Helicobacter pylori CagA interacts with E-cadherin and deregulates the beta-catenin signal that promotes intestinal transdifferentiation in gastric epithelial cells.

Infection with Helicobacter pylori cagA-positive strains is associated with gastric adenocarcinoma. Intestinal metaplasia is a precancerous lesion of the stomach characterized by transdifferentiation of the gastric mucosa to an intestinal phenotype. The H. pylori cagA gene product, CagA, is delivered into gastric epithelial cells, where it undergoes tyrosine phosphorylation by Src family kinases. Tyrosine-phosphorylated CagA specifically binds to and activates SHP-2 phosphatase, thereby inducing cell-morphological transformation. We report here that CagA physically interacts with E-cadherin independently of CagA tyrosine phosphorylation. The CagA/E-cadherin interaction impairs the complex formation between E-cadherin and beta-catenin, causing cytoplasmic and nuclear accumulation of beta-catenin. CagA-deregulated beta-catenin then transactivates beta-catenin-dependent genes such as cdx1, which encodes intestinal specific CDX1 transcription factor. In addition to beta-catenin signal, CagA also transactivates p21(WAF1/Cip1), again, in a phosphorylation-independent manner. Consequently, CagA induces aberrant expression of an intestinal-differentiation marker, goblet-cell mucin MUC2, in gastric epithelial cells that have been arrested in G1 by p21(WAF1/Cip1). These results indicate that perturbation of the E-cadherin/beta-catenin complex by H. pylori CagA plays an important role in the development of intestinal metaplasia, a premalignant transdifferentiation of gastric epithelial cells from which intestinal-type gastric adenocarcinoma arises.

Methylglyoxal, an endogenous aldehyde, crosslinks DNA polymerase and the substrate DNA.

Methylglyoxal, a known endogenous and environmental mutagen, is a reactive alpha-ketoaldehyde that can modify both DNA and proteins. To investigate the possibility that methylglyoxal induces a crosslink between DNA and DNA polymerase, we treated a 'primed template' DNA and the exonuclease-deficient Klenow fragment (KF(exo-)) of DNA polymerase I with methylglyoxal in vitro. When the reaction mixtures were analyzed by SDS-PAGE, we found that methylglyoxal induced a DNA-KF(exo-) crosslink. The specific binding complex of KF(exo-) and 'primed template' DNA was necessary for formation of the DNA-KF(exo-) crosslink. Methylglyoxal reacted with guanine residues in the single-stranded portion of the template DNA. When 2'-deoxyguanosine was incubated with Nalpha-acetyllysine or N-acetylcysteine in the presence of methylglyoxal, a crosslinked product was formed. No other amino acid derivatives tested could generate a crosslinked product. These results suggest that methylglyoxal crosslinks a guanine residue of the substrate DNA and lysine and cysteine residues near the binding site of the DNA polymerase during DNA synthesis and that DNA replication is severely inhibited by the methylglyoxal-induced DNA-DNA polymerase crosslink.

Formation of 5-formyl-2'-deoxycytidine from 5-methyl-2'-deoxycytidine in duplex DNA by Fenton-type reactions and gamma-irradiation.

5-methyl-2'-deoxycytidine (5-Me-dC) is formed by the enzymatic methylation of dC, primarily in CpG sequences in DNA, and is involved in the regulation of gene expression. In the present study, 5-Me-dC and double-stranded DNA fragments containing 5-Me-dC were either gamma-irradiated or aerobically treated with Fenton-type reagents, Fe(II)-EDTA, Fe(II)-nitrilotriacetic acid, Fe(III)-EDTA-H(2)O(2)-catechol or ascorbic acid-H(2)O(2) under neutral conditions. The formation of 5-formyl-2'-deoxycytidine (5-CHO-dC) was observed upon treatment of both 5-Me-dC and DNA fragments containing 5-Me-dC. The yields of 5-CHO-dC from 5-Me-dC and those of 5-formyl-2'-deoxyuridine from dT were comparable. These results suggest that 5-Me-dC in DNA is as susceptible to oxidation as dT in cells, and raise the possibility that 5-CHO-dC may contribute to the high mutagenic rate observed in CpG sequences in genomic DNA.

Types of mutations induced by glyoxal, a major oxidative DNA-damage product, in Salmonella typhimurium.

We have analyzed the types of mutations induced by glyoxal, a major oxidative DNA-damage product, in Salmonella typhimurium. A set of six strains, TA7001 to TA7006, was used to detect base-pair substitutions, and the TA98 strain was employed to detect frameshift mutations. Glyoxal did not induce mutations at A:T base pairs. The majority of the mutations induced by glyoxal were base-pair substitutions at G:C base pairs, and a small level of frameshift mutations was detected in the TA98 strain.

Deficient nucleotide excision repair increases base-pair substitutions but decreases TGGC frameshifts induced by methylglyoxal in Escherichia coli.

To investigate the mutation spectrum of a well-known mutagen, methylglyoxal, and the influence of nucleotide excision repair (NER) on methylglyoxal-induced mutations, we treated wild-type and NER-deficient (uvrA or uvrC) Escherichia coli strains with methylglyoxal, and analyzed mutations in the chromosomal lacI gene. In the three strains, the cell death and the mutation frequency increased according to the dose of methylglyoxal added to the culture medium. The frequencies of methylglyoxal-induced base-pair substitutions were higher in the NER-deficient strains than in the wild-type strain, in the presence and absence of mucAB gene. Paradoxically, the frequency of methylglyoxal-induced TGGC frameshifts was higher in the wild-type strain than in the NER-deficient strains. When the methylglyoxal-induced mutation spectra in the presence and absence of mucAB gene are compared, the ratios of base-pair substitutions to frameshifts were increased by the effects of mucAB gene. In the three strains, more than 75% of the base-pair substitutions occurred at G:C sites, independent of the mucAB gene. When the mucAB gene was present, G:C-->T:A transversions were predominant, followed by G:C-->A:T transitions. When the mucAB gene was absent, the predominant mutations differed in the three strains: in the wild-type and uvrC strains, G:C-->A:T transitions were predominant, followed by G:C-->T:A transversions, while in the uvrA strains, G:C-->T:A transversions were predominant, followed by G:C-->A:T transitions. These results suggest that NER may be involved in both the repair and the fixation of methylglyoxal-induced mutations.

Methylglyoxal induces G:C to C:G and G:C to T:A transversions in the supF gene on a shuttle vector plasmid replicated in mammalian cells.

We previously reported that the majority of base-pair substitutions induced by an endogenous mutagen, methylglyoxal, were G:C-->T:A transversions and G:C-->A:T transitions in wild-type and nucleotide excision repair (NER)-deficient (uvrA or uvrC) Escherichia coli strains. To investigate the mutation spectrum of methylglyoxal in mammalian cells and to compare the spectrum with those detected in other experimental systems, we analyzed mutations in a bacterial suppressor tRNA (supF) gene in the shuttle vector plasmid pMY189. We treated pMY189 with methylglyoxal and immediately transfected it into simian COS-7 cells. The cytotoxicity and the mutation frequency (MF) increased according to the dose of methylglyoxal. In the mutants induced by methylglyoxal, multi-base deletions were predominant (50%), followed by base-pair substitutions (35%), in which 89% of the substitutions occurred at G:C sites. Among them, G:C-->C:G and G:C-->T:A transversions were predominant. The overall distribution of methylglyoxal-induced mutations detected in the supF gene was different from that for the spontaneous mutations. These results suggest that methylglyoxal may take part in causing G:C-->C:G and G:C-->T:A transversions in vivo.

Mutational specificity of glyoxal, a product of DNA oxidation, in the lacI gene of wild-type Escherichia coli W3110.

To determine the mutation spectrum of glyoxal, which is produced from DNA by oxygen free-radicals, we analyzed the chromosomal lacI gene of mutants induced by treatment of a wild-type Escherichia coli strain with glyoxal. The cell death and the mutation frequency increased according to the concentration of glyoxal added to the culture medium. The majority of the spontaneous mutations (82%) and that of the glyoxal-induced mutations (50%) were the addition or deletion of a 5'-TGGC-3' sequence at positions 623-634, which was reported to be a mutational hot spot in the lacI gene. In the glyoxal-induced mutants, however, the ratio of base-pair substitutions was increased (35%). Although all types of base-pair substitutions were detected, 78% of the base-pair substitutions occurred at G:C sites. Among them, G:C-->A:T transitions were predominant, followed by G:C-->T:A transversions. These mutations appeared to be distributed randomly within the lacI gene. These results suggest that glyoxal-induced mutations may correlate to mutations induced by oxygen free-radicals.

Comparison of oxidation products from DNA components by gamma-irradiation and Fenton-type reactions.

The four 2'-deoxyribonucleosides were gamma-irradiated or were aerobically treated with Fenton-type-reagents, Fe(II)-EDTA or a renal carcinogen Fe(II)-nitrilotriacetic acid (NTA) under the neutral conditions. The reaction mixtures were immediately analyzed by reverse-phase HPLC. Major products detected were 2-hydroxydeoxyadenosine (2-OH-dA), 8,5'-cyclodeoxyadenosine (cyclo-dA), 8-hydroxydeoxyadenosine (8-OH-dA). 5-formyldeoxyuridine (5-CHO-dU), 5-hydroxydeoxycytidine (5-OH-dC), 8-hydroxydeoxyguanosine (8-OH-dG), 8,5'-cyclodeoxyguanosine (cyclo-dG), and glyoxal and its adduct with dG. Ratio of these oxidized products were dramatically changed depending upon the agents used. For example, 2-OH-dA was a modified nucleoside produced most efficiently by Fe(II)-EDTA, while 5-CHO-dU and 5-OH-dC were the major products by the Fe(II)-NTA treatment and gamma-irradiation, respectively. Glyoxal itself was estimated to be produced most frequently (13 folds of 8-OH-dG) when treated with Fe(II)-EDTA, but its formation was not detected by the treatment with Fe(II)-NTA or by gamma-irradiation. 8-OH-dA was not produced by Fe-EDTA or Fe-NTA but was produced by gamma-irradiation. In contrast, 2-OH-dA was not produced by gamma-irradiation. These results suggest that triphosphates of 2-OH-dA, cyclo-dA, 8-OH-dA, cyclo-dG, 5-CHO-dU, 5-OH-dC, and glyoxal-dG as well as 8-OH-dG may be produced in cells with different ratio by various types of oxidative stress and involved in mutagenesis and carcinogenesis.

Mutations induced by glyoxal and methylglyoxal in mammalian cells.

To investigate the mutation spectra of glyoxal and methylglyoxal in mammalian cells, we analyzed mutations in a bacterial suppressor tRNA (supF) gene in the shuttle vector plasmid pMY189. The cytotoxicity and the mutation frequency increased according to the doses of glyoxal and methylglyoxal. The majority of glyoxal-induced mutations (65%) were base-pair substitutions, in which G:C-->C:G transversions were predominant. In the mutants induced by methylglyoxal, multi-base deletions were predominant (50%), followed by base-pair substitutions (35%), in which G:C-->C:G and G:C-->T:A transversions were predominant.

Formation of a mutagen, glyoxal, from DNA treated with oxygen free radicals.

Oxygen free radicals cause extensive chemical changes in DNA, including base and sugar modifications and strand breaks. In the present study we found that a mutagen, glyoxal, is produced by exposure of DNA to an oxygen radical forming system (5 mM FeSO4-EDTA, 37 degrees C, 60 min). It was produced with 17 times higher efficiency than 8-hydroxydeoxyguanosine. Thus it is possible that the formation of glyoxal is one of the major types of damage in DNA exposed to oxygen free radicals.

Glyoxal, a major product of DNA oxidation, induces mutations at G:C sites on a shuttle vector plasmid replicated in mammalian cells.

Glyoxal is a major product of DNA oxidation in which Fenton-type oxygen free radical-forming systems are involved. To determine the mutation spectrum of glyoxal in mammalian cells and to compare the spectrum with those observed in other experimental systems, we analyzed mutations in a bacterial suppressor tRNA gene (supF) in the shuttle vector plasmid pMY189. We treated pMY189 with glyoxal and immediately transfected it into simian COS-7 cells. The cytotoxicity and mutation frequency increased according to the dose of glyoxal. The majority of glyoxal-induced mutations (48%) were single-base substitutions. Eighty three percent of the single-base substitutions occurred at G:C base pairs. Among them, G:C-->T:A transversions were predominant, followed by G:C-->C:G transversions and G:C-->A:T transitions. A:T-->T:A transversions were also observed. Mutational hotspots within the supF gene were detected. These results suggest that glyoxal may play an important role in mutagenesis induced by oxygen free radicals.

Nucleotide excision repair proteins may be involved in the fixation of glyoxal-induced mutagenesis in Escherichia coli.

To investigate the influence of nucleotide excision repair (NER) on glyoxal-induced mutations, we treated wild-type and NER-deficient (uvrC) Escherichia coli strains with glyoxal, and analyzed mutations in the chromosomal lacI gene. In both strains, the cell death and the mutation frequency increased according to the dose of glyoxal added to the culture medium, and cell death was induced to a similar level in both strains. Interestingly, the frequency of glyoxal-induced mutations in the wild-type strain was higher than that in the uvrC strain. Particularly, the frequency of base-pair substitutions was 4.7-fold higher in the wild-type strain. In the wild-type strain, G:C-->T:A transversions were predominant, followed by G:C-->A:T and A:T-->T:A mutations. In the uvrC strain, G:C-->A:T transitions were predominant, followed by G:C-->T:A transversions. All the base-pair substitutions except for G:C-->A:T transitions were >4-fold higher in the wild-type strain than in the uvrC strain. These results suggest that NER may be involved in the fixation of glyoxal-induced base-pair substitutions.

Hydrolysis of oxidized nucleotides by the Escherichia coli Orf135 protein.

To examine the possibility that the Orf135 protein of Escherichia coli functions as a hydrolyzing enzyme for a damaged DNA precursor (deoxyribonucleoside 5'-triphosphate), we purified the recombinant Orf135 protein and incubated it with oxidized deoxynucleotides. Of the nucleotides tested, 2-hydroxydeoxyadenosine 5'-triphosphate, and somewhat less efficiently, 8-hydroxydeoxyguanosine 5'-triphosphate, were hydrolyzed by this protein. These damaged deoxynucleotides elicit transversion mutations in E. coli (Inoue, M., Kamiya, H., Fujikawa, K., Ootsuyama, Y., Murata-Kamiya, N., Osaki, T., Yasumoto, K., Kasai, H. (1998) J. Biol. Chem. 273, 11069-11074). These results suggest that this protein may be involved in the prevention of mutations induced by these oxidized deoxynucleotides.

Mutagenicity of 5-formyluracil in mammalian cells.

5-Formyluracil, a major oxidized form of thymine, was incorporated into a predetermined site of one of the leading and lagging template strands of a double-stranded vector, and the DNA replication efficiency and the mutation frequency of 5-formyluracil in simian COS-7 cells were investigated. 5-Formyluracil did not block DNA replication and was weakly mutagenic in simian cells. 5-Formyluracil primarily elicited base substitutions at the modified positions.

8-Hydroxyguanine (7,8-dihydro-8-oxoguanine) in hot spots of the c-Ha-ras gene: effects of sequence contexts on mutation spectra.

We previously reported that 8-hydroxyguanine (7,8-dihydro-8-oxoguanine) at the second position of codon 12 of the c-Ha-ras gene induces many types of mutations in NIH3T3 cells. In this study we incorporated the modified base into the first and second positions of codon 12 in the coding strand and into the first position of codon 61 in the non-coding strand of the gene using a new 8-hydroxyguanine phosphoramidite as a building block during oligonucleotide synthesis. The ras genes with 8-hydroxyguanine were transfected into NIH3T3 cells and the mutations induced were analyzed. 8-Hydroxyguanine residues at the first positions of codons 12 and 61 induced mutations to T at the modified sites almost exclusively. On the other hand, the DNA lesion at the second position of codon 12 induced a G-->A transition in addition to a G-->T transversion, confirming our previous results. Mutations in 5'-flanking sites were observed with 8-hydroxyguanine at the second position of codon 12 or the first position of codon 61. These results indicate that 8-hydroxyguanine in mammalian cells mainly induces a G-->T transversion at the modified site, but that other types of mutations are also elicited.

Determination of carcinogenic potential of mineral fibers by 8-hydroxydeoxyguanosine as a marker of oxidative DNA damage in mammalian cells.

8-Hydroxydeoxyguanosine (8-OH-dG) is a typical form of oxidative DNA damage, which causes mutations in vitro and in vivo. To develop a simple method of testing the carcinogenicity of fibrous materials, the formation of 8-OH-dG was determined in the DNA of J774 cells, an established reticulum cell sarcoma line, after treatment with various natural and man-made mineral fibers. The amount of 8-OH-dG was determined using high-pressure liquid chromatography (HPLC) equipped with an electrochemical detector (ECD). We tested three natural mineral fibers (crocidolite, amosite, and chrysotile) and three man-made mineral fibers (ceramic, glass, and potassium octatitanate). Among them, a significant increase in 8-OH-dG formation was observed in the crocidolite- and amosite-treated cells. We also measured the amount of tumor necrosis factor (TNF) produced by J774 cells incubated with the fibrous materials. Cellular TNF production increased after treatment with all the fibers tested, but it was not statistically significant except in the case of chrysotile. Therefore, these results indicate that the mechanism of TNF production is different from that of 8-OH-dG formation, and that the carcinogenicity of various fibrous materials can be better evaluated by measuring the 8-OH-dG level in J774 cellular DNA after treatment with these fibers.

Induction of chromosomal gene mutations in Escherichia coli by direct incorporation of oxidatively damaged nucleotides. New evaluation method for mutagenesis by damaged DNA precursors in vivo.

We have developed a new strategy for the evaluation of the mutagenicity of a damaged DNA precursor (deoxyribonucleoside 5'-triphosphate) in Escherichia coli. 8-Hydroxydeoxyguanosine triphosphate (8-OH-dGTP) and 2-hydroxydeoxyadenosine triphosphate (2-OH-dATP) were chosen for this study because they appear to be formed abundantly by reactive oxygen species in cells. We introduced the oxidatively damaged nucleotides into competent E. coli and selected mutants of the chromosomal lacI gene. Both damaged nucleotides induced lacI gene mutations in a dose-dependent manner, whereas unmodified dATP and dGTP did not appear to elicit the mutations. The addition of 50 nmol of 8-OH-dGTP and 2-OH-dATP into an E. coli suspension induced 12- and 9-fold more substitution mutations than the spontaneous event, respectively. The 8-OH-dGTP induced A.T --> C.G transversions, and the 2-OH-dATP elicited G.C --> T.A transversions. These results indicate that the two oxidatively damaged nucleotides are mutagenic in vivo and suggest that 8-OH-dGTP and 2-OH-dATP were incorporated opposite A and G residues, respectively, in the E. coli DNA. This new method enables the evaluation and comparison of the mutagenic potentials of damaged DNA precursors in vivo.

Comparison of incorporation and extension of nucleotides in vitro opposite 8-hydroxyguanine (7,8-dihydro-8-oxoguanine) in hot spots of the c-Ha-ras gene.

DNA templates with 8-hydroxyguanine (7,8-dihydro-8-oxoguanine, oh8Gua) at a site corresponding to the first or second position of codon 12 of the c-Ha-ras gene were prepared, and the nucleotides inserted opposite the modified base were compared. The Klenow fragment (KF) of Escherichia coli DNA polymerase I inserted C opposite oh8Gua at both positions. Taq DNA polymerase incorporated C and A opposite oh8Gua, and the ratio of C to A was higher at the first position than at the second position. DNA polymerase alpha (pol alpha) inserted A and C at the first position, and A at the second position of codon 12, indicating that the ratio of C to A was higher at the first position. Moreover, we studied the extensions of bases paired with oh8Gua by DNA polymerases with or without 3'-5' exonuclease activity. G and T opposite oh8Gua were removed, and subsequently C was inserted by KF. We found that an oh8Gua:A pair was recognized by the exonuclease activity of the enzyme and that A was partially substituted by C. On the other hand, pol alpha extended only C and A opposite oh8Gua. No difference was observed with oh8Gua at the two positions. These results indicate that the ratio of nucleotides incorporated opposite oh8Gua depends on the sequence context, while there is no particular difference in the extension of base pairs involving oh8Gua by DNA polymerases.

8-Hydroxyadenine (7,8-dihydro-8-oxoadenine) induces misincorporation in in vitro DNA synthesis and mutations in NIH 3T3 cells.

An oligodeoxyribonucleotide containing 8-hydroxyadenine (OH8Ade) was chemically synthesized and single- and double-stranded c-Ha-ras gene fragments with OH8Ade at the second position of codon 61 were prepared. The single-stranded ras gene fragment was used as a template for in vitro DNA synthesis with the Klenow fragment of Escherichia coli DNA polymerase I, Taq DNA polymerase, rat DNA polymerase beta and mouse DNA polymerase alpha. The former two enzymes exclusively incorporated dTMP opposite OH8Ade. The DNA polymerases alpha and beta misinserted dGMP, and dAMP and dGMP, respectively. The c-Ha-ras gene was constructed using the double-stranded ras gene fragment containing OH8Ade and was transfected into NIH 3T3 cells. The gene with OH8Ade induced focus formation, indicating that OH8Ade elicited point mutations in cells. When c-Ha-ras genes present in transformed cells were analyzed, an A-->G transition and an A-->C transversion were detected. These results indicate that OH8Ade induced misincorporation in in vitro DNA synthesis and mutations in mammalian cells.