Cationic core–shell nanoparticles with carmustine contained within O-benzylguanine shell for glioma therapy

The application of carmustine (BCNU) for glioma treatment is limited due to its poor selectivity for tumor and tumor resistance caused by O⁶-methylguanine-DNA-methyl transferase (MGMT). To improve the efficacy of BCNU, we constructed chitosan surface-modified poly (lactide-co-glycolides) nanoparticles (PLGA/CS NPs) for targeting glioma, loading BCNU along with O⁶-benzylguanine (BG), which could directly deplete MGMT. With core–shell structure, PLGA/CS NPs in the diameter around 177 nm showed positive zeta potential. In vitro plasma stability of BCNU in NPs was improved compared with free BCNU. The cellular uptake of NPs increased with surface modification of CS and decreasing particle size. The cytotoxicity of BCNU against glioblastoma cells was enhanced after being encapsulated into NPs; furthermore, with the co-encapsulation of BCNU and BG into NPs, BCNU + BG PLGA/CS NPs showed the strongest inhibiting ability. Compared to free drugs, PLGA/CS NPs could prolong circulation time and enhance accumulation in tumor and brain. Among all treatment groups, F98 glioma-bearing rats treated with BCNU + BG PLGA/CS NPs showed the longest survival time and the smallest tumor size. The studies suggested that the co-encapsulation of BCNU and BG into PLGA/CS NPs could remarkably enhance the efficacy of BCNU, accompanied with greater convenience for therapy.     

Bone marrow-derived cells exhibiting lung epithelial cell characteristics are enriched in vivo using methylguanine DNA methyltransferase-mediated drug resistance

Previous studies have suggested that donor bone marrow-derived cells can differentiate into lung epithelial cells at low frequency. We investigated whether we could enrich the number of donor-derived hematopoietic cells that have type II pneumocyte characteristics by overexpression of the drug resistance gene methylguanine DNA methyltransferase (MGMT). MGMT encodes O(6)-alkylguanine DNA alkyltransferase (AGT), a drug resistance protein for DNA damage induced by N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU), and the mutant P140K MGMT confers resistance to BCNU and the AGT inactivator O(6)-benzylguanine (BG). For this study, we used two MGMT selection models: one in which donor cells had a strong selection advantage because the recipient lung lacked MGMT expression, and another in which drug resistance was conferred by gene transfer of P140K MGMT. In both models, we saw an increase in the total number of donor-derived cells in the lung after BCNU treatment. Analysis of single-cell suspensions from 28 mice showed donor-derived cells with characteristics of type II pneumocytes, determined by surfactant protein C (SP-C) expression. Furthermore, an increase in the percentage of donor-derived SP-C cells was noted after BCNU or BG and BCNU treatment. This study demonstrates that bone marrow cells expressing MGMT can engraft in the lung and convert into cells expressing the type II pneumocyte protein SP-C. Furthermore, these cells can be enriched in response to alkylating agent-mediated lung injury. These results suggest that expression of MGMT could enhance the capacity of bone marrow-derived cells to repopulate lung epithelium, and when used in combination with a gene of interest, MGMT could have therapeutic applications.     

Does increase in DNA repair allow “Tolerance‐to‐Insult” in chemical carcinogenesis? Skin tumor experiments with MGMT‐overexpressing mice

Several genotoxicity endpoints have been evaluated to define nonlinear dose‐responses for S_N1 and S_N2 alkylating genotoxicants. Dose‐response studies acknowledging the process of multistage tumorigenesis are important; however, data pertaining nonlinearity are not yet available. In this communication, the role of DNA repair in the dose‐response relationship for benign papillomas was examined using the two‐stage skin carcinogenesis protocol. The data obtained with O⁶‐methylguanine‐DNA methyltransferase (MGMT) overexpressing mice in which papillomas were induced by a single topical treatment with N‐methyl‐N‐nitrosourea (MNU) followed by promotion with 12‐O‐tetradecanoylphorbol‐13‐acetate are reported. As MGMT efficiently protects cells from mutations by repairing O⁶‐methylguanine, a miscoding lesion induced by MNU, the question whether MGMT is able to nullify carcinogenic lesions to an extent where they would be considered nonhazardous has been addressed. It is shown here that MGMT overexpression significantly protects against, but does not completely nullify, the effect of MNU in tumor initiation. The possible mechanisms involved have also been discussed. Environ. Mol. Mutagen. 55:145–150, 2014. © 2013 Wiley Periodicals, Inc.     

DNA修复蛋白MGMT基因的克隆及转导脐血CD23^＋细胞后耐药特征的研究

目的：增强脐血ＣＤ３４＾＋造血细胞对化疗药物的耐药表型,探讨逆转录病毒介导的基因转移效率和耐药基因特性,以及在脐血造血干细胞保护性基因治疗中的作用和意义。方法：应用逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）从人肝细胞中获得编码六氧甲基鸟嘌呤－ＤＮＡ－甲基转移酶（Ｏ＾６－ｍｅｔｈｙｌｇｕａｎｉｎｅ－ＤＮＡ－ｍｅｔｈｙｌｔｒａｎｓｆｅｒａｓｅ,ＭＧＭＴ）ｃＤＮＡ;利用基因重组技术,将其克隆于ｐＧＥＭ－Ｔ质粒载体并构建了逆转录病毒载体Ｇ１Ｎａ－ＭＧＭＴ;应用脂质体ＬｉｐｏｆｅｃｔＡＭＩＮＥ基因转移法将后者导入ＧＰ＋Ｅ８６和ＰＡ３１７病毒包装细胞,以卡氮芥１,３－Ｂｉｓ（２－Ｃｈｌｏｒｏｅｔｈｙｌ）－１－Ｎｉｔｒｏｓｏｕｒｅａ（ＢＣＮＵ）加压筛选后的阳性克隆上清经乒乓效应后继而感染脐血ＣＤ３４＾＋细胞。应用ＰＣＲ,Ｓｏｕｔｈ     

Contribution of O6-alkylguanine and N-alkylpurines to the formation of sister chromatid exchanges,chromosomal aberrations,and gene mutations: New insights gained from studies of genetically engineered mammalian cell lines

O⁶-methyl- and O⁶-ethylguanine are the major premutagenic and precarcinogenic lesions induced in DNA by monofunctional alkyiating agents, albeit formed in minor amounts. The involvement of these lesions in SCE and aberration formation is less clear. We have analyzed the contribution of O⁶-alkylguanine to SCE and aberration formation, as well as its toxic and point mutation inducing effect in transgenic Chinese hamster ovary (CHO) cell lines that express variable amounts of human O⁶-methylguanine-DNA methyltransferase (MGMT). Cells that overexpress MGMT (or the bacterial Ada protein) gained resistance to the formation of alkylation-induced SCEs and aberrations, as compared to MGMT deficient cells. A correlation was apparent between the level of protection for SCEs and cell killing, indicating that both phenomena are interrelated. The protective effects were dependent on the level of MGMT expression, the agent used for alkylation, and cell cycle progression. Our data suggest that at least 2 kinds of lesions are responsible for SCE and aberration formation, namely, O⁶-alkylguanine and one or various N-alkylation products. The probability that O⁶-methylguanine is converted into cytogenetic effects has been estimated to be about 1:30 for SCEs, and 1:147,000 and 1:22,000 for chromosomal aberrations in the first and second post-treatment mitosis, respectively. The induction of SCEs and likely also of aberrations by O⁶-methylguanine requires two replication cycles and is supposed to involve the formation of secondary DNA lesions. Increased repair of 3-methyladenine and 7-methylguanine in CHO cells that overexpress the N-methylpurine-DNA glycosylase (MPG) after transfection with the human MPG-cDNA did not give rise to protection against methylation-induced SCEs and aberrations, probably because of incomplete excision repair. MPG overexpressing cells reacted even more sensitively to methylating agents, suggesting apurinic sites formed as a result of MPG action to be SCE and aberration-inducing lesions. © 1993 Wiley-Liss, Inc.     

Identification of a 116 kDa protein able to bind 1,3-bis(2-chloroethyl)-1-nitrosourea-damaged DNA as poly(ADP-ribose) polymerase

SKI-1 is a 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-resistant glioma cell line and SK-MG-1 is a BCNU-sensitive glioma cell line. Both cell lines do not express O⁶-methylguanine-DNA methyl transferase (MGMT) and exhibit comparable levels of 3-methyladenine DNA glycosylase. In order to detect DNA binding proteins involved in alternative DNA repair mechanisms of BCNU damage, we performed Southwestern analysis using a DNA proble damaged with BCNU and nuclear protein extracts from SKI-1 and SK-MG-1 cell lines. Both cell lines express a protein of M_r 116 000 that is able to bind to BCNU-damaged DNA with higher specificity than to undamaged DNA. This protein was identified as poly(ADP-ribose) polymerase (PARP). Using glioma extracts depleted of PARP or using antibody to block the DNA binding domain of PARP no other protein binding to BCNU-treated probe was observed. Addition of methoxyamine, an inhibitor of DNA strand breaks, led to a significant reduction of PARP binding to BCNU-treated DNA. BCNU treatment of both glioma cell lines led to reduced nicotinamide adenine dinucleotide levels, indicating activation of PARP. Thus, the recognition and binding of PARP to BCNU-induced DNA nicks with concomitant PARP activation may be important processes that are involved in the initial stage of DNA repair of BCNU lesions in glial cells.     

Differential micronucleus frequency in isogenic human cells deficient in DNA repair pathways is a valuable indicator for evaluating genotoxic agents and their genotoxic mechanisms

The micronucleus (MN) test has become an attractive tool both for evaluating the genotoxicity of test chemicals because of its ability to detect clastogenic and aneugenic events and for its convenience. As the MN assay has been mostly performed using only DNA repair‐proficient mammalian cells, we believed that the comparison of the MN frequency between DNA repair‐proficient and ‐deficient human cells may be an excellent indicator for detecting the genotoxic potential of test chemicals and for understanding their mode of action. To address this issue, the following five genes encoding DNA‐damage‐response (DDR) factors were disrupted in the TK6 B cell line, a human cell line widely used for the MN test: FANCD2, DNA polymerase ζ (REV3), XRCC1, RAD54, and/or LIG4. Using these isogenic TK6 cell lines, the MN test was conducted for four widely‐used DNA‐damaging agents: methyl methanesulfonate (MMS), hydrogen peroxide (H₂O₂), γ‐rays, and mitomycin C (MMC). The frequency of micronuclei in the double strand break repair‐deficient RAD54^−/−/LIG4^−/− cells after exposure to γ‐rays, H₂O₂, MMS and MMC was 6.2‐7.5 times higher than that of parental wild‐type TK6 cells. The percentages of cells exhibiting micronuclei in the base excision repair‐ and single strand break repair‐deficient XRCC1^−/− cells after exposure to H₂O₂, MMC and MMS were all ∼5 times higher than those of wild‐type cells. In summary, a supplementary MN assay using the combination of RAD54^−/−/LIG4^−/−, XRCC1^−/− and wild‐type TK6 cells is a promising method for detecting the genotoxic potential of test chemicals and their mode of action. Environ. Mol. Mutagen., 2018. © 2018 Wiley Periodicals, Inc.     

Immunohistochemical analysis of O6-methylguanine-DNA methyltransferase in human melanoma in comparison with skin squamous cell carcinoma

Alkylating agents, often used for chemotherapy in patients with melanoma, can produce O⁶-alkylguanine (O⁶AG) which is related to tumor cell killing after treatment with alkylating agents. O⁶AG is effectively eliminated by O⁶-methylguanine-DNA methyltransferase (O⁶MGMT) and its level is correlative to the resistance to alkylating agents. However, little is known about the relationship of O⁶MGMT to the characteristics of melanoma. This study investigated the expression of O⁶MGMT in 12 melanomas and compared it with that in 11 skin squamous cell cancers (SCCs) immunohistochemically to evaluate the O⁶MGMT activity in melanoma and its clinical significance. All of the SCC samples had high O⁶MGMT expression, while the expression of O⁶MGMT in melanoma was diverse and 4 out of 12 samples had no or extremely low O⁶MGMT activity. Out of 6 lesions obtained from metastasis, 4 had a high O⁶MGMT activity. Two out of 3 cases with a low O⁶MGMT activity in each primary lesion did not show any evidence of metastasis or local recurrence. The evaluation of O⁶MGMT activity in melanoma may, therefore, be useful to determine the characteristics of tumor in each melanoma case. In addition, the present study implies the possibility of selective cancer chemotherapy for melanoma in the near future.     

DNA‐PKcs‐dependent NHEJ pathway supports the progression of topoisomerase II poison‐induced chromosome aberrant cells

The role of DNA double strand break (DSB) repair pathways, non‐homologous end joining (NHEJ), and homologous recombination (HR) was evaluated to prevent the chromosome instability induced by the topoisomerase II (Top2) poisons, idarubicin, and etoposide in Chinese hamster cell lines. XR‐C1 (DNA‐PKcs deficient) and V‐C8 (BRCA2 deficient) showed higher sensitivity to increased concentrations of Top2 poisons compared with their normal counterparts, CHO9 and V79. Both proficient and deficient cells exhibited a marked DSB induction in all phases of the cell cycle. Additionally, deficient cells showed persistent DNA damage 24 hr post‐treatment. Chromosomal aberrations increased in the first mitosis following Top2 poison‐treatments in G1 or G2 in proficient and deficient cells. CHO9 and V79 demonstrated chromosome and chromatid exchanges following treatments in G1 and G2 phases, respectively. Deficient cells showed high frequencies of chromatid exchanges following treatments in G1 and G2. Simultaneously, we analyzed the micronuclei (MN) induction in interphase cells after treatments in G1, S, or G2 of the previous cell cycle. Both Top2 poisons induced an important increase in MN in CHO9, V79, and V‐C8 cells. XR‐C1 exhibited an increased MN frequency when cells were treated in G1 phase but not in S or G2. This MN reduction was due to a cell accumulation at G2/M and death in G2‐treated cells. Our data suggest that NHEJ and HR operate differentially throughout the cell cycle to protect from Top2 poison‐induced chromosome instability, and that DNA‐PKcs‐dependent NHEJ pathway allows the survival of chromosome damaged cells during S/G2 to the next interphase. Environ. Mol. Mutagen., 2012. © 2012 Wiley Periodicals, Inc.     

Induction of cytogenetic damage in rodents after short-term inhalation of benzene

Experiments were designed to investigate both the induction of sister chromatid exchanges (SCEs) in peripheral blood lymphocytes (PBLs) and micronuclei (MN) in bone marrow polychromatic erythrocytes (PCEs) of mice and rats after inhalation of benzene (BZ). Male DBA/2 mice (17-19 weeks old) were exposed to target concentrations of either 0, 10, 100, or 1,000 ppm BZ for 6 hr. Male Sprague-Dawley rats (11-14 weeks old) were exposed to target concentrations of either 0, 0.1, 0.3, 1, 3, 10, or 30 ppm BZ for 6 hr. Blood was obtained by cardiac puncture 18 hr after exposure, and PBLs were cultured in the presence of lipopolysaccharide (mouse B cells, 60 micrograms/ml) or concanavalin A (rat T cells, 30 micrograms/ml) to stimulate blastogenesis for SCE analysis. Femoral bone marrow smears from both species were analyzed for MN in PCEs 18 hr after BZ exposure. Mouse PBLs revealed a significant concentration-related increase in the SCE frequency over controls at 10, 100, or 1,000 ppm BZ. Mouse bone marrow showed a significant concentration-dependent increase in MN over controls after exposure to 10, 100, or 1,000 ppm BZ. Rat PBLs showed a significant increase in the SCE frequency after exposure to 3, 10, or 30 ppm BZ. The statistical significance of the 1 ppm BZ result was borderline and dependent on the statistical test chosen. Rat cells revealed a significant concentration-related increase in MN after inhalation of either 1, 3, 10, or 30 ppm BZ. PBLs from treated mice showed significant concentration-dependent decreases in mitotic indices; however, cell cycle kinetics and leucocyte counts remained unaffected. Rat PBLs showed significant decreases in mitotic activity only after exposure to 3 and 30 ppm BZ, whereas cell cycle kinetics and leucocyte counts were unaffected. These results show that BZ can induce statistically significant cytogenetic effects in PBLs and PCEs of both mice and rats after a 6-hr inhalation of BZ at low concentrations.     

Mutation analysis of K-ras and beta-catenin genes related to O6-methylguanin-DNA methyltransferase and mismatch repair protein status in human gallbladder carcinoma

O6-methylguanin-DNA methyltransferase (MGMT) is a DNA repair enzyme that transfers methyl groups from O6-methylguanine to itself. Alkylation of DNA at the O6 position of guanine is the first step by alkylating agents in inducing DNA mutations in an organism. When MGMT and the mismatch repair (MMR) system are impaired, O6-methylguanine mispairs with thymine during DNA replication, resulting in a G:C right curved arrow A:T transitional mutation in DNA. We obtained cancer lesions by manual micro-dissection (MMD) from 26 paraffin-embedded formalin-fixed gallbladder carcinoma and Laser Capture Micro-dissection (LCM) method from 10 fresh frozen specimens. Mutation analysis was performed on the micro-dissected samples for K-ras and beta-catenin genes. At codon 12 of the K-ras gene, the MMD and LCM methods detected mutations in 3 (11.5%) and 1 (10%) case, respectively. In exon 3 of beta-catenin gene, only 1 (3.8%) case revealed a mutation in MMD cancer foci. Two cases without MGMT or MMR expression revealed a G right curved arrow A transition mutation in the K-ras gene. The findings suggested that negative MGMT and MMR status contributed to a G:C right curved arrow A:T transitional mutation in the K-ras gene. However, K-ras and beta-catenin mutations were actually rare in GB carcinoma. Other gene mutations frequently occurring in gallbladder carcinoma might be affected by this negative MGMT and MMR status.     

Inactivation of the DNA-repair gene MGMT and the clinical response of gliomas to alkylating agents

BACKGROUND: The DNA-repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) inhibits the killing of tumor cells by alkylating agents. MGMT activity is controlled by a promoter; methylation of the promoter silences the gene in cancer, and the cells no longer produce MGMT. We examined gliomas to determine whether methylation of the MGMT promoter is related to the responsiveness of the tumor to alkylating agents. METHODS: We analyzed the MGMT promoter in tumor DNA by a methylation-specific polymerase-chain-reaction assay. The gliomas were obtained from patients who had been treated with carmustine (1,3-bis(2-chloroethyl)-1-nitrosourea, or BCNU). The molecular data were correlated with the clinical outcome. RESULTS: The MGMT promoter was methylated in gliomas from 19 of 47 patients (40 percent). This finding was associated with regression of the tumor and prolonged overall and disease-free survival. It was an independent and stronger prognostic factor than age, stage, tumor grade, or performance status. CONCLUSIONS: Methylation of the MGMT promoter in gliomas is a useful predictor of the responsiveness of the tumors to alkylating agents.     

Flow cytometric scoring of micronucleated reticulocytes as a possible high‐throughput radiation biodosimeter

Micronucleated reticulocyte (MN‐RET) scoring by flow cytometry (FCM) has been used in assessment of the clastogenic effects of chemicals. However, its dose–response to acute whole body irradiation (WBI) at moderate dose rates remains to be established. We show that FCM scoring of MN‐RET in peripheral blood from male ICR mice exposed to WBI X‐ray doses of 0.5–5 Gy at a dose rate of 0.488 Gy/min exhibits a linear dose–response relationship 24, 48, and 72 hr following WBI. Parallel microscopic counting of micronucleated polychromatic erythrocytes (MN‐PCE) in bone marrow smears from the same animals showed similar linear dose–response patterns at the same time points. Indeed, MN‐RET and MN‐PCE were highly correlated at all doses and time points. In view of the speed and accuracy of this method, in addition to the small blood sample size needed for the assay, the flow cytometric protocol for MN‐RET scoring may provide a minimally‐invasive, high throughput radiation biodosimeter. Environ. Mol. Mutagen., 2010. © 2009 Wiley‐Liss, Inc.     

Effect of O6‐alkylguanine‐DNA alkyltransferase on genotoxicity of epihalohydrins

The effect of O⁶‐alkylguanine‐DNA alkyltransferase (AGT) on the toxicity and mutagenicity of epihalohydrins was studied. AGT is a DNA repair protein that protects cells from agents that produce genotoxic O⁶‐alkylguanine lesions by transferring the alkyl group to an internal cysteine residue (Cys¹⁴⁵ in human AGT) in a single‐step. This cysteine acceptor site is highly reactive and epihalohydrins reacted readily with AGT at this site with a halide order of reactivity of Br > Cl > F. AGT expression in bacterial cells caused a very large increase in the mutagenicity and cytotoxicity of epibromohydrin. The mutations were almost all G:C to A:T transitions. Epichlorohydrin also augmented AGT‐mediated mutagenesis but to a lesser extent than epibromohydrin. In vitro experiments showed that AGT was covalently cross‐linked to DNA in the presence of epibromohydrin and that this conjugation occurred predominantly at Cys¹⁴⁵, and to a smaller extent at Cys¹⁵⁰, a less reactive residue also located within the active site pocket. Two pathways yielding the AGT–DNA adduct were found to occur. The predominant mechanism results in an AGT‐epihalohydrin intermediate, which, facilitated by the DNA binding properties of AGT, then reacts covalently with DNA. The second pathway involves an initial reactive DNA‐epihalohydrin intermediate that subsequently reacts with AGT. Our results show that the paradoxical AGT‐mediated increase in genotoxicity which has previously been shown to occur with dihaloalkanes, butadiene diepoxide and nitrogen mustards, also occurs with epihalohydrins and is likely to contribute to their toxicity and mutagenicity. Environ. Mol. Mutagen., 2009. © 2009 Wiley‐Liss, Inc.     

Molecular and cellular influences of butylated hydroxyanisole on Chinese hamster V79 cells treated with N-methyl-N'-nitro-N-nitrosoguanidine: antimutagenicity of butylated hydroxyanisole

The antioxidant butylated hydroxyanisole (BHA) is a rodent carcinogen that also reduces the mutagenicity and carcinogenicity of other agents. In this study, we have evaluated possible mechanisms for the antimutagenicity of BHA by investigating its effects on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-treated Chinese hamster V79 cells. Mutant frequency was determined using the hprt/V79 assay, while plating efficiency was used to measure cytotoxicity, and apoptosis was measured by flow immunofluorocytometry. In addition, DNA strand breaks and the kinetics of strand-break rejoining were investigated by the alkaline elution of DNA and by single-cell gel electrophoresis (SCGE). Although the higher concentration of BHA (0.5 mM) increased the cytotoxicity of MNNG and the lower concentration of BHA (0.25 mM) did not change it, both concentrations were antimutagenic in MNNG-treated cells, with the greater effect occurring at the lower BHA concentration. Neither BHA nor MNNG nor BHA + MNNG increased the level of apoptotic nuclei, and BHA did not change the level of MNNG-induced DNA strand breaks, though it did inhibit their rejoining. Determination of O(6)-methylguanine-DNA-methyltransferase (MGMT) activity confirmed that V79 cells do not synthesize active MGMT protein; MGMT activity was also undetectable after MNNG and BHA + MNNG treatment. The ability of BHA to reduce the level of MNNG-induced mutations did not correlate with cytotoxicity, induction of apoptosis, the level of DNA strand break induction, or MGMT activity. A modified SCGE assay showed that BHA significantly reduced the level of formamidopyrimidine-DNA-glycosylase + endonucleaseIII-sensitive sites, which at least partially are caused by oxidative DNA lesions. The results suggest that the protective effect of BHA on MNNG-induced mutagenicity is best explained by the antioxidative activity of BHA, which may scavenge free radicals that participate in MNNG-induced mutagenicity.     

New insights into estrogenic regulation of O6-methylguanine DNA-methyltransferase (MGMT) in human breast cancer cells: Co-degradation of ER-α and MGMT proteins by fulvestrant or O6-benzylguanine indicates fresh avenues for therapy

Endocrine therapy using estrogen receptor-α (ER-α) antagonists for attenuating horm2one-driven cell proliferation is a major treatment modality for breast cancers. To exploit any DNA repair deficiencies associated with endocrine therapy, we investigated the functional and physical interactions of ER-α with O6-methylguanine DNA methyltransferase (MGMT), a unique DNA repair protein that confers tumor resistance to various anticancer alkylating agents. The ER-α -positive breast cancer cell lines (MCF-7, T47D) and ER- negative cell lines (MDAMB-468, MDAMB-231), and established inhibitors of ER-α and MGMT, namely, ICI-182,780 (Faslodex) and O6-benzylguanine, respectively, were used to study MGMT- ER interactions. The MGMT gene promoter was found to harbor one full and two half estrogen-responsive elements (EREs) and two antioxidant-responsive elements (AREs). MGMT expression was upregulated by estrogen, downregulated by tamoxifen in Western blot and promoter-linked reporter assays. Similarly, both transient and stable transfections of Nrf-2 (nuclear factor-erythroid 2-related factor-2) increased the levels of MGMT protein and activity 3 to 4-fold reflecting novel regulatory nodes for this drug-resistance determinant. Of the different ER-α antagonists tested, the pure anti-estrogen fulvestrant was most potent in inhibiting the MGMT activity in a dose, time and ER-α dependent manner, similar to O6-benzylguanine. Interestingly, fulvestrant exposure led to a degradation of both ER-α and MGMT proteins and O6-benzylguanine also induced a specific loss of ER-α and MGMT proteins in MCF-7 and T47D breast cancer cells with similar kinetics. Immunoprecipitation revealed a specific association of ER-α and MGMT proteins in breast cancer cells. Furthermore, silencing of MGMT gene expression triggered a decrease in the levels of both MGMT and ER-α proteins. The involvement of proteasome in the drug-induced degradation of both proteins was also demonstrated. Fulvestrant enhanced the cytotoxicity of MGMT-targeted alkylating agents, namely, temozolomide and BCNU by 3 to 4-fold in ER-α positive cells, but not in ER–negative cells. We conclude that MGMT and ER-α proteins exist as a complex and are co-targeted for ubiquitin-conjugation and subsequent proteasomal degradation. The findings offer a clear rationale for combining alkylating agents with endocrine therapy.     

Genotoxicity of doxorubicin in F344 rats by combining the comet assay,flow‐cytometric peripheral blood micronucleus test,and pathway‐focused gene expression profiling

Doxorubicin (DOX) is an antineoplastic drug effective against many human malignancies. DOX's clinical efficacy is greatly limited because of severe cardiotoxicity. To evaluate if DOX is genotoxic in the heart, ~7‐week‐old, male F344 rats were administered intravenously 1, 2, and 3 mg/kg bw DOX at 0, 24, 48, and 69 hr and the Comet assays in heart, liver, kidney, and testis and micronucleus (MN) assay in the peripheral blood (PB) erythrocytes using flow cytometry were conducted. Rats were euthanized at 72 hr and PB was removed for the MN assay and single cells were isolated from multiple tissues for the Comet assays. None of the doses of DOX induced a significant DNA damage in any of the tissues examined by the alkaline Comet assay. Contrastingly, the glycosylase enzymes‐modified Comet assay showed a significant dose dependent increase in the oxidative DNA damage in the cardiac tissue (P ≤ 0.05). In the liver, only the top dose induced significant increase in the oxidative DNA damage (P ≤ 0.05). The histopathology showed no severe cardiotoxicity but non‐neoplastic lesions were present in both untreated and treated samples. A severe toxicity likely occurred in the bone marrow because no viable reticulocytes could be screened for the MN assay. Gene expression profiling of the heart tissues showed a significant alteration in the expression of 11 DNA damage and repair genes. These results suggest that DOX is genotoxic in the heart and the DNA damage may be induced primarily via the production of reactive oxygen species. Environ. Mol. Mutagen. 55:24–34, 2014. © 2013 Wiley Periodicals, Inc.^?     

γH2AX formation kinetics in PBMCs of rabbits exposed to acute and fractionated radiation and attenuation of focus frequency through preadministration of a combination of podophyllotoxin and rutin hydrate

DNA damage can be assessed by the quantitation of γH2AX foci that form at DSB sites. This study examines the generation and persistence of γH2AX foci, variability in foci size after acute and fractionated radiation exposure, and the effect of pretreatment with a safe radioprotective formulation termed G‐003M on foci generation and persistence. G‐003M contains a combination of podophyllotoxin and rutin hydrate, and was administered intramuscularly to rabbits 1 hr prior to Co⁶⁰ gamma irradiation. Rabbits were assigned to one of the following treatment groups: untreated, G‐003M alone, irradiated (single dose 8 Gy, fractionated 2 Gy/day for 4 days or single dose 2 Gy) or G‐003M preadministration followed by radiation exposure. Foci continuously persisted for a week in peripheral blood mononuclear cells of rabbits exposed to a single 8 Gy dose. However, the number of foci gradually decreased after reaching a maximum at 1 h. In rabbits exposed to fractionated radiation, foci detected 1 hr after the final exposure were significantly larger (P < 0.001) than in rabbits exposed to a single 8 Gy dose, but disappeared completely after 24 h. In both groups, foci reappeared on days 11‐15 in terminally ill animals. G‐003M pretreatment significantly (P < 0.05) attenuated the formation of γH2AX foci in all irradiated rabbits. This study reveals that γH2AX focus assessment could be used to confirm radiation exposure, that focus size reflects the type of radiation exposure (acute or fractionated), that the re‐appearance of foci is a strong indicator of imminent death in animals, and that G‐003M provides protection against radiation. Environ. Mol. Mutagen. 57:455–468, 2016. © 2016 Wiley Periodicals, Inc.     

Expression of O6-methylguanine DNA methyltransferase (MGMT) and its clinical significance in gastroenteropancreatic neuroendocrine neoplasm

O⁶-methylguanine-DNA methyltransferase (MGMT) is a widespread DNA repair enzyme defending against mutation caused by guanine O⁶-alkylating agents. Until now, we know only little about the expression of MGMT in gastroenteropancreatic neuroendocrine neoplasm (GEP-NEN). To study the expression of MGMT and its clinical significance in GEP-NEN, 174 specimens of GEP-NEN were examined, of which 152 specimens came from The First Affiliated Hospital, Sun Yat-sen University during October 1995 to November 2013, 22 specimens came from Peking Union Medical College Hospital during September 2004 to April 2010. MGMT protein was detected with EnVision immunohistochemical staining method. Clinicopathological factors were also collected and analyzed. We observed that the overall expression rate of MGMT was 83.9%. Over expression of MGMT protein was not associated with sex, age, functional status, primary tumor location, grading, classification, TNM stage and metastasis (P > 0.05). Kaplan-Meier analysis revealed that there was no significant difference in survival between MGMT-positive and MGMT-negative tumors of GEP-NEN patients (χ² = 0.887, P = 0.346). In multivariate analyses carried out by Cox proportional hazards regression model, MGMT expression was also not an independent predictors of survival. These results demonstrated that MGMT protein was highly expressed in GEP-NEN. MGMT deficiency rate was similar in pancreatic NEN and in gastrointestinal NEN. MGMT expression was not correlated with prognosis of GEP-NEN.     

Pig‐a gene mutation and micronucleated reticulocyte induction in rats exposed to tumorigenic doses of the leukemogenic agents chlorambucil,thiotepa, melphalan,and 1,3‐propane sultone

To evaluate whether blood‐based genotoxicity endpoints can provide temporal and dose‐response data within the low‐dose carcinogenic range that could contribute to carcinogenic mode of action (MoA) assessments, we evaluated the sensitivity of flow cytometry‐based micronucleus and Pig‐a gene mutation assays at and below tumorigenic dose rate 50 (TD50) levels. The incidence of micronucleated reticulocytes (MN‐RET) was used to evaluate chromosomal damage, and the frequency of CD59‐negative reticulocytes (RET^CD59?) and erythrocytes (RBC^CD59?) served as phenotypic reporters of mutation at the X‐linked Pig‐a gene. Several leukemogenic agents with a presumed genotoxic MoA were studied. Specifically, male Sprague Dawley rats were treated via oral gavage for 28 days with chlorambucil, thiotepa, melphalan, and 1,3‐propane sultone at doses corresponding to 0.33x, 1x, and 3x TD50, as well as at the maximum tolerated dose. Frequencies of MN‐RET were determined at Days 4 and 29, and RET^CD59? and RBC^CD59? data were collected pretreatment as well as Days 15/16, 29, and 56/57. Dose‐related increases were observed for each endpoint, and time to maximal effect was consistently: MN‐RET < RET^CD59? < RBC^CD59?. For each of the chemicals studied, the genotoxic events occurred long before tumors or preneoplastic lesions would be expected. Furthermore, in the case of Pig‐a gene mutation, the responses were observed at or below the TD50 dose for three out of the four chemicals studied. These data illustrate the potential for quantitative blood‐based analyses to provide dose‐response and temporality information that relates genetic damage to cancer induction. Environ. Mol. Mutagen. 55:299–308, 2014. © 2014 Wiley Periodicals, Inc.