNA alkyltransferase purified from human leukemic lymphoblasts

Cancer Res

Affinity purification and characterization of human O6-alkylguanine-DNA alkyltransferase complexed with BCNU-treated, synthetic oligonucleotide

Nucleic Acids Res

Sensitivity of human cell strains having different abilities to repair O6-methylguanine in DNA to inactivation by alkylating agents including chloroethylnitrosoureas

Cancer Res

Pretreatment of human colon tumor cells with DNA methylating agents inhibits their ability to repair chloroethyl monoadducts

Carcinogenesis

Increased repair of O6-alkylguanine DNA adducts in glioma-derived human cells resistant to the cytotoxic and cytogenetic effects of 1,3-bis(2-chloroethyl)-1-nitrosourea

Carcinogenesis

Repair of O6-methylguanine in DNA by demethylation is lacking in Mer− human tumor cell strains

Carcinogenesis

O6-Alkylguanine-DNA alkyltransferase activity correlates with the therapeutic response of human rhab-domyosareoma xenografts to 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea

Hypersensitivity of human tumor xenografts lacking O6-alkylguanine-DNA alkyl-transferase to the anti-tumor agent 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea

Carcinogenesis

Effects of streptozotocin/bis-chloroethylnitrosourea combination therapy on O6-methylguanine DNA methyltransferase activity and mRNA levels in HT-29 cells in vitro

Cancer Res

Synergistic efficacy of O⁶benzylguanine and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in a human colon cancer xenograft completely resistant to BCNU alone

Formation of the cross-link 1-[N³-deoxycyctidyl], 2-[N¹-deoxy-guanosinyl]-ethane in DNA treated with N,N-bis(2-chloroethyl)-N-nitrosourea

A system in mouse liver for the repair of O⁶-methylguanine lesions in methylated DNA

Mammalian O⁶-alkylguanine-DNA alkyl-transferase: Regulation and importance in response to alkylating carcinogenic and therapeutic agents

Formation of DNA interstrand cross-links by the novel chloroethylating agent 2-chloroethyl(methylsulfonyl)-methanesulfonate: Suppression by O⁶-alkylguanine-DNA alkyltransferase purified from human leukemic lymphoblasts

Affinity purification and characterization of human O⁶-alkylguanine-DNA alkyltransferase complexed with BCNU-treated, synthetic oligonucleotide

Sensitivity of human cell strains having different abilities to repair O⁶-methylguanine in DNA to inactivation by alkylating agents including chloroethylnitrosoureas

Increased repair of O⁶-alkylguanine DNA adducts in glioma-derived human cells resistant to the cytotoxic and cytogenetic effects of 1,3-bis(2-chloroethyl)-1-nitrosourea

Repair of O⁶-methylguanine in DNA by demethylation is lacking in Mer⁻ human tumor cell strains

O⁶-Alkylguanine-DNA alkyltransferase activity correlates with the therapeutic response of human rhab-domyosareoma xenografts to 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea

Hypersensitivity of human tumor xenografts lacking O⁶-alkylguanine-DNA alkyl-transferase to the anti-tumor agent 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea

Effects of streptozotocin/bis-chloroethylnitrosourea combination therapy on O⁶-methylguanine DNA methyltransferase activity and mRNA levels in HT-29 cells in vitro