O6-(4-bromothenyl)guanine reverses temozolomide resistance in human breast tumour MCF-7 cells and xenografts

Clemons, Mark; Kelly, Jane; Watson, Amanda J.; Howell, Anthony; Mcelhinney, R. S.; McMurry, T. Brian H.; Margison, Geoffrey P.

doi:10.1038/sj.bjc.6602833

Cited by 53 publications

(42 citation statements)

References 32 publications

(30 reference statements)

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“…TK6 and MT1 cells are MGMT-deficient; thus, they fail to remove methyl adducts from O 6 -G (Goldmacher et al, 1986). MCF-7, HCT116, and HCT116/ 3-6 cells are MGMT-proficient (Vernole et al, 2003;Clemons et al, 2005).…”

Section: Methodsmentioning

confidence: 99%

AKT Is Activated in an Ataxia-Telangiectasia and Rad3-Related-Dependent Manner in Response to Temozolomide and Confers Protection against Drug-Induced Cell Growth Inhibition

Caporali

Levati

Starace³

et al. 2008

Mol Pharmacol

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The phosphatidylinositol 3-kinase/AKT pathway is activated frequently in human cancer, and it has been implicated in tumor cell proliferation, survival, and chemoresistance. In this study, we addressed the role of AKT in cellular responses to the therapeutic methylating agent temozolomide (TMZ), and we investigated the possible link between TMZ-induced modulation of AKT function and activation of ataxia-telangiectasia and Rad3-related (ATR)-and ataxia telangiectasia mutated (ATM)-dependent signaling pathways. We found that clinically relevant concentrations of TMZ caused activation of endogenous AKT in lymphoblastoid cells, and in colon and breast cancer cells, and that this molecular event required a functional mismatch repair system. Transfection of a dominant-negative kinasedead form of AKT1 into breast cancer cells abrogated TMZinduced activation of endogenous AKT, and it markedly enhanced cell sensitivity to the drug. Likewise, exposure of the MMR-proficient cell lines to the AKT inhibitor D-3-deoxy-2-O-methyl-myo inositol 1-[(R)-2-methoxy-3-(octadecyloxy)-propyl hydrogen phosphate] (SH-5) impaired AKT phosphorylation in response to TMZ, and it significantly increased cell chemosensitivity. Furthermore, small interfering RNA (siRNA)-mediated reduction of AKT1 expression in colon cancer cells potentiated the growth inhibitory effects of TMZ. Inhibition of ATM expression in colon cancer cells by siRNA did not impair TMZ-induced activation of AKT, whereas siRNA-mediated inhibition of ATR prevented AKT activation in response to the drug and increased cell chemosensitivity. These results strongly support the hypothesis that clinical benefit could be obtained by combining TMZ with inhibitors of the AKT pathway. Moreover, they provide the first evidence of a novel function of ATR as an upstream activator of AKT in response to DNA damage induced by O 6 -guanine-methylating agents.The serine/threonine protein kinase AKT is a critical regulator of major cellular processes, including gene expression, glycogen metabolism, migration, proliferation, and survival (for review, see Bellacosa et al., 2005). Three isoforms of AKT, encoded by three different genes, are present in mammalian cells. Each isoform comprises an NH 2 -terminal pleckstrin homology domain, a catalytic protein kinase domain, and a short COOH-terminal region. All AKT isoforms contain two main regulatory phosphorylation sites, one within the kinase domain (Thr308/AKT1, Thr309/AKT2, Thr305/ AKT3), and one in the COOH-terminal region (Ser473/AKT1, Ser474/AKT2, Ser472/AKT3).This study was supported by the Italian Ministry of Health. Article, publication date, and citation information can be found at

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Section: Methodsmentioning

confidence: 99%

AKT Is Activated in an Ataxia-Telangiectasia and Rad3-Related-Dependent Manner in Response to Temozolomide and Confers Protection against Drug-Induced Cell Growth Inhibition

Caporali

Levati

Starace³

et al. 2008

Mol Pharmacol

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“…Potent, non-toxic inhibitors of MGMT, O6 -benzylguanine ( O6 -BG) [15] and O6 -(4-bromothenyl)guanine (PaTrin-2) [16] act as ‘pseudosubstrates’ that irreversibly inactivate MGMT. They covalently transfer benzyl (or 4-bromothenyl) to active site cysteines, potentiating alkylating agent efficacy.…”

Section: Introductionmentioning

confidence: 99%

Acquired Resistance to Temozolomide in Glioma Cell Lines: Molecular Mechanisms and Potential Translational Applications

Zhang¹,

Stevens²,

Laughton³

et al. 2010

Oncology

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Treatment for glioblastoma multiforme includes the alkylating agent temozolomide combined with ionizing radiation. Persistent O6-guanine methylation by temozolomide in O6-methylguanine methyl transferase negative tumors causes cytotoxic lesions recognized by DNA mismatch repair, triggering apoptosis. Resistance (intrinsic or acquired) presents obstacles to successful temozolomide treatment, limiting drug efficacy and life expectancy. Two glioma cell lines, SNB19 and U373, sensitive to temozolomide (GI₅₀ values 36 and 68 µM, respectively) were exposed to increasing temozolomide concentrations (1–100 µM). Variant cell lines (SNB19VR, U373VR) were generated that displayed acquired temozolomide resistance (GI₅₀ values 280 and 289 µM, respectively). Cross-resistance to mitozolomide was observed in U373VR cells only. In clonogenic and MTT assays, methylguanine methyltransferase (MGMT) depletion using O6-benzylguanine sensitized U373VR cells to temozolomide, indicating the resistance mechanism involves MGMT re-expression. Indeed, Western blot analyses revealed MGMT protein in cell lysates. In SNB19VR cells, down-regulation of MSH6 message and protein expression may confer temozolomide tolerance. Inhibition of poly(ADP-ribose) polymerase-1 (a key base excision repair (BER) enzyme) partially restored sensitivity, and DNA repair gene arrays demonstrated up-regulation (>5-fold) of BER gene NTL1 in SNB19VR cells. In conclusion, we have developed two glioma cell lines whose distinct mechanisms of acquired resistance to temozolomide, involving expression of MGMT, or inactivation of DNA mismatch repair and recruitment of BER enzymes, are consistent with clinical observations. These cell lines provide valuable models for the development of strategies to combat temozolomide resistance.

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“…Chronic exposure to alkylating/methylating agents can lead to increased MGMT activity (1), and MGMT expression protects from spontaneous G:C to A:T transition mutations (2). Inactivation of MGMT increases efficacy of alkylating agent against cancer (3)(4)(5). MGMT promoter methylation is present in approximately 30-40% of colorectal cancers, and may co-exist with widespread CpG methylation known as the CpG island methylator phenotype (CIMP) (6).…”

Section: Introductionmentioning

confidence: 99%

MGMT germline polymorphism is associated with somatic MGMT promoter methylation and gene silencing in colorectal cancer

Ogino¹,

Hazra²,

Tranah³

et al. 2007

Carcinogenesis

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O6-(4-bromothenyl)guanine reverses temozolomide resistance in human breast tumour MCF-7 cells and xenografts

Cited by 53 publications

References 32 publications

AKT Is Activated in an Ataxia-Telangiectasia and Rad3-Related-Dependent Manner in Response to Temozolomide and Confers Protection against Drug-Induced Cell Growth Inhibition

AKT Is Activated in an Ataxia-Telangiectasia and Rad3-Related-Dependent Manner in Response to Temozolomide and Confers Protection against Drug-Induced Cell Growth Inhibition

Acquired Resistance to Temozolomide in Glioma Cell Lines: Molecular Mechanisms and Potential Translational Applications

MGMT germline polymorphism is associated with somatic MGMT promoter methylation and gene silencing in colorectal cancer

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