Potentiation of N-methyl-N'-nitro-N-nitrosoguanidine-induced O6-methylguanine-DNA-methyltransferase activity in a rat hepatoma cell line by poly (ADP-ribose) synthesis inhibitors

The O6-methylguanine-DNA-methyltransferase (transferase) activity in a rat hepatoma cell line (H4 cells) is enhanced as a response to DNA damaging agents. To study whether poly (ADP-ribosylation) is involved in this induction, the cells were treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) that induces the transferase activity and stimulates poly (ADP-ribose) synthesis. Addition of poly (ADP-ribose) polymerase inhibitors enhanced the transferase increase induced by MNNG. The influence of the inhibitors on the transferase induction was dose and time-dependent. The results suggest that poly (ADP-ribose) is involved in the induction of this protein.     

O^6—甲基鸟嘌呤—DNA—甲基转移酶与肿瘤预见性化疗

以中国人肿瘤株和活检组织为材料,观察了Ｏ^6－甲基鸟嘌呤－ＤＮＡ－甲基转移酶（ＭＧ ＭＴ）酶活性与亚硝脲药物耐药笥之间的关系,证明ＭＧＭＴｍＲＮＡ高表达是产生耐药笥的原因;体外和临床实验证明链脲菌素和苄基鸟嘌呤可以抑制ＭＧＭＴ酶活笥,克服耐药性;体外实验证明反义寡聚核苷酸可以调节ＭＧＭＴ基因表达,逆转录病毒介导的反义ＲＮＡ可以调节ＭＧＭＴ     

cDNA cloning of the rat O6-methylguanine-DNA-methyltransferase

A cDNA expression library was constructed from a rat hepatoma cell line ( H4 cells ) and introduced into an Escherichia coli strain ( BK2110 ) deficient in the repair of O6-methylguanine residues. Following three exposures to N-methyl-N'-nitro-N-nitrosoguanidine, a resistant colony harboring a plasmid named RMGMT was isolated. Extracts of BK2210 cells hosting the RMGMT plasmid expressed a O6-methylguanine-DNA-methyltransferase (transferase) activity and this protein had the same molecular weight as the transferase from H4 cells. The cDNA sequence of 763 bp contains an open reading frame of 630 bp encoding a protein of 209 amino acids with a calculated molecular weight of 22.2 kd. The rat protein shows 68% homology with the human transferase.     

Different patterns of DNA methylation of the two distinct O6-methylguanine-DNA methyltransferase (O6-MGMT) promoter regions in colorectal cancer

Colorectal cancer (CRC) is the third most common cancer worldwide. Colorectal cancer incidence differs widely among different geographic regions. In addition to mutational changes, epigenetic mechanisms also play important roles in the pathogenesis of CRCs. O6-methylguanine-DNA methyltransferase (O ⁶ -MGMT) is a DNA repair protein and in the absence of MGMT activity, G-to-A transition may accumulate in the specific genes such as K-ras and p53. To identify which CpG sites are critical for its downregulation, we analyzed the methylation status of the MGMT gene promoter in two sites in CRC patients. Then we compared the frequency of their methylation changes with the results of our previously reported K-ras gene mutation, APC2 and p16 methylation. MGMT methylation was examined in 92 tumor samples. A methylation specific PCR (MSP) method was performed for two loci of MGMT gene which described as MGMT-A and MGMT-B. The prevalence of MGMT-A, and MGMT-B methylation was 49/91 (53.8 %), and 83/92 (90.2 %), respectively. We detected high frequency of MGMT-B but not MGMT-A methylation in tumor tissues with APC2 methylation. Our results showed that MGMT-B methylation is significantly associated with K-ras gene mutation rather than MGMT-A (p = 0.04). Simultaneously, an inverse correlation was found between p16 and MGMT-B methylation simultaneously (p = 0.02). Our study indicated that hypermethylation of the specific locus near the MGMT start codon is critical for cancer progression. MGMT-B assessment that is associated with K-ras mutation can have a prognostic value in patients with CRC.     

Promoter methylation of O(6)-methylguanine-DNA-methyltransferase in lung cancer is regulated by p53

Methylation of the O(6)-methylguanine-DNA-methyltransferase (MGMT) promoter is associated with G:C to A:T transitions in the p53 gene in various human cancers, including lung cancer. In tumors with p53 mutation, MGMT promoter methylation is more common in advanced tumors than in early tumors. However, in tumors with wild-type p53, MGMT promoter methylation is independent of tumor stage. To elucidate whether p53 participates in MGMT promoter methylation, we engineered three cell models: A549 cells with RNA interference (RNAi)-mediated knockdown of p53, and p53 null H1299 cells transfected with either wild-type p53 (WT-p53) or mutant-p53 (L194R, and R249S-p53). Knockdown of endogenous p53 increased MGMT promoter methylation in A549 cells, and transient expression of WT-p53 in p53 null H1299 cells diminished MGMT promoter methylation, whereas the MGMT promoter methylation status were unchanged by expression of mutant-p53. Previous work showed that p53 modulates DNA-methyltransferase 1 (DNMT1) expression; we additionally examined chromatin remodeling proteins expression levels of histone deacetylase 1 (HDAC1). We found that p53 knockdown elevated expression of both DNMT1 and HDAC1 in A549 cells. Conversely, expressing WT-p53 in p53 null H1299 cells reduced DNMT1 and HDAC1 expression, but the reduction of both proteins was not observed in expressing mutant-p53 H1299 cells. CHIP analysis further showed that DNMT1 and HDAC1 binding to the MGMT promoter was increased by MGMT promoter methylation and decreased by MGMT promoter demethylation. In conclusion, MGMT promoter methylation modulated by p53 status could partially promote p53 mutation occurrence in advanced lung tumors.     

Purification and properties of O6-methylguanine-DNA-methyltransferase in human hepatic tissue

O6-Methylguanine-DNA-methyltransferase was partially purified from human liver. The transferase activity was purified by means of ammonium sulfate fractionation, DEAE-cellulose, Sepharose 6B, and double-strand DNA-cellulose chromatography. The native enzyme showed a molecular weight of about 44,000 as determined by gel filtration and a minimal molecular weight of 22,000 as obtained from SDS-PAGE. The native enzyme was unstable and underwent dissociation and decrease of activity in the presence of detergents.     

O6-methylguanine-DNA-methyltransferase (MGMT) gene therapy targeting haematopoietic stem cells: studies addressing safety issues

As haematopoietic stem cell gene therapy utilizing O(6)-methylguanine-DNA-methyltransferase has reached the clinical stage, safety-related questions become increasingly important. These issues concern insertional mutagenesis of viral vectors, the acute toxicity of pre-transplant conditioning protocols and in vivo selection regimens as well as potential genotoxic side effects of the alkylating drugs administered in this context. To address these questions, we have investigated toxicity-reduced conditioning regimens combining low-dose alkylator application with sublethal irradiation and have analysed their influence on engraftment and subsequent selectability of transduced haematopoietic stem cells. In addition, a strategy to monitor the acute and long-term genotoxic effects of drugs with high guanine-O(6) alkylating potential, such as chloroethylnitrosoureas or temozolomide is introduced. For this purpose, assays were implemented which allow an assessment of the generation and fate of primary drug-induced adducts as well as their long-term effect on chromosomal integrity at the single cell level.     

Dominant sensitization variants of human O(6)-methylguanine-DNA-methyltransferase obtained by a mutational screen of surface residues

A scanning mutagenesis experiment was performed on human O(6)-methylguanine methyltransferase (hMGMT), directed largely at non-conserved surface residues that have not previously been studied. Variants typically contained two or more substitutions. Two of the 16 variants characterized in detail are inactive for methyltransfer, but increase the cytotoxicity and mutagenic effects of methylating agents. This phenotype is reminiscent of a variant (C145A) that has a mutation in the methyl-accepting cysteine. C145A is inactive, but reportedly binds methylated DNA and confers sensitivity to methylating agents. The sensitization phenotype of the two new variants is more striking in strains that are wild-type for DNA repair than in strains that are deficient for repair, suggesting that these proteins inhibit functional DNA repair proteins by competitively binding to methylated DNA. Both variants have multiple substitutions in the last helix of the protein. These results suggest that the C-terminal helix is necessary for methyltransfer activity, but not for methylguanine-specific binding.     

The P140K mutant of human O(6)-methylguanine-DNA-methyltransferase (MGMT) confers resistance in vitro and in vivo to temozolomide in combination with the novel MGMT inactivator O(6)-(4-bromothenyl)guanine

The O(6)-methylguanine-DNA-methyltransferase (MGMT) inactivator O(6)-benzylguanine (O(6)-beG) is currently under clinical investigation as a potential tumour-sensitising agent. In clinical trials its use has been associated with increased myelotoxicity and a reduced maximum tolerated dose (MTD) for BCNU. Thus the concept of myeloprotection by gene therapy with an O(6)-beG-insensitive mutant of MGMT is soon to be tested. Recently, an alternative inactivator has been described (O(6)-(4-bromothenyl)guanine, PaTrin-2), which shows potential advantages over O(6)-beG in terms of higher activity against wild-type MGMT and oral formulation. The use of PaTrin-2 has also been associated with increased myelotoxicity in clinical trials and thus PaTrin-2 may also be a candidate for use in conjunction with mutant MGMT gene transfer in genetic chemoprotective strategies. However, its activity against mutant MGMTs has not been reported. We show here that the P(140)K mutant of MGMT is highly resistant to inactivation by PaTrin-2. Furthermore, we show that a human haemopoietic cell line (K562) transduced with a retroviral vector encoding MGMT(P140K) is highly resistant to the cytotoxic effects of PaTrin-2 in combination with the methylating agent temozolomide, and that cells expressing MGMT(P140K) can be effectively enriched in vitro following challenge with this drug combination. Finally, we show that animals reconstituted with bone marrow expressing MGMT(P140K) exhibit haemopoietic resistance to PaTrin-2/temozolomide, which results in in vivo selection of gene-modified cells. All of these effects were comparable to those also achieved using O(6)-beG/temozolomide. Thus our data show that MGMT(P140K) is a suitable candidate for chemoprotective gene therapy where PaTrin-2 is being used in conjunction with temozolomide.     

Evolution of the elaborate male intromittent organ of Xiphophorus fishes

Internally fertilizing animals show a remarkable diversity in male genital morphology that is associated with sexual selection, and these traits are thought to be evolving particularly rapidly. Male fish in some internally fertilizing species have “gonopodia,” highly modified anal fins that are putatively important for sexual selection. However, our understanding of the evolution of genital diversity remains incomplete. Contrary to the prediction that male genital traits evolve more rapidly than other traits, here we show that gonopodial traits and other nongonopodial traits exhibit similar evolutionary rates of trait change and also follow similar evolutionary models in an iconic genus of poeciliid fish (Xiphophorus spp.). Furthermore, we find that both mating and nonmating natural selection mechanisms are unlikely to be driving the diverse Xiphophorus gonopodial morphology. Putative holdfast features of the male genital organ do not appear to be influenced by water flow, a candidate selective force in aquatic habitats. Additionally, interspecific divergence in gonopodial morphology is not significantly higher between sympatric species, than between allopatric species, suggesting that male genitals have not undergone reproductive character displacement. Slower rates of evolution in gonopodial traits compared with a subset of putatively sexually selected nongenital traits suggest that different selection mechanisms may be acting on the different trait types. Further investigations of this elaborate trait are imperative to determine whether it is ultimately an important driver of speciation.     

Characterization of the vasoactive intestinal polypeptide (VIP) in the gut of fishes

• 1.1. The presence of VIP was investigated in the dogfish, Scyliorhinus canicula, the ballanwrasse. Lubrus berggylta and the bib. Trisopterus luscus, using a specific radioreceptorassay.
• 2.2. Pure porcine VIP and gut extracts of fishes yielded similar dilution curves.
• 3.3. In the dogfish, the highest concentration of VIP was found in the hindgut. In contrast, in the two teleostei studied, the highest levels of VIP were in the first part of the gut.
• 4.4. The biologically active VIP measured by radioreceptorassay correlated well with the molecule determined using a specific radioimmunoassay.
• 5.5. Our results support the hypothesis of the appearance of VIP early in evolution.

     

Genome organization in Xiphophorus (Poeciliidae; Telostei)

Summary Xiphophorus represents a valuable model for studying genomic contributions to neoplasia. For analyzing these contributions at the molecular level, basic information about the genome organization is a prerequisite. This study presents data on the organization and complexity of the genomes of three species of Xiphophorus, maculatus, variatus and helleri, representative of the problem. Their diploid nuclei, as measured in the erythrocyte, contain 1.19 pg, 1.23 pg, and 1.27 pg DNA, these values representing approximately 50% of that of birds, 20% of that of mammals. The melting curves of native, high molecular weight DNA are homogeneous, the T_m was determined for maculatus as 85.0° C (corresponding to a mean GC-content of 38.3%) for variatus as 86.0° C (GC=40.7%), for helleri as 85.0° C (GC=39.3%). Reassociation of sheared denatured DNA indicated approximately 90% single copy sequences, the remaining 10% are predominantly multiple copy sequences. The complexity of single copy DNA was determined from reassociation kinetics for maculatus as 3.97×10⁸ base pairs, for variatus as 4.31×10⁸ base pairs, and for helleri as 4.49×10⁸ base pairs. The DNA of the three species upon isopycnic density gradient centrifugation in the presence of the fluorescence dye Hoechst 33258 shows in addition to the main band, two heavy (GC-rich) satellites, denoted in the order of increasing density, components I and II. Analytical centrifugation reveals for the main band DNA a buoyant density of 1.6980 gcm^-3 (GC=38.7%), for component I 1.7080 gcm^-3 (GC=48.9%), for component II 1.7150 gcm^-3 (GC=56.1%). Each of the components comprises approximately 0.38% of the total DNA. Complete digestion of components I and II with restriction enzymes EcoRI and BamHI yields a complex banding pattern upon agarose gel-electrophoresis. A 2.4 kb fragment of component I and a 5.3 kb fragment of component II of helleri, cloned and amplified in the pBR322/E. coli RR1 system, hybridize efficiently to purified nuclei of liver. Furthermore, restriction fragments of component II DNA, transferred to nitrocellulose by Southern-blotting, hybridize with 18S and 28S ribosomal DNA.     

Site-specific mutagenesis induced by single O6-alkylguanines (O6-n-propyl, O6-n-butyl, O6-n-octyl) in vivo.

The mutagenic activity of a series of longer chain O6-n-alkylguanine residues (O6-n-propyl, O6-n-butyl, O6-n-octyl) has been analyzed using a plasmid molecule (pUC 9) in which single O6-alkylguanines were positioned in the unique Pstl recognition site by shot gun ligation (Nucleic Acids Res. 13, 3305-3316 (1985)) of overlapping synthetic oligonucleotides. After transfection of these vectors into E. coli cells having normal DNA repair systems, progeny plasmids were produced, of which 2.6%, 2.8% and 4.3% were mutated in their Pstl site when containing O6-n-propylguanine, O6-n-butylguanine, O6-n-octylguanine, respectively. DNA sequence analysis of mutant plasmid genomes revealed that O6-n-propylguanine and O6-n-butylguanine induced exclusively G-->A transitions located specifically at the preselected site. O6-n-octylguanine induced apart from G-->A transitions (70%) also targeted G-->T transversions (30%). These results indicate that the mutation frequency of longer chain O6-alkylguanines can be substantial in cells with normal repair systems and that the mutation pattern depends on the nature of the alkyl group.     

Estimation of nuclear DNA content by flow cytometry in fishes of the genus Xiphophorus

1. By use of flow cytometry we measured nuclear DNA content in cells from 16 stocks representing 9 species of the genus Xiphophorus. 2. Significant differences were detected between certain stocks and species with respect to DNA content. 3. Male-female differences were apparent in 5 of 7 stocks in which males and females were studied. 4. Estimation of nuclear DNA content is of potential significance in connection with the genetics of sex determination and the study of taxonomic relationships.     

Host determinants of DNA alkylation and DNA repair activity in human colorectal tissue: O(6)-methylguanine levels are associated with GSTT1 genotype and O(6)-alkylguanine-DNA alkyltransferase activity with CYP2D6 genotype.

There is increasing evidence that alkylating agent exposure may increase large bowel cancer risk and factors which either alter such exposure or its effects may modify risk. Hence, in a cross-sectional study of 78 patients with colorectal disease, we have examined whether (i) metabolic genotypes (GSTT1, GSTM1, CYP2D6, CYP2E1) are associated with O(6)-methyldeoxyguanosine (O(6)-MedG) levels, O(6)-alkylguanine-DNA alkyltransferase (ATase) activity or K-ras mutations, and (ii) there was an association between ATase activity and O(6)-MedG levels. Patients with colon tumours and who were homozygous GSTT1(*)2 genotype carriers were more likely than patients who expressed GSTT1 to have their DNA alkylated (83 versus 32%, P=0.03) and to have higher O(6)-MedG levels (0.178+/-0.374 versus 0.016+/-0.023 micromol O(6)-MedG/mol dG, P=0.04) in normal, but not tumour, DNA. No such association was observed between the GSTT1 genotype and the frequency of DNA alkylation or O(6)-MedG levels in patients with benign colon disease or rectal tumours. Patients with colon tumours or benign colon disease who were CYP2D6-poor metabolisers had higher ATase activity in normal tissue than patients who were CYP2D6 extensive metabolisers or CYP2D6 heterozygotes. Patients with the CYP2E1 Dra cd genotype were less likely to have a K-ras mutation: of 55 patients with the wild-type CYP2E1 genotype (dd), 23 had K-ras mutations, whereas none of the 7 individuals with cd genotype had a K-ras mutation (P=0.04). No other associations were observed between GSTT1, GSTM1, CYP2D6 and CYP2E1 Pst genotypes and adduct levels, ATase activity or mutational status. O(6)-MedG levels were not associated with ATase activity in either normal or tumour tissue. However, in 15 patients for whom both normal and tumour DNA contained detectable O(6)-MedG levels, there was a strong positive association between the normal DNA/tumour DNA adduct ratio and the normal tissue/tumour tissue ATase ratio (r(2)=0.66, P=0.001). These results indicate that host factors can affect levels both of the biologically effective dose arising from methylating agent exposure and of a susceptibility factor, the DNA repair phenotype.